Search Results (826)

Background: Epidural procedures benefit from a pre-procedural informed estimation of epidural depth, as anticipating the approximate distance can support safer needle placement and reduce technical difficulties during analgesia or anesthesia procedures. The influence of ethnicity has been established across different populations worldwide; however, there is a lack of Saudi-specific MRI data on epidural depth among the adult population. Aim of this Study: To measure the skin to epidural space distance (SED) at the lumbar interspaces L3–L4 and L4–L5 in the Saudi adult population using magnetic resonance imaging (MRI) and to examine its correlations with age, sex, height, weight, and body mass index (BMI). Methods: In this retrospective cross-sectional study, sagittal T1-weighted lumbar MRI images of the spine of 169 adult Saudi patients were studied. The age group ranged from 20 to 70 years, with an equal distribution of males and females. The skin to epidural space distance (SED) was measured at the L3–L4 and L4–L5 interspaces, and its correlations with age, sex, height, weight, and BMI were analyzed. Results: The average measurement of skin to epidural space distance (SED) was 59.08 mm in L3–L4, and 63.21 in L4–L5. BMI and weight showed strong positive correlations with SED across both levels. Female sex was associated with longer SED values at L4–L5. There was no significant correlation between SED and age or height of the patients. Conclusions: MRI-based assessment of SED revealed strong correlations with weight and BMI, but no correlation with height, age, and sex. These findings support the individualized estimation of epidural depth and needle length selection to enhance procedural safety and reduce complications. Full article

Tumor initiation and metastatic progression are driven by context-dependent genetic alterations that disrupt tumor suppressor pathways, metabolic homeostasis, and signaling networks. However, the initial drivers that transform normal cells into malignant ones and their context dependency remain elusive. To address this, we aimed to systematically identify and characterize these drivers across cancer types, species, and microenvironments. We constructed customized clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout (KO) libraries targeting high-frequency mutated and downregulated genes associated with liver hepatocellular carcinoma (LIHC) and breast carcinoma (BRCA) and conducted parallel functional screens in non-cancerous mouse and human fibroblast cell lines under two-dimensional (2D), three-dimensional (3D), and in vivo conditions. Strikingly, TP53 and NF1 emerged as pan-context drivers consistently enriched across immortalization, tumorigenesis, and metastasis in both LIHC and BRCA settings, while most other identified drivers were largely species-, tissue-, and microenvironment-specific with limited cross-model overlap. Despite this heterogeneity, all drivers converge on core pathways including epigenetic regulation, metabolic reprogramming, and growth factor signaling. Unlike prior studies on established cancer cells, this work defines the genetic barriers restricting the malignant transformation of primary normal cells, offering a new framework for early cancer evolution. Full article

Glucosamine (GlcN) is an essential amino monosaccharide widely used in pharmaceuticals, nutraceuticals, and cosmetics. Microbial fermentation presents a sustainable alternative to its traditional chemical production. However, in Saccharomyces cerevisiae, competitive carbon flux towards ethanol significantly limits GlcN yields. In this study, an S. cerevisiae strain for GlcN biosynthesis was engineered by integrating heterologous GlmD (glucosamine-6-phosphate deaminase) and GlmP (glucosamine-6-phosphate phosphatase) genes. To redirect carbon flux, the pyruvate decarboxylase genes pdc1, pdc5, and pdc6 were sequentially knocked out using the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach, generating strains S. cerevisiaeGlmDP/pdc1Δ, GlmDP/pdc1Δpdc5Δ, and GlmDP/pdc1Δpdc5Δpdc6Δ. S. cerevisiae GlmDP/pdc1Δpdc5Δpdc6Δ achieved a GlcN titer of 2.20 ± 0.11 g/L, a 1.54-fold increase over the parental S. cerevisia GlmDP strain, while its ethanol yield decreased by 26%. This enhancement was achieved without significantly affecting cell growth or glucose consumption. Comparative transcriptomics between the triple-knockout and parental yeasts revealed 892 differentially expressed genes. Pathways related to glycolysis and ethanol formation were predominantly downregulated, whereas pathways potentially supporting GlcN synthesis were upregulated. The engineered strain demonstrated high genetic stability over 50 generations. Our findings demonstrate that disrupting ethanol formation is an effective strategy to enhance GlcN production in S. cerevisiae, providing valuable insights for carbon flux redistribution. Full article

Cancer immunotherapy has recently become an essential approach for treating cancer, showing considerable promise as a substitute for surgery, radiation therapy, and conventional chemotherapy. It primarily aims to boost the host’s natural defense system to combat cancer malignancies by utilizing components of immune checkpoint blockades (ICBs), mainly programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), along with elements of adoptive cellular therapies (ACTs) like Chimeric Antigen Receptor (CAR) therapy, T Cell Receptor (TCR) therapy and Tumor-Infiltrating Lymphocyte (TIL) therapy. However, cancer cells tend to undermine the effectiveness of cancer immunotherapeutic strategies by employing one or more immune evasion mechanisms. This review briefly highlights how key mechanisms of cancer immune evasion confer resistance to immunotherapy and how the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR)/Cas9 systems, as gene-editing tools, are poised to enhance cancer immunotherapy for treating challenging cancers. We emphasize that (CRISPR/Cas9) systems can be used to explore and positively alter the genes of the immune system, boosting the effectiveness of cancer immunotherapy by editing immune checkpoints, TILs, and CAR-T cells, and disrupting genes, facilitating tumors’ evasion of the immune system. Furthermore, we highlight the growing interest in emerging base editor technology to engineer natural killer (NK) cells to overcome NK-cell-based immunotherapy challenges, particularly human leukocyte antigens (HLA)-mediated limitations, and to engineer CAR-T cells for improved immunotherapy outcomes. Full article

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, a genome-editing technology pioneered in 2012, enables the precise correction of deleterious mutations or disruption of disease-causing genes through targeted double-strand breaks (DSBs), offering potential for treating genetic diseases. However, CRISPR/Cas9 can cause off-target cleavage at non-specific DNA sites, leading to unintended insertions or deletions (indels), which limit its safety and applicability despite ongoing improvements in specificity. Recently, prime editing (PE), an advanced CRISPR-derived technology, has been employed with a Cas9 nickase (Cas9n) fused with a reverse transcriptase and a prime editing guide RNA (pegRNA) to enable precise insertions, deletions, and transversions without inducing DSBs, thus reducing risks of indels and chromosomal aberrations. Furthermore, ongoing optimizations, such as improved pegRNA design and enhanced editing efficiency, have expanded the applications of PE in medical therapeutics, agriculture, and fundamental research. This review summarizes recent advancements in the PE system, including optimized pegRNA designs and enzyme engineering for enhanced efficiency and specificity, alongside novel delivery methods. It also evaluates cutting-edge delivery strategies, such as adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs) and novel extracellular vesicle (EV)-based systems, and explores PE applications in vitro and in vivo, including disease modeling and therapeutic gene correction. Full article

Plant-derived extracellular vesicles (PDEVs), engineered phytosomes, bioinspired polymeric plant-based nanoparticles (PBNPs), hybrid phyto-inorganic nanocomposites, green-synthesized metal nanoparticles, self-assembled nanoarchitectures, and multifunctional composites represent a rapidly advancing class of sustainable, nature-inspired nanocarriers. These platforms combine exceptional biocompatibility, negligible immunogenicity, and renewable sourcing with tunable drug loading, targeted delivery, and controlled release properties. This review synthesizes translational advances from 2020 to 2026, covering scalable isolation/bioprocessing (bioreactors, elicitation), multi-parametric physicochemical/multi-omics characterization, rational engineering/hybridization, and rigorous in vitro/in vivo assessments of uptake, biodistribution, pharmacokinetic (PK), and efficacy. Phytosomes and PBNPs markedly enhance oral bioavailability and targeted delivery of lipophilic phytochemicals, while PDEVs offer unique immunomodulatory, anti-inflammatory, and gene-regulatory activities. Hybrid and green-synthesized systems provide structural stability, redox modulation, and synergistic effects, and self-assembled/multifunctional composites address solubilization barriers with stimuli-responsive design. Early-phase human studies on grapefruit-, ginger-, turmeric-, and ginseng-derived PDEVs report excellent short-term safety, favorable PK, and preliminary bioactivity signals, with no observed immunogenicity or dose-limiting toxicities; however, these trials remain exploratory, constrained by small sample sizes and safety-focused endpoints. Despite challenges, including methodological heterogeneity, variable yields, long-term safety uncertainties (notably for inorganic hybrids), and regulatory ambiguities, emerging strategies such as clustered regularly interspaced short palindromic repeats (CRISPR)-engineered plant line; artificial-intelligence-driven process optimization; standardized guidelines, and integrated clinical, intellectual property, and commercialization frameworks are progressively addressing these barriers. Collectively, these advances position plant-derived nanocarriers as immunologically privileged, eco-friendly alternatives to synthetic and mammalian platforms, laying the foundation for a sustainable era of precision phytomedicine. Full article

Currently, most research on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing in edible fungi focuses on monokaryotic strains. However, the biological mechanisms in a monokaryotic state often do not accurately reflect the actual physiological and metabolic conditions of dikaryotic strains. Therefore, this study used two mating-type-compatible monokaryotic strains, L1 and L2, isolated from Ganoderma lucidum ‘Hunong No.1’ G0119, and employed an RNP (ribonucleoprotein)-based CRISPR/Cas9 system to successfully knock out the cyp512a3 gene in strain L2, resulting in the edited strain L2-KO-cyp512a3. The strain was single-crossed with the previously edited L1 strain L1-KO-cyp512a3 in our laboratory to obtain a dikaryotic editing strain that was homozygous at the cyp512a3 locus, named G0119-KO-cyp512a3. UPLC-MS (Ultra Performance Liquid Chromatography–Mass Spectrometry) analysis showed that compared to the starting strain G0119, the dikaryotic editing strain exhibited varying degrees of reduction in the content of eight types of ganoderic acids, including ganoderic acid Me, ganoderic acid P, ganoderic acid T1, etc., with the reduction ranging from 30.5% to 80.1%. To further validate the function of cyp512a3, we overexpressed this gene in the L1 strain. The results showed that the contents of ganoderic acid Mk, ganoderic acid S, ganoderic acid T, and ganoderic acid R in the mycelium were 0.548 ± 0.020, 1.780 ± 0.028, 2.416 ± 0.148, and 0.281 ± 0.016 mg/g (dry weight), which were 1.5 times, 1.3 times, 1.3 times, and 1.3 times that of G0119, respectively. By integrating the results of gene knockout and overexpression, it can be clearly established that cyp512a3 is a key cytochrome P450 gene regulating the biosynthesis of ganoderic triterpenoids in Ganoderma lucidum. This study not only establishes, for the first time, a homologous recombination-based gene editing system in dikaryotic strains of Ganoderma lucidum, but also provides a research paradigm based on a dikaryon-editing tool for investigating key life traits of other edible fungi. Full article

Phytoplasmas are obligate intracellular parasitic bacteria that infect over 1000 plant species globally, causing devastating diseases characterized by yellowing, witches’ broom, phyllody, and significant yield losses in economically important crops. The unculturable nature of these pathogens has historically hindered their study; however, advances in molecular biology and genomics have substantially accelerated progress over the past two decades. This review provides a comprehensive overview of current knowledge on phytoplasma diseases and control technologies. In terms of taxonomy, phytoplasmas are currently classified into 37 16Sr groups with over 150 subgroups based on 16S rRNA gene analysis, and approximately 50 ‘Candidatus Phytoplasma’ species have been formally named. Genomic studies have revealed that phytoplasmas possess highly reduced genomes (530–1350 kb) lacking many essential metabolic pathways, reflecting their obligate parasitic lifestyle. Regarding pathogenesis, secreted effector proteins such as SAP (Secreted Aster Yellows Witches’ Broom Protein), TENGU (tengu-su inducer), and SWP (Secreted Wheat Blue Dwarf Protein) manipulate plant hormone signaling and developmental processes, leading to characteristic disease symptoms. Detection technologies have evolved from traditional microscopy to molecular methods, including nested PCR, real-time quantitative PCR, loop-mediated isothermal amplification (LAMP), and CRISPR/Cas-based systems (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein), with AI-based image recognition and remote sensing emerging as promising tools for large-scale field monitoring. Integrated management strategies encompassing agricultural practices, insect vector control, biological control agents, induced resistance, and breeding for resistance are discussed. Finally, future research directions, including functional genomics, microbiome-based approaches, and precision agriculture technologies, are highlighted. This review aims to provide researchers and practitioners with a systematic reference for understanding phytoplasma biology and developing effective disease management strategies. Full article

Plant pathogens are a major cause of crop yield loss, making disease resistance breeding crucial for crop improvement. Plants have evolved innate immune systems, mediated by immune-related genes such as nucleotide-binding site leucine-rich repeat (NLR), pattern-recognition receptors (PRR) and susceptibility genes, which are essential resources for breeding disease-resistant plants. To identify immunity genes, extensive genetic approaches that examine the association between resistance phenotypes and genomic regions have been applied with great success. While genetic methods remain important for identifying immunity genes, novel strategies that rely on functional rather than genetic association with disease resistance offer unique advantages. For example, mutagenesis with R gene enrichment sequencing (MutRenSeq) enabled the identification of wheat resistance genes Sr22 and Sr45 by comparing the NLRomes of resistant and susceptible lines while single-cell RNA sequencing resolved cell-type-specific responses to pathogen infection and revealed ZmChit7, especially in maize epidermal and guard cells. These approaches reach beyond existing natural variation, can accelerate experimental timelines, reduce the experimental scale, and provide mechanistic insights into pathogen resistance. This review discusses emerging techniques that generate focused candidate immunity gene lists or accelerate their validation, as both are required to identify causal variants for resistance breeding. We consider advances in RenSeq-derived methods, spatial omics, proximity labelling, computational prediction, Clustered regularly interspaced short palindromic repeats (CRISPR) screens, and cell death assays. These approaches are reshaping resistance breeding pipelines beyond association-based discovery. By discussing the strengths and limitations of these emerging methods and their combinations, we outline current opportunities and future directions to help plant pathologists to more effectively identify and validate plant immunity genes. Full article

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a simple and powerful tool, which enables gene knockout or insertion of new gene cassettes. This method has been applied to various plants and is used for crop improvement. Cultivated strawberry (Fragaria× ananassa), a member of the Rosaceae family, is a high-value horticultural crop. However, its complex octoploid genome poses challenges for precise genome editing in polyploids. This study aimed to establish a protoplast-based, DNA-free genome-editing approach in the cultivated octoploid strawberry. We optimized the culture conditions and enzyme combinations to enable efficient protoplast isolation from the fully developed leaves. The highest protoplast yield was obtained with a Murashige and Skoog medium containing 1% sucrose and 2 mg/L 6-benzylaminopurine (BA), along with enzymatic digestion using 2% Viscozyme, 1% Celluclast, and 1% Pectinex. Transient transfection conditions were optimized using a green fluorescence protein (GFP) plasmid with the highest expression efficiency (up to 52.5%) observed using 40% PEG 4000 and 20 min incubation. Under these conditions, Cas9 ribonucleoproteins (RNPs) targeting the FaPDS and FaPG1 genes were introduced, and guide RNA (gRNA) screening was conducted by targeted deep sequencing. In conclusion, this study successfully demonstrated protoplast isolation and DNA-free CRISPR/Cas9 genome editing in cultivated strawberry. The optimized protoplast-based system provides a valuable platform for functional genomics and molecular breeding efforts in octoploid strawberries. Full article

Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean–Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed. Full article

High-grade serous ovarian carcinoma (HGSOC) is characterised by profound genomic instability and limited durable responses to standard therapy, leading to poor prognosis. The use of next-generation sequencing technologies has improved understanding of its molecular landscape, revealing consistent Tumour Protein p53 (TP53) mutations, homologous recombination defects, pathway alterations, and epigenetic dysregulation. Such genomic profiling now underpins the classification criteria between the ovarian cancer subtypes described by the Cancer Genome Atlas. Widespread chromosomal instability and pathogenic variants in multiple genes distinguish HGSOC from other subtypes of ovarian cancer and, further, from low-grade serous ovarian cancer. Importantly, the new-found understanding of the genomic landscape of HGSOC guides the use of platinum-based chemotherapies and Poly(ADP-ribose) Polymerase (PARP) inhibitors, with homologous recombination deficiency emerging as a cancer vulnerability that enhances treatment response. A combined multi-omics approach integrates transcriptomics, proteomics, metabolomics, and epigenomics to further the understanding of the characteristics, therapeutic targets and treatment resistance within HGSOC. Despite these advances, major challenges persist, including intratumoural heterogeneity and the poor diversity of genomic datasets. Artificial Intelligence (AI) technology, Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing, neoantigen-guided immunotherapy and ovarian cancer vaccination indicate a promising future for genomics-guided interventions and support the integration of genomics within multi-omic approaches to improve HGSOC outcomes. Full article

This study investigates the functional role of OsHSBP1, a heat shock factor-binding protein, in regulating abiotic stress tolerance in rice, with the aim of enhancing climate resilience in the elite indica cultivar Bacthom 7 (BT7). Using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing, we generated transgene-free homozygous knockout lines targeting OsHSBP1 and evaluated their physiological, biochemical, and agronomic responses under heat stress. Mutant lines exhibited markedly improved tolerance to both stresses, with survival rates reaching 43–46% under heat stress, compared to near-zero in wildtype plants. Enhanced tolerance was associated with significantly increased catalase and peroxidase activities and reduced oxidative damage, including lower malondialdehyde content and decreased superoxide accumulation. Despite these stress-related advantages, the knockout lines showed minimal differences in key agronomic traits under normal growing conditions, with comparable plant height, tillering ability, grain yield, and amylose content relative to the wildtype. These results demonstrate that OsHSBP1 functions as a negative regulator of abiotic stress tolerance in rice, and its knockout enhances resilience without compromising yield potential. The study highlights OsHSBP1 as a promising target for precision breeding of climate-resilient rice cultivars. Full article

Genome editing (GE) has transformed medicine by allowing precise changes to DNA, offering potential treatments for a range of inherited and acquired disorders. Several technologies support these advances, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, of which the latter has emerged as the most accessible, versatile, and popular. While GE holds great promise, its clinical use requires careful attention to safety, ethics and regulatory standards. Inadvertent on- and off-target DNA alterations and unintended modification of non-target cells pose major technical challenges, while bioethical considerations and the need for harmonized safety standards create regulatory challenges. The Food and Drug Administration (FDA) and European Medicines Agency (EMA), as regulatory agencies for key advanced therapy markets, provide detailed guidance on these aspects, emphasizing rigorous preclinical testing, patient monitoring, ethical consent, and compliance with legal frameworks. This concise review summarizes what is currently published in the scientific literature and recommended by regulatory agencies, providing an overview of the responsible clinical application of GE, with emphasis on patient safety, adherence to regulatory guidance, and ethical practice. Full article