Pornchai Kaewsapsak

Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A–D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R² = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance.

Scheme of peroxidase-catalyzed proximity-based labeling by a novel homo-disubstituted aniline biotin probe.

Helicobacter pylori is a gastric pathogen implicated in peptic ulcer disease and gastric cancer. The increasing prevalence of antibiotic-resistant strains underscores the urgent need for alternative therapeutic strategies. In this study, we investigated the chemical composition and antibacterial activity of an aqueous extract from Caulerpa lentillifera (sea grape), a farm-cultivated edible green seaweed collected from Krabi Province, Thailand. Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) revealed that the extract was enriched in bioactive nucleosides and phenolic compounds. In vitro assays demonstrated dose-dependent inhibition of H. pylori growth following exposure to sea grape extract. Furthermore, untargeted intracellular metabolomic profiling of H. pylori cells treated with the extract uncovered significant perturbations in central carbon and nitrogen metabolism, including pathways associated with the tricarboxylic acid (TCA) cycle, one-carbon metabolism, and alanine, aspartate, and glutamate metabolism. Pyrimidine biosynthesis was selectively upregulated, indicating a potential stress-induced shift toward nucleotide salvage and DNA repair. Of particular note, succinate levels were markedly reduced despite accumulation of other TCA intermediates, suggesting disruption of electron transport-linked respiration. These findings suggest that bioactive metabolites from C. lentillifera impair essential metabolic processes in H. pylori, highlighting its potential as a natural source of antimicrobial agents targeting bacterial physiology.

Purpose The epidermal growth factor receptor (EGFR) mutation is a widely prevalent oncogene driver in non–small cell lung cancer (NSCLC) in East Asia. The detection of EGFR mutations is a standard biomarker test performed routinely in patients with NSCLC for the selection of targeted therapy. Here, our objective was to develop a portable new technique for detecting EGFR (19Del, T790M, and L858R) mutations based on Nanopore sequencing.Materials and Methods The assay employed a blocker displacement amplification (BDA)–based polymerase chain reaction (PCR) technique combined with Nanopore sequencing to detect EGFR mutations. Mutant and wild-type EGFR clones were generated from DNA from H1650 (19Del heterozygous) and H1975 (T790M and L858R heterozygous) lung cancer cell lines. Then, they were mixed to assess the performance of this technique for detecting low variant allele frequencies (VAFs). Subsequently, formalin-fixed, paraffin-embedded (FFPE) tissue and cell-free DNA (cfDNA) from patients with NSCLC were used for clinical validation.Results The assay can detect low VAF at 0.5% mutant mixed in wild-type EGFR. Using FFPE DNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and Cobas real-time PCR were 98.46%, 100%, and 100%, respectively. For cfDNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and droplet digital PCR were 94.74%, 100%, and 100%, respectively.Conclusion The BDA amplicon Nanopore sequencing is a highly accurate and sensitive method for the detection of EGFR mutations in clinical specimens.

Coronavirus disease (COVID-19) caused by the SARS-CoV-2 has been an outbreak since late 2019 up to now. This pandemic causes rapid development in molecular detection technologies to diagnose viral infection for epidemic prevention. In addition to antigen test kit (ATK) and polymerase chain reaction (PCR), CRISPR-based assays for detection of SARS-CoV-2 have gained attention because it has a simple setup but still maintain high specificity and sensitivity. However, the SARS-CoV-2 has been continuing mutating over the past few years. Thus, molecular tools that rely on matching at the nucleotide level need to be reevaluated to preserve their specificity and sensitivity. Here, we analyzed how mutations in different variants of concern (VOC), including Alpha, Beta, Gamma, Delta, and Omicron strains, could introduce mismatches to the previously reported primers and crRNAs used in the CRISPR-Cas system. Over 40% of the primer sets and 15% of the crRNAs contain mismatches. Hence, primers and crRNAs in nucleic acid-based assays must be chosen carefully to pair up with SARS-CoV-2 variants. In conclusion, the data obtained from this study could be useful in selecting the conserved primers and crRNAs for effective detections against the VOC of SARS-CoV-2.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate target mRNA expression, and altered expression of miRNAs is associated with liver pathological conditions. Recent studies in animal models have shown neutrophil/myeloid-specific microRNA-223 (miR-223) as a key regulator in the development of various liver diseases including fibrosis, where hepatic stellate cells (HSCs) are the key player in pathogenesis. However, the precise roles of miR-223 in human HSCs and its therapeutic potential to control fibrosis remain largely unexplored. Using primary human HSCs, we demonstrated that miR-223 suppressed the fibrogenic program and cellular proliferation while promoting features of quiescent HSCs including lipid re-accumulation and retinol storage. Furthermore, induction of miR-223 in HSCs decreased cellular motility and contraction. Mechanistically, miR-223 negatively regulated expression of smooth muscle α-actin (α-SMA) and thus reduced cytoskeletal activity, which is known to promote amplification of fibrogenic signals. Restoration of α-SMA in miR-223-overexpressing HSCs alleviated the antifibrotic effects of miR-223. Finally, to explore the therapeutic potential of miR-233 in liver fibrosis, we generated co-cultured organoids of HSCs with Huh7 hepatoma cells and challenged them with acetaminophen (APAP) or palmitic acid (PA) to induce hepatotoxicity. We showed that ectopic expression of miR-223 in HSCs attenuated fibrogenesis in the two human organoid models of liver injury, suggesting its potential application in antifibrotic therapy.