Abstract Background BAP1 is a tumor-suppressive deubiquitinase essential for DNA repair, and missense mutations in BAP1 are common in clear cell renal cell carcinoma (ccRCC). We previously showed that precise correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 activity, reinstated anchorage-dependent growth, and re-sensitized cells to anoikis. Here, we asked the converse question: whether disrupting Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells. Methods Using the same adenine base-editing strategy, we introduced an inactivating Glu31Gly mutation into HK-2 normal kidney epithelial cells, generating two independent isogenic BAP1-mutant clones. As an additional control, we created a BAP1-knockout HK-2 clone via CRISPR/Cas9. Parental, mutant, and knockout cells were assessed for BAP1 enzymatic activity, DNA repair capacity, viability, proliferation, cell cycle status, anchorage-independent growth, and anoikis resistance. Migration and invasion of HK-2 mutants and knockouts were compared with KMRC-20 revertant clones in which endogenous Glu31Lys had been corrected. Results The Glu31Gly HK-2 mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair—phenotypes comparable to BAP1-knockout cells—confirming successful functional inactivation. Despite this, both mutant and knockout HK-2 cells maintained parental-like morphology, viability, and proliferation. Surprisingly, Glu31Gly did not confer anchorage-independent growth or anoikis resistance: upon detachment, both mutant and knockout cells showed increased apoptosis. In contrast, in KMRC-20 cells, restoration of BAP1 activity enhanced both migration and invasion. Conversely, BAP1 inactivation or loss in HK-2 cells increased invasion but paradoxically reduced migration. These opposite outcomes indicate that BAP1 regulates motility through distinct mechanisms in normal versus malignant renal cells, likely reflecting differences in lineage state, cytoskeletal organization, and downstream signaling. Conclusions Although BAP1 restoration suppresses anchorage-independent growth and anoikis resistance in KMRC-20 ccRCC cells, BAP1 inactivation alone is insufficient to induce these oncogenic traits in normal HK-2 epithelial cells, implying that additional oncogenic alterations are required for anchorage-independent survival during kidney tumorigenesis. The divergent effects of BAP1 gain versus loss on migration and invasion further underscore the context-dependent nature of BAP1 function. These base-editing studies demonstrate that BAP1 differentially regulates adhesion, anoikis, and motility in normal and malignant renal cells and highlight the utility of precise base editing for dissecting clinically relevant mutations.

Neospora caninum, the causative agent of abortion in cattle, has a major economic impact worldwide. This review aims to provide an overview of key advances of the last 5-8 years in understanding host-pathogen interactions, molecular mechanisms, and emerging control strategies. Epidemiological studies have revealed the influence of environmental, genetic, and ecological factors on parasite transmission dynamics, and emphasized the importance of integrated "One Health" strategies. Characteristics of different Neospora strains have been elucidated through animal models and molecular tools such as clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-based gene editing, high-throughput sequencing and advanced proteomics, aiming to shed light on stage-specific gene regulation and virulence factors, contributing to the development of interventions against neosporosis. Insights into immune modulation, immune evasion and parasite persistence contributed to the efforts towards vaccine development. In terms of therapeutics, repurposed drugs but also more targeted inhibitors have shown promising efficacy in reducing parasite burden and mitigating vertical transmission in laboratory models. Here, more recent innovations in nanoparticle-based drug delivery systems and immunomodulatory strategies are prone to enhance therapeutic outcomes. However, a significant challenge remains the integration of molecular and immunological insights into practical applications.

Abstract Genetic inactivation of SKP2 has been shown to effectively prevent cancer initiation and block tumorigenesis. However, direct in vivo evidence for SKP2 on cancer initiation and prostatic microenvironment is still lacking and a SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting SKP2. We therefore have established a prostate-specific human SKP2 knock-in mouse model driven by an endogenous mouse probasin promoter. Overexpression of hSKP2 induces PIN and low-grade carcinoma. RNA-sequencing analysis revealed significant gene expression alterations in EMT, extracellular matrix, and interferon signaling. Single cell deconvolution showed an increase of fibroblast population and a decrease of CD8+ T cell and B cell populations. Consistently with these results from the SKP2 humanized mouse, SKP2 protein is overexpressed in human prostatic hyperplasia, PIN and prostate adenocarcinoma compared to normal prostate tissues. Overexpression of SKP2 markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decreased viability and altered the morphologies of organoids of hSKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific hSKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.

As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve an Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI) (asymptomatic plants), as well as in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37-39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in wheat blast hotspot district, Meherpur in Bangladesh. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as a modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers.

Abstract Myotonic dystrophy type 1 (DM1) is caused by toxic CTG repeat expansions in the 3′UTR of the DMPK gene, leading to pathogenic RNA gain-of-function effects and widespread splicing abnormalities. RNA-targeting strategies such as antisense oligonucleotides (ASOs) and CRISPR-Cas13 hold strong therapeutic promise, but require reproducible design frameworks that balance specificity with potency. Here, we present a transparent computational pipeline for candidate identification and evaluation at the DMPK locus. The pipeline integrates off-target searches, RNA structure predictions, and composite scoring metrics to generate 50 ASO and 50 Cas13 candidates. ASOs achieved absolute specificity with zero off-targets, clustering tightly around moderate composite scores (mean 57.71), while Cas13 guides consistently carried a single off-target that mapped uniquely to the DMPK gene, with no additional genes affected yet delivered superior thermodynamic properties and higher corrected scores (62.12 vs. 57.71). By anchoring design to the pathogenic 3′UTR region and releasing complete candidate listings, this framework ensures methodological rigor, reproducibility, and translational relevance. Overall, it advances readiness for DM1 therapy by combining ASO safety with Cas13 potency, and establishes a reproducible foundation for precision RNA therapeutics across both monogenic and complex diseases.

Abstract Background Fulfilling the promise of human genetics in elucidating disease requires identifying causal variants and genes underlying genetic association signals. Molecular quantitative trait locus (molQTL) analyses, e.g. expression QTL (eQTL) and splicing QTL (sQTL), link genetic variants to intermediate molecular phenotypes, but pinpointing causal variants and their regulatory effects remains challenging. Here, we integrate sQTL analysis with deep-learning-based splicing effect annotation to identify causal genetic variants and elucidate their functional mechanisms affecting human phenotypes. Results Using a single-cell GWAS method (scHi-HOST) on 96 lymphoblastoid cell lines (LCLs) with and without influenza A virus (IAV) infection, we discovered ~ 43,000 sQTLs associated with 217 genes after IAV infection. Integrating sQTLs with AI splice prediction, we uncovered 76 likely causal variants that affect cis-acting molecular splicing components (5’ donor, 3’ acceptor), supported by further computational analysis. Among these, we experimentally validated a causal sQTL signal affecting poly (ADP-ribose) polymerase 2 (PARP2). The causal variant, rs2297616, alters the 5’ splice donor site in the second intron of PARP2 , resulting in two protein isoforms differing by 13 amino acids. The derived A allele was associated with the longer protein isoform and increased IAV levels in LCLs. CRISPR editing validated the causal effect of this variant on both protein length and IAV infection. Lastly, these 76 putative causal sQTLs were further linked to over a hundred GWAS traits, including many variants associated with autoimmune diseases. Conclusions Our work provides a catalog of causal sQTL with direct splicing impacts, providing causal mechanistic insights from genotype to disease susceptibility.

Spinal cord regeneration requires a transformative strategy capable of rewriting inhibitory genetic programs while orchestrating real-time electrical communication with regenerating neural tissues. Recent advancements in precision CRISPR genome editing effectively silence or activate crucial molecular gatekeepers such as PTEN, SOCS3, and various epigenetic repressors, thereby reactivating dormant intrinsic regenerative pathways and enabling robust axonal growth. Concurrently, cutting-edge bioelectronic technologies utilizing piezoelectric, triboelectric, and magnetoelectric scaffolds have emerged, adeptly harnessing the body's inherent biomechanical energy. These innovative materials convert subtle physiological micromotions into finely tuned electrical stimuli, precisely guiding neuronal regeneration without external power sources, addressing limitations associated with traditional implants such as infection risks and mechanical incompatibility.Integrating these genetic modifications with bioelectric innovations creates a potent synergy. Genome-level reprogramming amplifies neuronal responsiveness to bioelectrical signals, markedly enhancing axonal regeneration. Simultaneously, autonomous electrical stimulation sustains and stabilizes cellular, metabolic, and synaptic improvements induced by genomic interventions, forming a closed-loop, self-sustaining therapeutic platform. This advanced system significantly transcends conventional transient recovery approaches, moving toward durable, personalized outcomes. Such convergence of advanced genetic engineering and intelligent biomaterial design represents a groundbreaking shift in regenerative neurology.Despite promising preclinical outcomes, significant translational challenges remain. Critical hurdles include ensuring precise delivery of CRISPR tools, mitigating off-target genomic effects, enhancing biocompatibility and scaffold stability, and navigating rigorous regulatory pathways. Addressing these challenges necessitates integrating next-generation gene-editing technologies, comprehensive genomic surveillance, advanced biomaterial sciences, and meticulous preclinical evaluations. Future directions in spinal cord injury research encompass multiplex genome editing, AI-driven scaffold optimization via digital twins, and tailored immune-evasive biomaterials. Collectively, this innovative approach has the potential to redefine regenerative medicine's boundaries, offering unprecedented hope for sustained, personalized recovery and dramatically improving quality of life for individuals affected by spinal cord injuries.

Abstract Background Heterozygous variants in CTNND2 , encoding the brain-specific protein δ-catenin, are associated with a broad spectrum of neurodevelopmental disorders, including dyslexia, attention deficit hyperactivity disorder, intellectual disability, and autism. Despite its clinical significance, the full phenotypic spectrum of CTNND2 -associated disorders and the neurodevelopmental role of δ-catenin, a key component of the cadherin-catenin cell adhesion complex, remain poorly defined. Methods Through international collaboration, we assembled the phenotypic and molecular information for 57 individuals, 42 previously unpublished, carrying heterozygous CTNND2 variants. All individuals were evaluated by local clinicians, and the variants were identified through exome or genome sequencing, clinical microarray, or karyotyping. To investigate the effects of δ-catenin loss on early neurogenesis, we performed neural differentiation and transcriptomic profiling in three patient-derived neural stem cell lines and three CRISPR-Cas9-generated CTNND2 knockout lines. In one patient-derived line, we further analyzed cerebral organoid development and performed pathway modulation to assess phenotypic rescue. Results The 41 CTNND2 variants included 12 previously reported loss-of-function- and one missense variant, and 28 novel variants comprising 10 missense and 18 predicted loss-of-function changes. Eight of the novel variants occurred de novo , and 12 were inherited from a parent with a neurodevelopmental phenotype. The most common clinical features were developmental delay (90%), intellectual disability (74%), and behavioral abnormalities (79%). Functional studies revealed impaired early neurogenesis in one patient-derived line, characterized by aberrant neural rosette formation. Transcriptome analysis showed dysregulated WNT signaling, and partial rescue of these defects was achieved by modulating the WNT pathway, highlighting δ-catenin's role in early neural development. Conclusions This study defines the clinical symptoms of CTNND2 -related neurodevelopmental disorders, outlining a recognizable yet variable phenotype that overlaps with other forms of intellectual disability and autism. Our findings provide preliminary evidence of genotype–phenotype correlations and highlight δ-catenin's critical role in modulating WNT signaling during early neural development. These insights advance our understanding of CTNND2 -associated disorders and support the importance of mechanistic studies to inform personalized diagnostics and therapies.

Background: Aging brains are shaped by a persistent dialogue between declining neurogenesis and rising neuroinflammation. Neural stem cells progressively lose regenerative capacity, while microglia and astrocytes shift toward maladaptive states that erode synaptic plasticity and cognition. This convergence defines inflammaging, a slow yet relentless process that undermines resilience. However, the field remains hampered by critical gaps: incomplete mapping of microglial heterogeneity, poorly understood epigenetic scars from inflammasome signaling, lack of longitudinal data, unclear niche-specific immune mechanisms, and uncertain cross-species relevance. This review addresses these pressing barriers, aiming to transform fragmented insights into actionable strategies. Summary: I chart how neurogenesis and neuroinflammation operate in continuous dialogue, identify five major knowledge gaps, and evaluate strategies to reprogram this interaction. Approaches include longitudinal imaging, niche-focused immunomodulation, glial subtype reprogramming, brain-penetrant inflammasome inhibitors, and CRISPR-based epigenetic editing. Each strategy is mapped against translational potential, short-term feasibility, and long-term vision, with emphasis on how mechanistic precision can guide clinical innovation. Conclusion: Here I highlight that neurogenic potential is not entirely lost with age but may be preserved or restored by tuning immune and epigenetic environments. This review proposes a roadmap for reshaping the aging brain’s fate, offering mechanistically grounded strategies to delay cognitive decline. Beyond neurology, the work underscores a broader principle: by integrating cellular plasticity with immune modulation, science edges closer to re-engineering resilience across the lifespan.

The Hedgehog (Hh) signaling pathway is a key regulator of adipogenesis and lipid metabolism. However, the specific role of its receptor, Patched2 (Ptch2), in these processes remains unknown. Here, using a CRISPR/Cas9-mediated ptch2 homozygous mutation model in Nile tilapia (Oreochromis niloticus), we found that Ptch2 deficiency induced visceral and perirenal lipomatosis characterized by small, multinucleated adipocytes. Comparative adipose transcriptomics revealed pronounced adipogenic reprogramming, with marked upregulation of genes governing de novo lipogenesis (e.g., acaca, fasn), fatty acid desaturation (e.g., scd, fadsd6), and triglyceride synthesis (e.g., dgat2, lpl). Biochemically, mutants exhibited elevated blood glucose and liver transaminases (alanine aminotransferase, aspartate aminotransferase) with reduced alkaline phosphatase, indicating systemic metabolic dysregulation and hepatic stress. Our findings demonstrate that loss of Ptch2 triggers lipoma formation and adipogenic transcriptome reprogramming, highlighting its essential role in maintaining adipose tissue homeostasis.

A/T(U) and G/C nucleobase pair formation in DNA and RNA are crucial to numerous fundamental biological processes, including replication, transcription, and translation. The specificity of A/T(U) and G/C base pairing is used to recognize complementary sequences in medical and biotechnological applications, such as PCR, nucleic acid drugs, and CRISPR–Cas9-based gene editing. To understand the fidelity of biological reactions and improve the accuracy and efficacy of applications, particularly by avoiding off-target binding, clarifying the mechanism of recognition of complementary bases or sequences is essential. Despite the prevailing view that Watson-Crick hydrogen bonding is a primary mechanism for complementary base recognition, several experiments have shown that DNA polymerase does not require hydrogen bonding to select complementary bases. Other factors—such as the geometry of bases and base stacking—appear to be involved in the selection. Artificial base pairs lacking hydrogen bonds but recognized by DNA polymerase were successfully designed solely based on base-pair geometry. However, hydrogen bonding also contributes to recognition. Furthermore, the accuracy of selecting a complementary nucleobase or sequence varies across reactions, suggesting the existence of multiple selection mechanisms. This review provides an overview of biological processes and applications involving base pairing and discusses the molecular mechanism underlying complementary base recognition.

Background: NOTCH receptors play a pivotal role in carcinogenesis. Upon ligand binding, a cascade of proteolytic cleavages mediated by ADAM proteases and the γ-secretase complex activates the receptor, culminating in the release of the NOTCH intracellular domain (NICD). NICD translocates to the nucleus, where it modulates gene expression. The review aims to assess γ-secretase inhibitors (GSIs) as anticancer agents in preclinical and clinical settings, focusing on their ability to block tumor progression, target cancer stem cells, and overcome resistance to standard therapies. Methods. A systematic search was conducted in the ISI Web of Science, PubMed and Scopus databases, following the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines. The review included preclinical in vitro and in vivo studies and clinical trials on GSIs as monotherapy or in combination with other therapies for TNBC, metastatic melanoma, PDAC, GC, and NSCLC. Excluded were duplicates, non-English articles, articles with unknown authors in Scopus, studies before 2010, studies on non-cancer conditions, studies unrelated to NOTCH signaling, and studies outside the cancer types under study. Risk of bias was assessed through study design factors like randomization, blinding, and data handling. Findings were synthesized qualitatively across the selected cancer types. Results. This review analyzes therapeutic advances using GSIs in cancers driven by oncogenic NOTCH signaling, based on 61 sources covering NSCLC, TNBC, metastatic melanoma, gastric cancer, PDAC, and global epidemiology data. Preclinical studies show GSIs synergize with chemotherapy, radiotherapy, and novel agents, especially in NSCLC, melanoma, and TNBC, blocking EMT, overcoming resistance, and improving prognosis. Common GSIs include DAPT and RO4929097, which enhance drugs like gemcitabine (PDAC), paclitaxel, osimertinib, erlotinib (NSCLC), and 5-FU (GC, TNBC). Promising combinations include GSIs with SAHA, ATRA, and CB103 in TNBC. Clinical trials remain limited: PDAC trials with gemcitabine showed no benefit; melanoma trials had modest results; TNBC trials showed partial responses but overall low efficacy and severe adverse events. RO4929097 is the most clinically tested GSI. Discussion. Clinical trials with γ-secretase inhibitors (GSIs) have underperformed despite promising preclinical results, largely due to tumor heterogeneity, dosing challenges, and non-selective inhibition. Future strategies should prioritize receptor-specific GSIs, patient stratification based on NOTCH pathway activation, and combination therapies administered at optimized doses. Emerging approaches include integrating immunotherapy with advanced techniques such as CRISPR, CAR-T cells, and bispecific antibodies, alongside targeted delivery systems to enhance efficacy and minimize toxicity. Addressing the tumor microenvironment, which contributes to therapy resistance, and inhibiting tumor angiogenesis are also active areas of investigation. Finally, leveraging artificial intelligence and big data for personalized medicine, including male- and female-specific considerations, will be critical for improving patient outcomes.

Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterisation of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differ significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterisation based on ancestral spacer (AS) and TR sequences indicate that E. faecalis with the same CRISPR1-AS genotype do not always harbour same CRISPR2-AS genotypes, and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to multilocus sequence typing (MLST), suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in the Southeast Asia region.

Abstract Background Resistance to carbapenems and third-generation cephalosporins is increasing in Klebsiella pneumoniae globally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely used to circumvent β-lactamase-mediated resistance. In 2021, an unusual K. pneumoniae clinical isolate, KpMVR1, was recovered from a hospitalised patient in England, exhibiting resistance to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. To investigate this phenomenon, we characterised the genome and antimicrobial susceptibility of KpMVR1 alongside two clonally related isolates susceptible to all three β-lactam/β-lactamase inhibitor combinations: KpMVS1, collected from the same patient 42 days earlier, and KpMVS2, from another patient in the same hospital. Methods Illumina and MinION whole-genome sequencing were conducted for these three isolates, followed by hybrid genome assembly. Annotated genome assemblies were compared to identify genetic variation. Mutagenesis experiments were performed to verify predicted functional alterations. Results All isolates belonged to clone ST8134 and carried bla KPC−2 alleles (KpMVR1: bla KPC−157 ; KpMVS1 and KpMVS2: bla KPC−2 ) in presumptively conjugative plasmids. Insertion sequence IS Ec68 caused a frameshift mutation in KpMVR1’s ompK36 gene, reducing susceptibility to meropenem-vaborbactam and imipenem-relebactam. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. Conclusions KpMVR1 carried multiple resistance-associated genetic alterations and likely developed its resistance profile through within-patient evolution. This study highlights the importance of routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy and the need to characterise underlying resistance mechanisms to assess the risk of onward dissemination.

Abstract Background Cold stress severely limits the productivity and geographical distribution of mango, a tropical fruit crop of significant economic importance. We combined transcriptomic profiling and functional genomics to investigate the molecular mechanisms underlying cold tolerance in mango. Results Comparative analysis of the cold-tolerant Jinhuang (JH) and cold-sensitive Guiqi (GQ) cultivars identified 7,757 differentially expressed genes, with significant enrichment in plant hormone signal transduction pathways. Notably, the DELLA protein GAIP-B (LOC123214451) was markedly upregulated in JH under cold stress. Functional validation through CRISPR/Cas9-mediated knockout and overexpression in Nicotiana benthamiana demonstrated that GAIP-B is both necessary and sufficient for cold tolerance. The knockout lines exhibit extreme cold sensitivity, reduced antioxidant enzyme activity, increased oxidative damage, and impaired osmotic adjustment. Conversely, the overexpression lines showed enhanced cold tolerance, with superior antioxidant capacity, maintenance of photosynthetic pigments, and increased soluble sugar accumulation. Conclusions These findings establish GAIP-B as a central regulator that integrates antioxidant defense, photosynthetic protection, and osmotic adjustment in the cold stress response. This study provides valuable insight into molecular breeding strategies aimed at enhancing cold tolerance in mango and other economically important fruit crops.

Abstract K. pneumoniae strains that combine multidrug resistance, hypervirulence and persistence are spreading worldwide, causing a severe threat to public health. Unraveling the genetics that underlies these high-risk clones is critical for the development of countermeasures. We isolated K. pneumoniae JU-BAEC-01 from treated effluent of antibiotic-manufacturing pharmaceutical facilities in Bangladesh. Herein, we report a comprehensive genomic analysis of the K. pneumoniae strain JU-BAEC-01 using whole-genome sequencing, comparative genomics, and various bioinformatics tools, including CARD, ResFinder, VFDB, PADLOC, Defense Finder, and CRISPRCas Finder, to outline its phylogenetic position, antibiotic resistance profile, virulence potential, mobile genetic elements, and antiviral defense systems. JU-BAEC-01 belongs to a phylogenetically distinct lineage, serotype O3b:KL150, unrelated to globally dominant high-risk clones. This isolate shows resistance to nearly all clinically relevant antibiotic classes except carbapenems and colistin, mediated by an extensive acquired resistome, including tmexCD3-toprJ3 (tigecycline), armA, aac(6')-Ib-cr, qnrB4, oqxAB, blaDHA-1, blaSHV-182, and blaTEM-1B, mostly carried on conjugative IncC, IncFIB, IncHI1B, and IncR plasmids. Classical hypervirulence markers are present: complete aerobactin (iucABCD-iutA) and salmochelin (iroBCDEN) clusters, rmpA2, type 1 and type 3 fimbriae, T6SS, and pgaABCD. Six prophage regions and multiple insertion elements further enhance genomic plasticity. Notably, the strain encodes one of the most elaborate anti-phage defense arsenals reported in Klebsiella to date, comprising functional Type I-E, III-A, and IV-A CRISPR-Cas systems, multiple restriction-modification systems, BREX Type I, abortive infection systems (AbiE, AbiU), and additional novel defenses that coexist with phage-derived anti-CRISPR (AcrIE9) and anti-restriction (ArdA) proteins. Klebsiella pneumoniae JU-BAEC-01 is a "perfect storm" pathogen that combines pan-drug resistance (PDR), hypervirulence, and a multilayered, highly developed defense against bacteriophages. This genomic convergence confounds treatment options and emphasizes the evolutionary capability of this priority pathogen to resist both the antimicrobial and natural predatory pressures. The presence of phage anti-defense systems underlines a dynamic co-evolutionary arms race with significant implications for the potential failure of phage therapy against such robustly defended isolates.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) and next-generation al-logeneic T cell therapies are curative options for hematologic malignancies, but their effi-cacy is often limited by graft-versus-host disease (GvHD), a complex syndrome resulting from dysregulated donor–host immune interactions. GvHD is mediated by interconnected signaling pathways that regulate T cell activation, metabolic and epigenetic program-ming, tissue-specific migration, stromal-immune interactions, and fibrotic remodeling. Therapeutic agents targeting key pathways, including ruxolitinib (JAK1/2), ibrutinib (BTK/ITK), and belumosudil (ROCK2), can modulate inflammatory responses while pre-serving graft-versus-tumor (GvT) activity. Emerging technologies, such as CRISPR-based genome editing, single-cell and spatial multi-omics, and AI-driven network modeling, enable patient-specific mapping of signaling hierarchies, prediction of disease trajectories, and identification of actionable targets. Integration of these approaches supports precise modulation of immune circuits, offering alternatives to broad immunosuppression. These insights provide a framework for next-generation, individualized interventions that pro-mote durable immune tolerance without compromising anti-tumor immunity and high-light rational combination strategies to improve outcomes in allo-HSCT and allogeneic T cell therapies.

Background: /Objectives: Bacterial biofilms formed by Escherichia coli pose a significant challenge in veterinary medicine due to their intrinsic resistance to antibiotics. Antimicrobial peptides (AMPs) represent a promising alternative. AMPs exert their bactericidal activity by binding to negatively charged phospholipids in bacterial membranes via electrostatic interactions, leading to membrane disruption and rapid cell lysis. Methods: In vitro assays included MIC determination, biofilm eradication testing (crystal violet, colony counts, CLSM), swimming motility, and EPS quantification. CRISPR/Cas9 was used to construct and complement a kduD mutant. A transposon mutagenesis library was screened for biofilm-defective mutants. In vivo, a murine excisional wound infection model was treated with CRAMP-34, with wound closure and bacterial burden monitored. Gene expression changes were analyzed via RT-qPCR. Results: The mouse-derived AMP (abbreviation CRAMP-34) effectively eradicates pre-formed biofilms of a clinically relevant, porcine-origin E.coli strain and promotes wound healing in a murine infection model. We conducted a genome-wide transposon mutagenesis screen, which identified kduD, as a critical gene for robust biofilm formation. Functional characterization revealed that kduD deletion drastically impairs flagellar motility and alters exopolysaccharide production, leading to defective biofilm architecture without affecting growth. Notably, the anti-biofilm activity of CRAMP-34 phenocopied aspects of the kduD deletion, including motility inhibition and transcriptional repression of a common set of biofilm-related genes. Conclusions: The research highlight CRAMP-34 as a potent anti-biofilm agent and unveil kduD as a previously unrecognized regulator of E.coli biofilms development, whose associated pathway is implicated in the mechanism of action of CRAMP-34.

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. 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, 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.

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal haematopoietic stem cell disease, characterized primarily by intravascular hemolysis, thrombosis, and bone marrow failure. Complement inhibitors are commonly used in clinical treatment, showing limited efficacy, thus highlighting the urgent need to identify new therapeutic targets and explore alternative treatment strategies to provide theoretical guidance for clinical practice. Methods: We established a PNH cell model and constructed a miRNA–mRNA regulatory network to identify key miRNAs and core target genes. Single-cell sequencing data were analyzed to further clarify the critical genes. Drug prediction was then performed using multiple databases to identify potential therapeutic agents for PNH, which were subsequently validated through molecular docking and molecular dynamics simulations. Results: Using CRISPR/RNP technology, we successfully constructed a PIGA-knockout (PIGA-KO) THP-1 cell model. Differential expression analysis identified 1979 differentially expressed mRNAs (DEmRNAs) and 97 differentially expressed miRNAs (DEmiRNAs). The multiMiR package in R was used to predict the target genes of DEmiRNAs, from which those experimentally validated through dual-luciferase reporter assays were selected. After integrating with the DEmRNAs, a miRNA–mRNA regulatory network was constructed, comprising 26 miRNAs and 38 mRNAs. Prediction of miRNA pathway enrichment analysis identified hsa-miR-23a-3p as a key miRNA, with CXCL12, CXCL8, HES1, and TRAF5 as core target genes. Integration of single-cell sequencing datasets (PRJNA1061334 and GSE157344) was performed, followed by cell communication and enrichment analysis. This approach, combined with clinical relevance, identified neutrophil cluster as a key cluster. Intersection analysis of neutrophil cluster differential analysis result with key modules from hdWGCNA further clarified the critical genes. Drug prediction using the EpiMed, CMap, and DGIdb identified Leflunomide, Dipyridamole, and Pentoxifylline as potential therapeutic agents. Molecular docking and molecular dynamics simulations showed stable binding of these potential drugs to the critical molecules, indicating a strong molecular interaction foundation. Conclusion: Leflunomide, Dipyridamole, and Pentoxifylline may serve as promising therapeutic agents for PNH, and the hsa-miR-23a-3p/CXCL8 regulatory axis could play a pivotal role in the pathogenesis and progression of PNH.

Soybean is usually grown at large scales, with pest control based on insecticides. However, the overuse of chemicals has led to several adverse effects. Thus, integrated pest management (IPM) is the best way to protect yield through integrating different pest control tools, based on plant resistance (including Bt cultivars), adoption of economic thresholds (ETs), scouting procedures, use of selective insecticides, biological control, and other sustainable tools, which help maintain environmental quality in an ecological and economical manner. Soon, those tools will also include RNAi, CRISPR based control strategies, among other sustainable alternatives. In Brazil, results from the Soybean-IPM Program indicate that adopters of the technology have reduced insecticide use by approximately 50% relative to non-adopters, with yields comparable to or slightly higher than those of non-adopters. This reduction can be explained not only by the widespread adoption of Bt soybean varieties across the country but also by the adoption of ETs in Soybean-IPM, which has reduced insecticide use, thereby increasing natural biological control in the agroecosystem. However, low refuge compliance has led to the first cases of pest resistance to Cry1Ac, thereby growing reliance on chemical control and posing an additional challenge for integrated pest management practitioners. The obstacles to adopting IPM programs for commodity crops, such as soybean, may be mitigated by recent economic incentives within the new global agenda for decarbonized agriculture and the increase of bioinputs available in the Brazilian market. Such incentives can support the broader adoption of IPM, thereby reducing dependence on chemical inputs to achieve high yields.

Programmable organoids are emerging as a powerful new class of engineered developmental systems in which genetic circuits, epigenetic memory architectures, synthetic organizers, and closed-loop control frameworks converge to enable precise regulation of morphogenesis. Traditional organoids rely on spontaneous self-organization, but this intrinsic variability limits reproducibility, causal inference, and translational relevance. Recent advances in CRISPR-based transcriptional and epigenetic engineering, optogenetic and chemogenetic patterning technologies, reaction–diffusion design, and real-time biosensing now allow developmental trajectories to be scripted with increasing precision. This review synthesizes these developments into a unified framework spanning genetic circuit construction, epigenetic programming, synthetic morphogenesis, multi-scale sensing, adaptive regulation, and AI-guided design. Applications across human developmental biology, disease modeling, and regenerative medicine are highlighted, alongside the technical, biosafety, and ethical considerations associated with building increasingly autonomous, self-regulating developmental systems. Collectively, these advances establish programmable organoids as a foundation for developmental synthetic biology and outline a roadmap toward fully engineered human developmental architectures.

Abstract WNT5A-driven Wnt/Planar Cell Polarity (Wnt/PCP) signaling is essential for vertebrate limb morphogenesis, and pathogenic WNT5A variants cause autosomal dominant Robinow Syndrome (RS). The recurrent Cys83Ser (C83S) substitution is among the best-studied RS-associated alleles and has been variably described as hypomorphic, loss-of-function, or dominant-negative based largely on overexpression in non-mammalian systems. However, a physiologically relevant mammalian model and robust in vivo readouts that distinguish loss- from gain-of-function Wnt/PCP perturbations have been lacking. Here, we introduce a quantitative image-based metric, the local misalignment score (LMS), which visualizes and measures cell orientation in situ across long bones during late embryonic development. LMS captures local coherence of chondrocyte alignment independently of embryo age, sectioning depth, or canonical Wnt activity, and selectively recognizes Wnt/PCP defects in Wnt5a and Wntless conditional knockouts, but not in Lrp5/6-deficient limbs. Using LMS, we show that a CRISPR/Cas9-engineered Wnt5a-C83S mouse exhibits profound and uniform chondrocyte orientation defects and limb shortening that are mechanistically distinct from the spatially patterned phenotypes in Wnt5a loss-of-functon(Wnt5a-cKO) and ectopic-expression (Wnt5a-LSL) models, and occur without SOX9 downregulation or severe distal digit defects. Complementary in vitro analyses using a KIF26B-NanoLuc Wnt/PCP reporter and biochemical readouts demonstrate that WNT5A-C83S has markedly reduced signaling activity but does not inhibit wild-type WNT5A in autocrine, paracrine, or juxtracrine contexts, arguing against a dominant-negative mechanism. Together, these data support a model in which WNT5A-C83S behaves as a hypomorphic, non–dominant-negative variant that perturbs Wnt/PCP gradient-dependent limb development through altered, rather than simply reduced, signaling output. More broadly, LMS provides a scalable morphological readout for spatially resolved interrogation of Wnt/PCP-associated defects in complex tissues.

Abstract Foxtail millet is a small diploid C4 crop that possesses superior nutritional properties. It provides a high content of essential amino acids, vitamins, and minerals. In this study, we used CRISPR/Cas9 to simultaneously edit two α-prolamin genes in foxtail millet. Analysis of the derived homozygous lines revealed a decrease in prolamin content. Surprisingly, the content of FAAs such as lysine, taurine, GABA, tryptophan, and sialic acid as well as glucose, fructose, sucrose, and maltose, was significantly increased. Additionally, peak viscosity, final viscosity, trough viscosity, and other parameters showed significant differences, suggesting that the food quality of the gene editing lines may have been altered. The editing lines grew normally, with only a slight decrease in 1000-grain weight compared to the wild type. Overall, we successfully created new germplasm with significantly increased FAAs content through gene editing, providing a valuable reference for the selection of functional foxtail millet varieties.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a highly deadly cancer with limited treatment options. The base excision repair (BER) pathway, crucial for fixing DNA abasic sites, is driven by apurinic/apyrimidinic endonuclease 1 (APE1). While APE1’s redox function has been extensively studied, its endonuclease activity in PDAC homeostasis and therapeutic response remains poorly understood. We created stable, homozygous APE1 endonuclease-reduced PDAC cell lines to examine the effects of impaired BER activity on pancreatic cancer growth, progression, and response to treatment. Methods CRISPR/Cas9-mediated editing was used to introduce an E96A mutation into the Pa03C PDAC cell line, generating three clonal mutant cell lines: E96A B1, E96A B4, E96A G8. APE1 expression and activity were verified in vitro through biochemical assays. Cellular responses to genotoxic stress were examined using cytotoxicity, colony formation, and mtDNA damage assays. Transcriptomic changes were evaluated via RNA sequencing. In vivo tumor growth and metastatic dissemination were studied in orthotopic PDAC mouse models, with and without temozolomide (TMZ) treatment. Results The E96A mutant cell lines exhibited significantly decreased endonuclease activity but had no changes to redox signaling and protein expression. Short-term cytotoxic assays revealed no enhancement in acute sensitivity; however long-term assessment demonstrated a proliferative defect and a vulnerability to genotoxic stress. Transcriptomic analysis revealed that the mutant cell lines maintain a stressed phenotype at baseline, which becomes more pronounced following genotoxic stress. In vivo , E96A mutants had notably lower tumor burden and metastasis at baseline, and the mutation potentiated the effect of the alkylating drug temozolomide, which further inhibited tumor growth and metastasis in a dose-dependent manner. Conclusion We established the first stable human PDAC cell models deficient in APE1 endonuclease activity. Our findings demonstrate that selective impairment of APE1’s DNA repair function expands therapeutic options by lowering the threshold for effective DNA damage validating combination treatments with targeted inhibitors and DNA-damaging agents. Targeting APE1 endonuclease activity represents a promising therapeutic strategy for PDAC, capable of suppressing metastatic spread and enhancing tumor responsiveness to alkylating and other genotoxic therapies.