Search Results (1,317)

Alkaloids have garnered significant interest as potential anticancer agents. Vitamin D receptor (VDR) plays a role in preventing the progression of colorectal cancer (CRC) and may be a crucial mediator of the anticancer effects produced by certain alkaloids. The search for novel anticancer drugs that induce VDR expression and act through the VDR could improve the clinical outcomes of CRC patients. The anticancer and pro-apoptotic effects of coclaurine and reticuline were investigated using CRISPR/Cas9-edited VDR/knockout (KO) and wild-type (WT) CRC HCT116 cell lines. Western blotting, RT-qPCR, confocal microscopy, cell viability, scratch assays, and flow cytometry were employed to assess VDR expression and cellular localization, cell growth, wound-healing, cytotoxicity, apoptotic status, cell cycle progression, and VDR-mediated gene expression. Coclaurine and reticuline dose-dependently inhibited HCT116-WT cell viability, decreased wound-healing, and increased VDR nuclear localization and gene expression while downregulating the oncogenic genes SNAIL1 and SNAIL2. Both alkaloids induced late apoptosis in HCT116-WT cells, increased the cleavage of PARP and caspase-3, and upregulated Bax and TP53 while decreasing BCL-2. Both alkaloids caused HCT116-WT cell growth arrest in the S-phase, which is associated with cyclin A1 overexpression. Coclaurine and reticuline lost their anticancer effects in HCT116-VDR/KO cells. Docking studies revealed that both alkaloids occupied the VDR’s active site. These findings demonstrate that coclaurine and reticuline exert anti-CRC and pro-apoptotic activities via the VDR, suggesting them as natural therapeutic candidates. The use of in vivo CRC models is needed to validate the anticancer activities of coclaurine and reticuline. Full article

Plants have evolved a complex, multilayered immune system that integrates molecular recognition, signaling pathways, epigenetic regulation, and small RNA-mediated control. Recent studies have shown that DNA-level regulatory mechanisms, such as RNA-directed DNA methylation (RdDM), histone modifications, and chromatin remodeling, are critical for modulating immune gene expression, allowing for rapid and accurate pathogen-defense responses. The epigenetic landscape not only maintains immunological homeostasis but also promotes stress-responsive transcription via stable chromatin modifications. These changes contribute to immunological priming, a process in which earlier exposure to pathogens or abiotic stress causes a heightened state of preparedness for future encounters. Small RNAs, including siRNAs, miRNAs, and phasiRNAs, are essential for gene silencing before and after transcription, fine-tuning immune responses, and inhibiting negative regulators. These RNA molecules interact closely with chromatin features, influencing histone acetylation/methylation (e.g., H3K4me3, H3K27me3) and guiding DNA methylation patterns. Epigenetically encoded immune memory can be stable across multiple generations, resulting in the transgenerational inheritance of stress resilience. Such memory effects have been observed in rice, tomato, maize, and Arabidopsis. This review summarizes new findings on short RNA biology, chromatin-level immunological control, and epigenetic memory in plant defense. Emerging technologies, such as ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing), ChIP-seq (Chromatin Immunoprecipitation followed by Sequencing), bisulfite sequencing, and CRISPR/dCas9-based epigenome editing, are helping researchers comprehend these pathways. These developments hold an opportunity for establishing epigenetic breeding strategies that target the production of non-GMO, stress-resistant crops for sustainable agriculture. Full article

Secondary metabolites, including alkaloids, flavonoids, and tannins, are crucial for human health, agriculture, and ecosystem functioning. Their synthesis is often species-specific, influenced by both genetic and environmental factors. The increasing demand for these compounds across various industries highlights the need for advancements in plant breeding and biotechnological approaches. Transcription activator-like effector nucleases (TALENs) have emerged as a powerful tool for precise genome editing, offering significant potential for enhancing the synthesis of secondary metabolites in plants. However, while plant genome editing technologies have advanced significantly, the application of TALENs in improving secondary metabolite production and expanding genetic diversity remains underexplored. Therefore, this review aims to provide a comprehensive analysis of TALEN-mediated genome editing in plants, focusing on their role in enhancing secondary metabolite biosynthetic pathways and improving genetic diversity. The mechanisms underlying TALENs are examined, including their ability to target specific genes involved in the synthesis of bioactive compounds, highlighting comparisons with other genome editing tools such as CRISPR/Cas9. This review further highlights key applications in medicinal plants, particularly the modification of pathways responsible for alkaloids, flavonoids, terpenoids, and phenolic compounds. Furthermore, the role of TALENs in inducing genetic variation, improving stress tolerance, and facilitating hybridization in plant breeding programs is highlighted. Recent advances, challenges, and limitations associated with using TALENs for enhancing secondary metabolite production are critically evaluated. In this review, gaps in current research are identified, particularly regarding the integration of TALENs with multi-omics technologies and synthetic biology approaches. The findings suggest that while underutilized, TALENs offer sustainable strategies for producing high-value secondary metabolites in medicinal plants. Future research should focus on optimizing TALEN systems for commercial applications and integrating them with advanced biotechnological platforms to enhance the yield and resilience of medicinal plants. Full article

The BEST1 gene encodes a transmembrane protein in the retinal pigment epithelium (RPE) in the eye, that functions as a calcium-dependent chloride channel (CaCC). Pathogenic variants in BEST1 are the underlying cause for bestrophinopathies, a group of inherited retinal disorders that vary in their pattern of inheritance, clinical appearance, and underlying molecular disease mechanisms. Currently, there are no treatments available for any of the bestrophinopathies, and gene therapy represents an attractive strategy due to the accessibility of the eye and slow disease progression. While gene augmentation may be effective for a subset of bestrophinopathies, others require allele-specific silencing or correction of the disease-causing variant to reconstitute expression of the BEST1 protein. This review aims to give an overview of the clinical diversity of bestrophinopathies and proposes the molecular disease mechanism of the pathogenic BEST1 variant as an important parameter for the choice of treatment strategy. Furthermore, we discuss the potential of different mutation-specific and mutation-independent CRISPR/Cas9-based gene editing strategies as a future treatment approach for bestrophinopathies. Full article

Gene editing enables a better understanding of protein functions. The genome of the protozoan parasite Entamoeba histolytica contains a 4500 bp DNA fragment comprising the ehcp112, ehadh, and ehrabb genes, which together form the V1 virulence locus. Studying these genes has been challenging due to the lack of suitable methodologies. Here, we report the first in vitro and in vivo knockout in E. histolytica (ehcp112 gene) using a modified CRISPR-Cas9 strategy and explore its effects on the other V1 locus genes. Confocal and transmission electron microscopy were used to detect the RNP pathway formed by the Cas9 enzyme and the crRNA–tracrRNA complex, from their entry into the trophozoites until their arrival at the nucleus and heterochromatin. Scanning electron microscopy revealed that the mutant cells (EhCP112-KO) were smaller, with fewer pseudopodia and plasma membrane depressions. DNA sequencing and RT-qPCR assays identified a four-base deletion in the ehcp112 gene in the mutant trophozoites. Western blot assays of EhCP112-KO extracts revealed the absence of the EhCP112 protein. While the EhCP112-KO lysates digested gelatin more efficiently than the HM1:IMSS extracts, their secreted products showed poor enzymatic activity. The ehcp112 knockout also affected the transcription of the ehadh and ehrabb genes, probably due to their genomic position. In conclusion, the implementation of the CRISPR-Cas9 strategy in E. histolytica evidenced the coordinated expression of the ehcp112 gene and the other members of the V1 locus. Full article

Background: Cardiac amyloidosis (CA) is an increasingly recognized but historically underdiagnosed cause of restrictive cardiomyopathy and heart failure with preserved ejection fraction (HFpEF). It results from the extracellular deposition of misfolded protein fibrils, most commonly transthyretin (ATTR) or immunoglobulin light chains (AL), leading to progressive myocardial dysfunction and multi-organ involvement. Objective: This review provides a comprehensive, cardiology-centered overview of cardiac amyloidosis, with an emphasis on early recognition, diagnostic strategies, subtype differentiation, and the evolving therapies. Content: We summarize the epidemiology, pathophysiology, and clinical manifestations of both ATTR and AL subtypes. Key diagnostic tools, including echocardiography, cardiac magnetic resonance imaging, bone scintigraphy, monoclonal protein screening, and endomyocardial biopsy, are reviewed in the context of a stepwise diagnostic approach. Special attention is given to clinical presentation, electrocardiographic and imaging “red flags,” and to differentiating CA from mimickers such as hypertrophic cardiomyopathy, hypertension-induced left ventricular hypertrophy, and aortic stenosis. Staging systems are detailed, highlighting the prognostic role of cardiac biomarkers. Therapeutic strategies are explored, including subtype-specific regimens (e.g., daratumumab-based therapy for AL; tafamidis and gene silencers for ATTR), the judicious use of conventional heart failure medications, and emerging therapies such as CRISPR-based gene editing. Conclusions: Timely recognition and accurate diagnosis of cardiac amyloidosis are critical to improving outcomes. As diagnostic tools and disease-modifying therapies evolve rapidly, cardiologists must remain at the forefront of multidisciplinary care. A structured biomarker- and imaging-guided approach can enhance diagnostic yield, inform prognosis, and optimize patient-specific management. Full article

Chimeric Antigen Receptor (CAR)-T cell therapy has transformed the treatment landscape of cancer, yet major challenges remain in enhancing efficacy, reducing adverse effects, and expanding accessibility. Autologous CAR-T cells, derived from individual patients, have achieved remarkable clinical success in hematologic malignancies; however, their highly personalized nature limits scalability, increases costs, and delays timely treatment. Allogeneic CAR-T cells generated from healthy donors provide an “off-the-shelf” alternative but face two critical immune barriers: graft-versus-host disease (GvHD), caused by donor T-cell receptor (TCR) recognition of host tissues, and host-versus-graft rejection, mediated by recipient immune responses against donor HLA molecules. Recent advances in genome engineering, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, allow precise modification of donor T cells to overcome these limitations. For example, TRAC gene knockout eliminates TCR expression, preventing GvHD, while disruption of HLA molecules reduces immunogenicity without impairing cytotoxicity. Beyond hematologic cancers, CRISPR-edited allogeneic CAR-T cells targeting the NKG2D receptor have shown promise in preclinical studies and early-phase trials. NKG2D CAR-T cells recognize stress ligands (MICA/B, ULBP1–6) expressed on over 80% of diverse solid tumors, including pancreatic and ovarian cancers, thereby broadening therapeutic applicability. Nevertheless, the genomic editing process carries risks of off-target effects, including potential disruption of tumor suppressor genes and oncogenes, underscoring the need for stringent safety and quality control. This review examines the distinguishing features of allogeneic versus autologous CAR-T therapy, with a particular focus on NKG2D-based allogeneic CAR-T approaches for solid tumors. We summarize current strategies to mitigate immune barriers, discuss practical manufacturing challenges, and analyze available clinical data on NKG2D CAR-T trials. Collectively, these insights underscore both the promise and the hurdles of developing safe, universal, and scalable allogeneic CAR-T therapies for solid malignancies. Full article

Acute respiratory distress syndrome (ARDS) is a life-threatening condition with high mortality. A central driver in its pathogenesis is alveolar epithelial cell (AEC) dysfunction, which leads to disruption of the epithelial barrier, impaired fluid clearance, and dysregulated inflammatory responses. This review summarizes the key mechanisms underlying AEC injury, including programmed cell death (apoptosis, pyroptosis, necroptosis, ferroptosis), oxidative stress, mitochondrial dysfunction, epigenetic reprogramming (DNA methylation, histone modifications), metabolic rewiring (succinate accumulation), and spatiotemporal heterogeneity revealed by single-cell sequencing and spatial transcriptomics. Multicellular crosstalk involving epithelial–immune–endothelial networks and the gut-lung axis further shapes disease progression. Building on these mechanistic foundations, we evaluate emerging AEC-targeted interventions such as pharmacologic agents (antioxidants, anti-inflammatories), biologics (mesenchymal stem cells and engineered exosomes), and gene-based approaches (adeno-associated virus and CRISPR-Cas9 systems delivered via smart nanocarriers). Complementary strategies include microbiome modulation through probiotics, short-chain fatty acids, or fecal microbiota transplantation, and biomarker-guided precision medicine (e.g., sRAGE, exosomal miRNAs) to enable promise individualized regimens. We also discuss translational hurdles, including nanotoxicity, mesenchymal stem cell (MSC) heterogeneity, and gene-editing safety, and highlight future opportunities involving AI-driven multi-omics, lung-on-chip platforms, and epithelium-centered regenerative therapies. By integrating mechanistic insights with innovative therapeutic strategies, this review aims to outline a roadmap toward epithelium-targeted, precision-guided therapies for ARDS. Full article

The document is an updated review, starting from the Special Issue “Molecular Breeding for Abiotic Stress Tolerance in Crops” published in the Int. J. Mol. Sci. It reviews molecular breeding strategies to enhance abiotic stress tolerance in crops, addressing challenges like drought, salinity, temperature extremes, and waterlogging, which threaten global food security. Climate change intensifies these stresses, making it critical to develop resilient crop varieties. Plants adapt to stress through mechanisms such as hormonal regulation (e.g., ABA, ethylene), antioxidant defense (e.g., SOD, CAT), osmotic adjustment (e.g., proline accumulation), and gene expression regulation via transcription factors like MYB and WRKY. Advanced tools, such as CRISPR/Cas9 genome editing, enable precise modifications of stress-related genes, improving tolerance without compromising yield. Examples include rice (OsRR22, OsDST) and wheat (TaERF3, TaHKT1;5). Epigenetic regulation, including DNA methylation and histone modifications, also plays a role in stress adaptation. Specific studies focused on polyamine seed priming for improved germination and stress resistance, cadmium detoxification mechanisms, and genome-wide association studies (GWAS) to identify genetic markers for salt tolerance and yield. Research on salinity tolerance in wheat emphasizes sodium exclusion and tissue tolerance mechanisms. Future perspectives focus on genetic engineering, molecular markers, epigenetic studies, and functional validation to address environmental stress challenges, including the use of AI and machine learning to manage the large amount of data. The review underscores the importance of translating molecular findings into practical applications to ensure sustainable crop production under changing climates. Full article

Plant viruses have evolved from being viewed exclusively as pathogens into versatile and powerful tools for modern biotechnology. Among them, Tomato bushy stunt virus (TBSV) holds a special place due to its well-studied molecular biology and unique structural properties. This review systematizes the knowledge on TBSV’s dual role as a multifunctional platform. On one hand, we cover its application as a viral vector for the highly efficient expression of recombinant proteins in plants, as well as a tool for functional genomics, including Virus-Induced Gene Silencing (VIGS) and the delivery of CRISPR/Cas9 gene-editing components. On the other hand, we provide a detailed analysis of the use of the stable and monodisperse TBSV virion in nanobiotechnology. Its capsid serves as an ideal scaffold for creating next-generation vaccine candidates, platforms for targeted drug delivery to tumor cells, and as a building block for the programmable self-assembly of complex nanoarchitectures. In conclusion, key challenges limiting the widespread adoption of the platform are discussed, including the genetic instability of vectors and difficulties in scalable purification, along with promising strategies to overcome them. Full article

Rice (Oryza sativa), particularly the japonica subspecies, is a vital global food source but often suffers from short grain length and heat sensitivity, highlighting the need for genetic improvement. This study employed CRISPR/Cas9 technology to investigate the effects of Grain Size3 (GS3) gene editing in the japonica cultivar, ‘Nipponbare’. Successful GS3 editing increased grain size across stable T₃ and T₄ generations. Importantly, different GS3-edited lines, even when all targeted within exon 1, resulted in varied effects on grain length and other yield components. Transcriptomic analyses revealed unique gene expression profiles for each edited line, highlighting the fact that subtle GS3 mutations trigger diverse transcriptional cascades. While common differentially expressed genes (DEGs) were enriched in ethylene signaling and chitinase activity, line-specific KEGG analyses showed distinct pathway enrichments. Crucially, the CR-L5 line exhibited significantly enhanced heat tolerance at heading stage. Under high-temperature stress, CR-L5 maintained a higher relative seed setting rate and a 15% greater grain yield than the wild type. This enhanced thermotolerance in CR-L5 correlated with differing expressions of several wax biosynthesis and chitinase-related genes. Our study provides evidence that specific gs3 mutations can confer enhanced reproductive-stage thermotolerance, offering a strategy for breeding climate-resilient japonica rice with improved grain quality and yield under stress. Full article

CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens. Full article

Human papillomavirus (HPV) is a major etiological factor in cervical, anal, and oropharyngeal cancers. Although prophylactic vaccines have substantially reduced infection rates, effective therapeutic options for established HPV-associated malignancies remain limited. This review provides an up-to-date overview of emerging strategies to treat HPV-driven tumours. Key approaches include immune checkpoint inhibitors, therapeutic vaccines such as VGX-3100 and PRGN-2012, and gene-editing tools like CRISPR/Cas9. Epigenetic drugs, particularly histone deacetylase inhibitors, show promise in reactivating silenced tumour suppressor genes and enhancing antitumour immunity. In addition, natural bioactive compounds and plant-derived molecules are being explored as complementary anti-HPV agents, while drug repurposing and combination therapies offer cost-effective opportunities to broaden treatment options. We also highlight the role of patient-derived organoid models as powerful platforms for personalized drug screening and functional assessment. By integrating these therapeutic innovations with precision oncology approaches, this review outlines a multidimensional framework aimed at improving clinical outcomes and quality of life for patients with HPV-associated cancers. Full article

The rapid evolution of CRISPR technology has revolutionized molecular biology, and among the various systems, CRISPR/Cas12a stands out for its high specificity and efficient collateral cleavage activity. This review article focuses on the recent advancements and applications of split technology within the CRISPR/Cas12a framework, highlighting its transformative role in molecular diagnostics and biosensing. Split technology innovatively divides functional nucleic acid components into modular segments that are activated by specific targets, significantly enhancing the specificity and sensitivity of biosensors. This design addresses the inherent limitations of traditional sensor systems, enabling the direct detection of ultrashort nucleic acids and improved discrimination of single-nucleotide variants, thereby facilitating the simultaneous detection of multiple biomolecules. The versatility of split-enabled biosensors extends beyond genetic testing, making them valuable tools in diagnostics, therapeutics, and environmental science. Despite challenges such as crRNA degradation and reassembly kinetics, ongoing research and engineering solutions continue to enhance the stability and performance of these systems. This review synthesizes the foundational principles, recent advancements, and potential applications of split technology while also identifying challenges and opportunities for future exploration. Ultimately, our insights provide a comprehensive resource to leverage the full potential of CRISPR/Cas12a-based split technology in advancing biosensing methodologies and clinical applications. Full article

Twenty causative genes have been reported that cause non-syndromic childhood glaucoma associated with anterior segment dysgenesis. FOXC1, PAX6 and PITX2 are the most well-known, but cases linked to SLC4A11, PITX3 and SOX11 have also been reported. As genetic testing becomes increasingly widespread and rates of molecular diagnosis rise, the extent of phenotypic overlap between the different genetic causes of non-syndromic glaucoma associated with anterior segment dysgenesis is becoming more evident. Taking aniridia as an example, whilst PAX6 mutations remain the predominant cause, variants in CYP1B1, FOXC1, PXDN and SOX11 have also been reported in patients with childhood glaucoma and aniridia. Developments in molecular-based therapies for retinal and corneal disease are advancing rapidly, and pre-clinical studies of gene-based treatments for glaucoma and aniridia are showing promising results. Use of adeno-associated viral vectors for gene delivery is most common, with improvements in intraocular pressure and retinal ganglion cell survival in Tg-MYOC^Y437H mouse models of glaucoma, and successful correction of a germline PAX6^G194X nonsense variant in mice using CRISPR-Cas9 gene editing. This review will explore the actions and interactions of the genetic causes of non-syndromic glaucoma associated with anterior segment dysgenesis and discuss the current developments in molecular therapies for these patients. Full article