Search Results (35,059)

Genome-editing technology has advanced significantly since the 2020 Nobel Prize in Chemistry was awarded for the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). While CRISPR–Cas9 has become widely used in academic research, its social implementation has lagged due to unresolved patent disputes and slower progress in gene function analysis. To address this, new approaches bypassing direct gene function analysis are needed, with bioinformatics and next-generation sequencing (NGS) playing crucial roles. NGS is essential for sequencing the genome of target species, but challenges such as data quality, genome heterogeneity, ploidy, and small individual sizes persist. Despite these issues, advancements in sequencing technologies, like PacBio high-fidelity (HiFi) long reads and high-throughput chromosome conformation capture (Hi-C), have improved genome sequencing. Bioinformatics contributes to genome editing through off-target prediction and target gene selection, both of which require accurate genome sequence information. In this review, I will give updates on the development of genome editing and bioinformatics technologies with a focus on the rapid progress in genome sequencing. Full article

Functional U6 promoters are widely utilized in CRISPR gene editing systems for crops. The identification of endogenous U6 promoter activity and the establishment of CRISPR/Cas9 gene editing systems in various crops can enhance the efficiency and accuracy of gene editing in molecular breeding. In this study, four U6 snRNAs were identified in the genome of the oil flax (Linum usitatissimum L.) cultivar Longya 10, which exhibit high homology with the promoter regions of Arabidopsis thaliana U6 snRNA. We cloned and constructed fusion expression vectors with U6 promoter-driven dual-luciferase reporter genes. Transient transformation of flax and Nicotiana benthamiana was performed to measure the relative activity of dual luciferase. The U6-4 on chromosome 14 showed the highest transcriptional activity. Truncations of varying lengths from the 5′ end of this promoter were tested, revealing that a 342 bp U6 promoter fragment possesses high transcriptional activity and an optimal length. Subsequently, we constructed a CRISPR/Cas9 gene editing vector with LuU6-5P/AtU6-P driving LusPDS sgRNA. Agrobacterium-mediated infection of flax hypocotyls yielded transgenic albino flax shoots. DNA from these shoots was used as a template to amplify LusPDS fragments, which were then sequenced. Sequencing analysis revealed that CRISPR/Cas9 vectors using Lu14U6-4-5P achieved higher editing frequencies at LusPDS compared to AtU6-P-driven systems. Full article

Cholangiocarcinoma (CCA) is an aggressive malignancy with limited methods for early detection, necessitating the development of reliable biomarkers for diagnosis and management. However, conventional tumor markers, such as CA19-9 and CEA, exhibit insufficient diagnostic accuracy. Recent advancements in molecular genetics have identified several actionable mutations in CCA, enabling molecularly targeted therapies that improve survival in patients harboring these genetic alterations. Cancer panels, which facilitate multiplex genetic profiling, are critical for identifying these mutations. Studies indicate that several actionable mutations are detected in CCA cases, with patients receiving mutation-guided therapies achieving markedly better outcomes. Liquid biopsies, including cell-free DNA and circulating tumor DNA, offer real-time, non-invasive approaches to monitoring tumor dynamics, heterogeneity, and treatment responses. Furthermore, numerous studies have identified non-coding RNAs in serum and bile as promising biomarkers for the diagnosis and management of CCA. On the other hand, immunotherapy, particularly immune checkpoint inhibitors, has shown efficacy in subsets of CCA patients. However, the success of these therapies is often affected by the status of the tumor immune microenvironment (TME), underscoring the need for comprehensive TME analysis to predict responses to immune checkpoint inhibitors. Despite these advances, no single biomarker currently demonstrates sufficient sensitivity or specificity for clinical application. The integration of multi-omics approaches with cutting-edge technologies holds promise for enhancing diagnostic accuracy, optimizing treatment stratification, and advancing precision medicine in CCA. These developments highlight the transformative potential of biomarkers to improve early detection, prognostic assessment, and personalized therapeutic interventions for CCA. Full article

By leveraging machine learning insights from prior perovskite studies and employing the sol–gel method, we successfully synthesized two novel perovskite nanoceramics—M_0.5 Ca_0.25Mg_0.25MnO₃ (M = La, Pr)—as multifunctional nanomaterials. X-ray diffraction (XRD) confirmed their orthorhombic Pnma crystal structure. The Williamson–Hall method estimated average particle sizes of 59.5 nm for PCMMO and 21.8 nm for LCMMO, while the Scherrer method provided corresponding values of 32.59 nm and 20.43 nm. SEM, UV-Vis, and FTIR analyses validated the chemical composition, homogeneity, and optical properties of the synthesized compounds, revealing band gaps of 3.25 eV (LCMMO) and 3.71 eV (PCMMO) with Urbach energies of 0.29 eV and 0.26 eV, respectively. These findings provide valuable insights into the structural and optical properties of LCMMO and PCMMO, highlighting their potential as multifunctional materials for advanced device applications. Full article

Pepper (Capsicum annuum L.), recognized as a globally preeminent vegetable, holds substantial economic and nutritional value. The BTB (broad-complex, tramtrack, and bric-a-brac) family of proteins, characterized by a highly conserved BTB domain, also denoted as the POZ domain, are intricately involved in a diverse array of biological processes. However, the existing corpus of research regarding pepper BTB genes remains relatively meager. In this study, a total of 72 CaBTB gene members were meticulously identified from the entire genome of pepper. Phylogenetic analysis illuminated the presence of conspicuous collinear relationships between the CaBTB genes and those of its closely affiliated species. Gene expression profiling and RT-qPCR analysis revealed that multiple CaBTB genes exhibited pronounced differential expression under diverse treatment regimens. Expression pattern analysis unveiled that CaBTB25 manifested a remarkably elevated abundance in leaves. Moreover, its promoters were replete with an abundance of light-responsive cis-elements. Our comprehensive and in-depth explorations into subcellular localization revealed that CaBTB25 was predominantly detected to localize within the nucleus and lacked transcriptional activation. This research provides a crucial theoretical edifice, enabling a more profound understanding of the biological functions of the BTB gene family in pepper, thereby underscoring its potential significance within the intricate network of gene–environment interactions. Full article

Background/Objectives: The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme, encoded by OsGAPDHC6, plays a crucial role in glycolysis while participating in various physiological and stress response pathways. Methods: In this study, the expression levels of the OsGAPDHC1 and OsGAPDHC6 genes were investigated over time by treating various abiotic stresses (ABA, PEG, NaCl, heat, and cold) in rice seedlings. Results: As a result, the expression levels of both genes in the ABA-treated group increased continuously for 0–6 h and then de-creased sharply from 12 h onwards. The mutational induction of the GAPDHC6 gene by the CRISPR/Cas9 system generated a stop codon through a 1 bp insertion into protein production. The knockout (KO) lines showed differences in seed length, seed width, and seed thickness compared to wild-type (WT) varieties. In addition, KO lines showed a lower germination rate, germination ability, and germination index of seeds under salt treatment compared to WT, and leaf damage due to 3,3′-diaminobenzidine (DAB) staining was very high due to malondialdehyde (MDA) accumulation. The KO line was lower regarding the expression level of stress-related genes compared to WT. Conclusions: Therefore, the OsGAPDHC6 gene is evaluated as a gene that can increase salt resistance in rice as it actively responds to salt stress in the early stages of growth, occurring from seed germination to just before the tilling stage. Full article

Houpoea officinalis (H. officinalis) flowers are rich in a spectrum of bioactive compounds and mineral nutrients. The availability and balance of mineral elements directly impact the morphogenesis of flower organs, which play pivotal roles in various physiological and biochemical processes that drive flower development. However, relatively little is known about the changes in mineral elements composition that occur during flower development in H. officinalis. The objective of this study is to analyze the variations of 22 mineral elements contents in pistil, stamens, and petals of H. officinalis flower at four development stages. The amount of mineral elements (Na, Mg, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Sr, Sn, Al, Ti, Ga, Cd, Ba, Tl, Pb, and Bi) in these samples was determined using atomic absorption spectroscopy and inductively coupled plasma mass spectrometry. Results showed that H. officinalis flowers are rich in macroelements such as potassium (K, 25.80–48.06 mg/g) and calcium (Ca, 17.27–31.00 mg/g), as well as microelements like zinc (Zn, 445.17–1553.16 μg/g) and iron (Fe, 324.27–622.31 μg/g). Notably, the pistil part is found to harbor a more significant concentration of mineral elements during the early developmental stages of flowers. Correlation analysis and PCA have effectively exposed a pronounced association between the accumulation patterns of mineral elements in H. officinalis flowers and their corresponding developmental stages and organs. These findings will provide more detailed information about the accumulation and distribution of mineral elements in H. officinalis flowers at different development stages and organs, which help to encourage researchers to enhance the flower quality for human consumption. Full article

Changes in microRNA (miRNA) levels are closely associated with the pathological processes of many diseases. The sensitive and fast detection of miRNAs is critical for diagnosis and prognosis. Here, we report a platform employing CRISPR/Cas12a to recognize and report changes in miRNA levels while avoiding complex multi-thermal cycling procedures. A non-enzyme-dependent hybridization chain reaction (HCR) was used to convert the miRNA signal into double-stranded DNA, which contained a Cas12a activation sequence. The target sequence was amplified simply and isothermally, enabling the test to be executed at a constant temperature of 37 °C. The detection platform had the capacity to measure concentrations down to the picomolar level, and the target miRNA could be distinguished at the nanomolar level. By using photonic crystal microarrays with a stopband-matched emission spectrum of the fluorescent-quencher modified reporter, the fluorescence signal was moderately enhanced to increase the sensitivity. With this enhancement, analyzable fluorescence results were obtained in 15 min. The HCR and Cas12a cleavage processes could be conducted in a single tube by separating the two procedures into the bottom and the cap. We verified the sensitivity and specificity of this one-pot system, and both were comparable to those of the two-step method. Overall, our study produced a fast and sensitive miRNA detection platform based on a CRISPR/Cas12a system and enzyme-free HCR amplification. This platform may serve as a potential solution for miRNA detection in clinical practice. Full article

Citrus alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is one of the most severe fungal diseases affecting citrus crops. Currently, there is a critical need for rapid and visual detection techniques to identify the tangerine pathotype of A. alternata. In this study, a novel detection system was developed by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology, targeting the ACTT3 gene specific to the tangerine pathotype of A. alternata. Through optimization of reaction time and component concentrations, the assay demonstrated a detection sensitivity of 1 pg μL⁻¹ within 40 min at a constant temperature of 37 °C. The results can be visually interpreted using nucleic acid test strips, offering advantages in specificity, sensitivity, and speed. This system has been successfully validated for the rapid detection of the pathogen within plant tissues, including leaves and fruits, providing an efficient and practical solution for real-time field detection of the tangerine pathotype of A. alternata. Full article

The elastic modulus of basalt fibers is closely associated with their chemical composition. In this study, eight machine learning models were developed to predict the elastic modulus, with hyper-parameter tuning implemented through the GridSearchCV technique. Model performance was evaluated using the coefficient of determination (R²), root-mean-square error (RMSE), and mean absolute error (MAE). SHAP analysis was employed to uncover the relevance of oxide compositions and their interactions with the elastic modulus. Among these models, the Categorical Boosting algorithm exhibited the best results, with an R² of 0.9554, an RMSE of 4.7556, and an MAE of 2.0323. SHAP analysis indicated that CaO had the most significant influence on elastic modulus predictions. The importance of other oxides was ranked as follows: SiO₂, Al₂O₃, MgO, K₂O, Na₂O, Fe₂O₃, FeO, and TiO². Additionally, SHAP analysis determined oxide ranges for positive elastic modulus prediction. This research provides new insights into leveraging machine learning to optimize the mechanical properties of basalt fibers. Full article

Ultraviolet B (UVB) radiation is a key factor contributing to photodamage in epidermal cells. This study investigated the protective effects of Thua Nao, a Thai fermented soybean product, against UVB-induced damage in human epidermal keratinocytes (HaCaT) and the underlying mechanisms. Thua Nao extract fractions were prepared using a solvent partition method. We found that the dichloromethane fraction (TN-DC), along with its isoflavones daidzein and glycitein, significantly protected against UVB-induced HaCaT cell death. This protection involved inhibiting caspase-9 and caspase-3 activation, thus preventing apoptosis. Additionally, treatment with TN-DC, daidzein, and glycitein suppressed the UVB-induced production of inflammatory mediators, including interleukin-6 (IL-6), IL-8, inducible nitric oxide synthase, and cyclooxygenase-2. These protective effects were associated with reduced intracellular reactive oxygen species and enhanced the levels of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase 4. Signaling pathway analysis revealed that TN-DC activated the pro-survival ERK1/2 and Akt pathways while decreased the phosphorylation of JNK in UVB-exposed cells. On the other hand, daidzein and glycitein enhanced ERK1/2 activation and reduced the phosphorylation of JNK and p38 MAPKs. The involvement of ERK1/2 and Akt activation in cell survival was confirmed using specific inhibitors. Thus, TN-DC and its isoflavones protects keratinocytes from UVB-induced oxidative damage and inflammation by modulating MAPKs and Akt signaling. Full article

Objectives: Pancreatic cancer remains a therapeutic challenge due to its immunosuppressive microenvironment and treatment resistance. This study aimed to develop a novel recombinant oncolytic vaccinia virus (VVL-GL7) co-expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-7 (IL-7), designed to enhance anti-tumor immunity and synergize with immune checkpoint inhibitors. Methods: VVL-GL7 was constructed through CRISPR/Cas9-mediated knockout of TK and A49 genes, combined with the simultaneous insertion of dual cytokine-encoding cassettes. Anti-tumor efficacy was evaluated in vitro and in vivo using C57BL/6 mouse and Syrian hamster pancreatic cancer models. Comprehensive immune profiling evaluated CD8⁺ T-cell and macrophage infiltration dynamics while simultaneously assessing memory T-cell differentiation patterns using flow cytometry. Preclinical combination studies of VVL-GL7 and the PD-1 immune checkpoint inhibitor were systematically evaluated in a syngeneic pancreatic cancer model. Results: VVL-GL7 exhibited potent oncolytic activity, inducing significant tumor regression in both preclinical models. VVL-GL7 therapy significantly augmented CD8⁺ T-cell and macrophage infiltration within the tumor microenvironment, while concomitantly driving memory T-cell differentiation. The synergistic effects of VVL-GL7 and the PD-1 blockade further improved therapeutic outcomes, resulting in significantly higher tumor remission rates compared to monotherapy and achieving complete tumor regression in pancreatic cancer models. Conclusions: VVL-GL7 reprograms the immunosuppressive tumor microenvironment and synergizes with anti-PD-1 antibodies to overcome resistance in pancreatic cancer. Full article

Amending saline–alkali soils to improve agricultural productivity is critical for addressing global food security challenges. Biochar is a promising soil amendment, and its modified composites offer significant potential for soil remediation. In this study, we developed a novel phosphoric acid–mineral-comodified biochar composite for saline–alkali soil improvement. SEM and XRD analyses indicate that chemical interactions between phosphoric acid, minerals, and biochar result in the formation of distinct mineral phases on the composite surface. Furthermore, FTIR analysis reveals that these interactions give rise to functional groups such as Si-O-Si, and thermogravimetric analysis demonstrates that the modified biochar composite exhibited enhanced stability. Compared with raw biochar, the modified biochar composites exhibited significant decreases in pH, EC, and base cation content (especially Na⁺), with maximum reductions of 7.26 pH units, 639.5 μS/cm, and 3.69 g/kg, respectively. In contrast, the contents of P, Si, and Ca increased significantly, with maximum increases of 140.04 g/kg, 90.32 g/kg, and 114.27 g/kg, respectively. In addition, the specific surface area and pore volume of the modified biochar composite increased by up to 5.2 and 15 times, respectively. Principal component analysis indicates that mineral type was the primary factor influencing the properties of the composites: hydroxyapatite enhanced porosity and phosphorus levels, whereas kaolinite and montmorillonite increased silicon content. Pot experiments show that the modified biochar composite increased alfalfa plant height by 17.36–20.27% and shoot biomass by 107.32–125.80% in saline–alkali soils. Overall, the newly developed phosphoric acid–mineral–biochar composites were evaluated to have high application potential for saline–alkali soil amendment. Full article

Upfront Next-Generation Sequencing (NGS) is increasingly recommended in advanced NSCLC to guide targeted therapy. This prospective single-center study in Romania evaluated routine, upfront NGS in advanced NSCLC at baseline (tissue and/or liquid) and progression (liquid). Baseline FoundationOne NGS (tissue/liquid) was performed in 119 consecutive stage IV NSCLC patients, along with PD-L1 immunohistochemistry (IHC, SP263). Liquid biopsy was repeated at progression. Turnaround time (TAT), the prevalence of actionable targets, and clinical utility were assessed. Patients were predominantly male (68.1%) with a median age of 62 years (range 30–86). Most had ECOG PS 0–1 (79%) and non-squamous histology (67.2%). Never-smokers accounted for 25.2%. The median TAT for the NGS results was 9 days (range 5–21). Overall, 671 genetic alterations were detected in 149 genes. The mean number of distinct mutations per patient dropped from 5.6 at baseline to 4.3 at progression. Tissue samples yielded more alterations (6 per patient) than baseline liquid biopsies (4.6). Squamous tumors had more alterations (7.1 vs. 4.8 in non-squamous), and the number of smokers exceeded that of never-smokers (6 vs. 4.5). TP53 was the most frequent (70.59%). Actionable variants were found in 74.8% of patients, though only 35.3% received personalized therapy, largely due to performance status deterioration, reimbursement, or trial availability barriers. Common targets in non-squamous tumors included EGFR (21%), KRAS G12C (11%), NF1 (11%), and ERBB2 (6%); in squamous tumors, common targets included NF1 (24%), PIK3CA (18%), and ERBB2 (8%). Among smokers, driver mutations were often NF1 (15%), PIK3CA (11%), KRAS G12C (9%), and ERBB2 (8%); never-smokers were dominated by EGFR (45%), NF1 (15%), and KRAS G12C (8%). TMB ≥ 10 mut/Mb was seen in 26.9%; no patients were MSI-H. PD-L1 TPS was <1% in 33% of patients, 1–49% in 20%, ≥50% in 18%, and unknown in 29%. Upfront NGS offers rapid, comprehensive genomic data, guiding tailored therapies and trials in advanced NSCLC. Liquid rebiopsy at progression further refines treatment decisions. Full article

CRISPR/Cas-based diagnostics offer unparalleled specificity, but their reliance on fluorescently labeled probes and complex nucleic acid extraction limits field applicability. To tackle this problem, we have developed a label-free, equipment-free platform integrating FTA card-based extraction, CRISPR/Cas12a, and pre-folded G-quadruplex (G4)–Thioflavin T (ThT) signal reporter. This system eliminates costly fluorescent labeling by leveraging G4-ThT structural binding for visible fluorescence output, while FTA cards streamline nucleic acid isolation without centrifugation. Achieving a limit of detection (LOD) to 10¹ CFU/mL for Escherichia coli O157:H7 in spiked food samples, the platform demonstrated 100% concordance with qPCR and standard fluorescent probe-based CRISPR/Cas12a system. Its simplicity, minimal equipment (portable heating/imaging), and cost-effectiveness make it a revolutionary tool for detecting foodborne pathogens in resource-limited environments. Full article