Sulfur metabolism and stress responses are fundamental to plant growth and seed development. ETHE1 encodes a mitochondrial sulfur dioxygenase that plays important roles in these processes; however, its function in Jatropha curcas remains unknown. In this study, we report the cloning and in silico characterization of a putative JcETHE1 gene from J. curcas. Bioinformatic analysis revealed that JcETHE1 has an open reading frame of 726 bp encoding a 241-amino-acid protein, which is predicted to localize in mitochondria and possesses a typical sulfur dioxygenase conserved domain. Because the transcript ends were not experimentally confirmed and the predicted protein is shorter than related plant homologues, the possibility that the cloned sequence is partial cannot be excluded. Phylogenetic analysis showed that JcETHE1 clusters within the Euphorbiaceae clade, showing high similarity to ETHE1 from Manihot esculenta and Hevea brasiliensis. In silico promoter analysis suggested that, in addition to core elements, the JcETHE1 promoter contains multiple putative regulatory elements associated with light response, hormone responses (ABA, ethylene, gibberellin), floral development (CArG-box, AAGAA-motif), stress responses, and endosperm development. These findings provide a molecular basis for future functional studies of JcETHE1 and suggest that it may serve as a potential candidate gene for further investigation into reproductive development and stress responses in J. curcas. However, these hypotheses require experimental validation.
Glycosyltransferases (GTs) are key enzymes that catalyze the transfer of sugar moieties to diverse acceptor molecules and play important roles in a wide range of biological processes, including plant growth, development, and environmental adaptation. However, glycosyltransferase family 4 (GT4), the second-largest GT family, remains poorly characterized in soybean. In this study, we identified 60 GT4 family members in the soybean genome based on sequence information from the CAZy database and systematically characterized their phylogenetic relationships, genomic organization, and expression patterns by integrating genomic and multidimensional transcriptomic data. Furthermore, using a previously established soybean ethyl methanesulfonate (EMS) mutant population, we identified GmSPS8 as a candidate gene potentially involved in the regulation of seed weight. Notably, natural variation and haplotype analyses revealed that GmSPS8 is located within a genomic region subject to domestication selection, suggesting its potential involvement in the evolutionary selection of soybean seed size. In sum, we systematically characterized the soybean GT4 gene family and identified GmSPS8 as a potential domestication-associated candidate gene for seed weight regulation, providing new insights into the functional characterization of soybean GT4 family members and the identification and utilization of genes associated with soybean yield-related traits.
Endocrine disorders and inherent flaws in oocyte quality are traditional causes of female infertility. Increasing research indicates that structural and biomechanical alterations in the ovarian microenvironment may significantly contribute to reproductive decline. Progressive extracellular matrix remodeling, stromal fibrosis, chronic inflammation, and heightened tissue stiffness seem to affect follicular homeostasis, granulosa–oocyte communication, and ovarian function. Fibro-inflammatory pathways, including transforming growth factor-β (TGF-β), Hippo/YAP-TAZ signaling, oxidative stress, macrophage activation, and mechanotransduction, are increasingly associated with ovarian ageing, polycystic ovary syndrome, reduced ovarian reserve, and unfavourable reproductive outcomes. Here, the term ‘fibro-inflammatory ovary’ is used descriptively to unify these mechanisms rather than as an established clinical entity. Mechanical alterations of the ovarian stroma may affect follicular activation, vascularization, and oocyte competence by altering cellular tension and extracellular matrix dynamics. Our review examines growing findings about ovarian fibrosis and stromal remodeling in female infertility, focusing on molecular causes, mechanobiology, and translational implications for assisted reproduction. Potential diagnostic uses, such as ovarian elastography, and prospective anti-fibrotic therapy techniques are also examined.
Colorectal cancer (CRC) motivates the investigation of complementary molecular markers, although tissue-transcriptomic discrimination does not by itself establish early-detection performance. We identified differentially expressed genes in 98 paired CRC and adjacent-normal samples from GSE44076 using gene-level limma analysis (|log2FC| > 1; Benjamini–Hochberg-adjusted p < 0.05), screened candidates through a STRING protein–protein interaction network, and evaluated diagnostic performance with independent validation in GSE9348. Functional characterization included MSigDB KEGG_LEGACY over-representation analysis and preranked GSEA, immune-marker correlations, mRNA stemness index analysis, exploratory topology-filtered cysteine assessment, exploratory TCGA-COAD prognostic modelling, and literature-curated single-cell contextualization. Seven candidates—AQP8, CA7, GUCA2A, GUCA2B, BEST4, TMIGD1, and OTOP2—showed AUC values of 0.986–0.995 in the discovery cohort; five genes showed AUC values of 0.999–1.000 in the retrospective GSE9348 tissue cohort. AQP8 and GUCA2B showed weak nominal inverse correlations with mRNAsi that did not remain significant after correction across six tests (BH-FDR = 0.078), and the genes were predominantly associated with differentiated colonic epithelial lineages. The exploratory prognostic model was non-significant. These results identify seven tissue-transcriptomic candidates with strong dataset-specific discriminative performance and provide a multidimensional biological framework warranting experimental validation and prospective clinical evaluation.
Chili pepper consumption has been linked to lower cardiovascular disease (CVD) risk, although underlying mechanisms remain unclear. We investigated the association between long-term changes in chili pepper consumption and changes in circulating inflammatory markers. This longitudinal analysis included 573 adults (≥35 years) from the Italian Moli-sani Study with dietary, health, and biological data collected at baseline (2005–2010) and follow-up (2017–2020). The exposure was the 12.7-year change in chili pepper consumption frequency. Outcomes included changes in serum concentrations of C-reactive protein, eight other inflammatory factors (IL-6, IL8, IL-10, TNF-α, IFN-γ, MCP-1, IP-10, and VEGF), and the INFLA-score. Multivariable-adjusted linear regression models accounted for changes in dietary and lifestyle factors. Overall, 41.2%, 20.6%, and 38.2% of participants reduced, increased, or maintained their chili pepper consumption, respectively. A 1-unit increase in the delta of chili pepper consumption was associated with lower serum TNF-α concentrations (β = −0.85; 95% CI: −1.67, −0.03; p = 0.041). No associations were observed with the other inflammatory markers, although most showed downward trends. Among women, increased consumption was associated with different MCP-1 and IP-10 levels. An increase in chili pepper consumption over 12.7 years was associated with lower circulating TNF-α concentrations in this population. Although no consistent associations were observed across the inflammatory markers evaluated, these findings suggest a potential modulation of selected inflammatory pathways by sustained chili pepper consumption and warrant confirmation in larger prospective studies.
The G protein-coupled receptor, CXC chemokine receptor 5 (CXCR5), is predominantly expressed on B cells located in the secondary lymphoid tissues, follicular helper T cells, and lymphoma cells. Binding to its ligand, CXCL13, mediates cell migration and regulates lymphocyte trafficking. Aberrant CXCL13/CXCR5 expression and signaling have been implicated in tumor progression, autoimmune diseases, and chronic inflammatory disorders. Therefore, specific mAbs against CXCR5 are expected to be useful for diagnosis and therapeutic applications. In this study, novel anti-human CXCR5 mAbs (Cx5Mabs) were developed through flow cytometry-based high-throughput screening. One clone, Cx5Mab-6 (IgG2b, κ), recognized CXCR5-overexpressed Chinese hamster ovary (CHO)-K1 cells but did not react with the other five CXCR receptors-overexpressed CHO-K1 cells in flow cytometry. Additionally, Cx5Mab-6 recognized endogenous CXCR5 in the human Burkitt lymphoma Raji cell line. The dissociation constant (KD) values of Cx5Mab-6 for CHO/CXCR5 and Raji were 3.4 × 10−9 M and 1.2 × 10−10 M, respectively. Furthermore, Cx5Mab-6 is useful for Western blotting and can detect CXCR5 in human lymphoma tissue by immunohistochemistry. These findings suggest that Cx5Mab-6 is versatile for basic research and has potential applications in clinical diagnosis.
Background: Hepatocellular carcinoma (HCC) remains a leading cause of cancer mortality. The c-Met and VEGFR2 pathways synergistically drive HCC progression. Marine natural products offer chemically diverse drug reservoirs; however, conventional activity-guided isolation faces labor intensity, low throughput, and frequent compound rediscovery, limiting marine drug development. Objective: To pioneer an artificial intelligence-driven marine drug discovery workflow integrating deep learning virtual screening for identifying dual c-Met/VEGFR2 promising in silico candidate from marine natural product repositories. Methods: UniSite predicted binding pockets in c-Met (PDB: 4R1V) and VEGFR2 (PDB: 2XIR). Drug-likeness filtering of 695,000 compounds from COCONUT and CMNPD databases yielded 84,730 candidates. DiffDock-based screening identified dual-target binders, validated through 200 ns molecular dynamics simulations, MM-GBSA calculations, and DFT analyses. Results: The marine phthalide CMNPD30506 [(S)-3-ethyl-5,6-dihydroxyphthalide] emerged as the lead candidate, engaging VEGFR2 via four hydrophobic contacts and one π-cation interaction with LYS868, while binding c-Met through four hydrophobic interactions, two hydrogen bonds, and π-π stacking. Molecular dynamics demonstrated stable RMSD profiles and dynamic hydrogen bond enrichment. MM-GBSA revealed binding free energies of −14.79 and −13.28 kcal/mol for VEGFR2 and c-Met, respectively, driven by van der Waals forces. DFT calculations indicated a HOMO-LUMO gap of 2.410 eV. Conclusions: This AI-augmented workflow successfully identified CMNPD30506 as a promising dual c-Met/VEGFR2 HCC therapeutic from marine libraries, overcoming traditional discovery bottlenecks through integrated deep learning and physics-based simulations, exemplifying AI’s potential in marine pharmacological research.
Methotrexate (MTX) remains central to the treatment of rheumatoid arthritis and several malignancies, yet its use is complicated by dose-dependent toxicity, variable oral exposure, photolability, and pH-dependent solubility. Cyclodextrins (CDs) can alter the molecular environment of MTX, but the literature often conflates true inclusion complexes with formulations in which CD merely forms part of a larger carrier. This review critically distinguishes direct MTX–CD complexes, dosage forms built from a preformed complex, CD-containing carriers without direct evidence of cavity occupancy, and covalent MTX–CD conjugates. Particular attention is given to binding stoichiometry, apparent association constants, guest orientation, preparation methods, and the evidence needed to establish inclusion. Both solution-state host–guest association and isolated solid products are considered; however, solid-state changes are treated as supportive evidence rather than as stand-alone proof of cyclodextrin cavity occupancy. Among the limited head-to-head comparisons of native cyclodextrins, β-CD generally showed more favorable MTX recognition than α- or γ-CD, although the magnitude of this difference is method- and condition-dependent. Complexation can improve dissolution, photostability, and oral or local delivery; however, greater solubilization does not necessarily enhance membrane transport. In carrageenan hydrogels, β-CD increased MTX loading and release while reducing membrane permeation, illustrating the importance of the equilibrium between complexed and freely permeating drug. The most promising systems remain preclinical. Progress toward translation will require clearer nomenclature, orthogonal structural characterization, mechanism-resolving controls, and standardized pharmacokinetic and safety studies.
The peroxisome has been implicated in the processes of aging and longevity regulation. Nevertheless, the mechanism by which peroxisomes regulate longevity remains far from fully elucidated. Here, we compared reactive oxygen species (ROS) levels among five long-lived Caenorhabditis elegans mutants using cross-validation with four different ROS probes and observed an increase in hydrogen peroxide (H2O2) levels in rsks-1(ok1255) mutants. Transcriptomic analysis revealed that the genes involved in peroxisomal fatty acid β-oxidation were upregulated in the rsks-1(ok1255) mutants. The imbalance of H2O2 metabolism in the rsks-1(ok1255) mutants was found to be regulated by the peroxisomal fatty acid β-oxidation enzyme ACOX-1.2. Furthermore, lifespan assays and genetic analysis of transgenic strains demonstrated that the increases in autophagy and lifespan observed in long-lived rsks-1(ok1255) mutants were suppressed by acox-1.2 RNAi. These findings uncover a previously unknown role of peroxisomal fatty acid β-oxidation in the regulation of aging and longevity.
Rabbit monoclonal antibodies (RabMAbs) are valuable for biomedical research and diagnostic applications because of their high affinity, specificity, and broad epitope recognition; here, we established an integrated phenotype-linked workflow for rapid RabMAb discovery using serum-derived polyclonal mouse IgG as a proof-of-concept model antigen. Three Big-Eared White rabbits were immunized in parallel, and the rabbit exhibiting the highest serum endpoint titer was selected for the complete downstream single-B-cell discovery workflow. Approximately 5 × 105 activated B cells were subjected to polydisperse oblate dispersion system (POD)-based screening, yielding 13,266 antigen-positive POD events. Following recovery and 10× Genomics single-cell V(D)J sequencing, 4294 B-cell barcodes yielded valid/interpretable V(D)J data, of which 2099 contained at least one complete, productive, and translatable heavy-chain/light-chain pair, generating 2199 functional VH/VL pairing records. AbFinder™-assisted prioritization generated a computationally recommended pool of 158 candidates, and the five highest-ranked VH/VL pairs within this pool were selected for recombinant expression and validation. All five yielded antigen-reactive RabMAbs with an endpoint ELISA titer of 1:256,000 and BLI-derived apparent KD values ranging from 4.19 × 10−10 to 9.79 × 10−9 M. The antibodies showed differential concentration-dependent reactivity toward mouse IgG1, IgG2a, IgG2b, and IgG3 preparations, weak reactivity toward human IgG, and clone-dependent reactivity toward rat IgG. The workflow from spleen collection to functional validation was completed within approximately three weeks. Because only five prioritized candidates from one selected responder rabbit were evaluated, the 5/5 validation outcome should not be interpreted as an overall platform hit rate or definitive validation of the prioritization algorithm. These findings support the feasibility of this workflow for research-grade and diagnostic antibody discovery, while broader evaluation will require larger candidate cohorts, independent biological validation, and additional antigen classes.
Hypoxia is a hallmark of the tumor microenvironment. Under hypoxia, HIF-1α accumulates and promotes both epithelial–mesenchymal transition (EMT) and glycolysis depending on glucose levels. However, how EMT and glycolysis are coordinated by oxygen and glucose abundance is still not well understood. Here, we developed an integrated model to investigate the mechanism underlying the regulation of EMT and glycolysis at varying oxygen and glucose levels. We focused on how the interplay between EMT and glycolysis maintains cell phenotypes. Our results show that hypoxia and sufficient glucose facilitate the transition of cells toward an invasion-associated mesenchymal–glycolytic phenotype. Moreover, enhanced glycolysis promotes the completion of EMT and reinforces the intermediate states. Under glucose-sufficient conditions, the reciprocal promotion between EMT and glycolysis may convert transient hypoxia into persistent mesenchymal memory that maintains the mesenchymal phenotype after reoxygenation. Our work clarifies how metabolic microenvironmental fluctuations are transformed into durable invasion-associated phenotypic states. Our work may provide insights into therapies that target both the EMT and glycolysis pathways.
Localized phytochemical formulations may provide complementary strategies for controlling mucosal colonization by Streptococcus agalactiae and Streptococcus pyogenes, but computational prioritization requires orthogonal biological validation. This study integrated molecular docking against the redox-sensing transcriptional repressor Rex of S. agalactiae and a protein tyrosine phosphatase target of S. pyogenes (SP-PTP) with in vitro broth microdilution, biofilm, target-bridging, and epithelial-tolerance experiments. Isoflavone showed the most favorable natural-compound interaction with Rex (ΔG = −8.3 kcal/mol), whereas secoisolariciresinol diglucoside (SDG) led the natural-ligand ranking for SP-PTP (ΔG = −5.2 kcal/mol). The complete formulation produced MIC50 values of 0.031% and 0.063% v/v against S. agalactiae and S. pyogenes, respectively; inhibited biofilm formation by 89.2% and 80.1%; and preserved >92% epithelial viability at 1× MIC. Among the three natural candidates with complete matched broth data, target-normalized docking and composite MIC50/MIC90/MBC50 ranks were perfectly concordant in both species; pooled species-stratified Spearman analysis yielded ρ = 1.000 (exact p = 0.0556; n = 6). In contrast, the extended panel including antibacterial comparators showed negligible concordance (ρ = 0.081; p = 0.7826; n = 14). Principal component analysis assigned 74.8% of variance to an in vitro potency axis and 24.6% to a largely orthogonal docking axis. Thus, the experiments confirmed the target-specific ordering of the natural candidates but did not support extrapolation of docking rank across mechanistically heterogeneous antibacterial classes. Intracellular target inhibition, genetic causality, and component synergy remain unestablished.
The increasing demand and limited availability of fishmeal (FM) have intensified the need for sustainable alternative protein sources in shrimp aquafeeds. This study evaluated high FM replacement in juvenile Penaeus vannamei reared under biofloc technology (BFT), integrating in vitro protein digestibility, growth performance, nutrient utilization and microbiome analyses. Six isoproteic and isolipidic diets combined two FM levels (0% and 7.5%) with animal (A), plant (P), or mixed (AP) protein sources, plus a 15% FM Control diet were assayed. Plant-based diets showed higher in vitro protein digestibility and amino acid release than animal-based diets. However, these differences were not reflected in shrimp performance, as final body weight, SGR and survival were similar among dietary treatments. Apparent feed conversion ratio, protein efficiency, and economic performance were improved in shrimp fed the A7.5 and AP7.5 diets, whereas complete FM replacement resulted in poorer performance. Biofloc and shrimp intestinal microbial community composition was primarily driven by temporal succession, whereas dietary protein source modulated the abundance of specific bacterial families. Biofloc and intestinal communities shared limited taxonomic overlap at the family level, suggesting restricted microbial exchange together with strong habitat and host mediated selection. Overall, these findings indicate that a diet containing 7.5% fishmeal (FM), supplemented with either animal-derived proteins or a combination of animal- and plant-derived protein sources, represents an optimal strategy for juvenile Penaeus vannamei cultured under BFT conditions, maintaining performance while preserving intestinal microbial stability and health.
Regular bathing is a common habit worldwide, yet bath additives have been characterized almost exclusively at the physiological level, while their molecular effects on human cells remain largely unexplored. We previously showed by RNA sequencing (RNA-seq) that the complex bath additive Karada Totonou ProBath (KTPB) induced the expression of EGR1 and hyaluronic acid synthase genes in human keratinocytes and fibroblasts, which represent the vascular endothelial growth factor (VEGF)-producing side of the cutaneous angiogenic axis; whether the VEGF-receiving endothelium responds to KTPB was unknown. Here, human vascular endothelial cells were exposed to KTPB and profiled by RNA-seq, and cytotoxicity was assessed using resazurin and Hoechst assays. KTPB altered gene expression in a time-dependent manner, and the differentially expressed genes were classified into three clusters with distinct temporal profiles: a late-repressed cluster, a transiently induced cell cycle-associated cluster, and a late-induced cluster enriched for blood vessel development and VEGFA-VEGFR2 signaling. The angiogenesis-related genes ID1, ID3, and EDN1 were markedly upregulated, peaking at 1–2 h. KTPB showed no detectable cytotoxicity in this cell model at any of the tested concentrations, which spanned the recommended use range. These results indicate that KTPB elicits an angiogenesis-associated transcriptional program in vascular endothelial cells without compromising viability and highlight transcriptomics’ value for characterizing bath additives.
Background/Aims: Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia and is a risk factor for stroke. Our previous studies demonstrated that adipocyte-derived exosomes (Ad-EXs) mediate adipose–brain communication. This study investigated the effects of hyperglycemic subcutaneous and visceral Ad-EXs on brain microvascular endothelial cell (BMEC) dysfunction during ischemic injury. Hypothesis: Hyperglycemic Ad-EXs exacerbate stroke injury via inducing oxidative stress and lipid peroxidation in BMECs. Methods: EXs were isolated from primary human subcutaneous and visceral adipocytes cultured in normal glucose (NG): NG-S-Ad-EXs and NG-V-Ad-EXs, or high-glucose (HG, 25 mM) media: HG-S-Ad-EXs and HG-V-Ad-EXs by ultracentrifugation and characterized by a nanoparticle tracking analysis system. PKH26-labeled Ad-EXs were used to evaluate uptake mechanisms in human BMECs (HBMECs). Functional effects were assessed in hypoxia/reoxygenation (H/R)-injured HBMECs treated with different Ad-EXs. Oxidative stress and lipid peroxidation markers (NOX2/4, MDA, 4-HNE, and GPX4) were analyzed. Results: HG increased Ad-EX release from both adipocyte depots. HG-derived Ad-EXs increased HBMEC cytotoxicity and permeability while reducing angiogenesis after H/R injury. Mechanistically, HG-Ad-EXs promoted oxidative stress through increased NOX2/4 and lipid peroxidation via elevated MDA/4-HNE and reduced GPX4 expression. Conclusions: Hyperglycemic Ad-EX triggered oxidative stress via NOX2/4 and lipid peroxidation via MDA/4HNE/GPX4, leading to impaired HBMEC functioning, which was exacerbated in the H/R injury condition.
As a non-volatile diterpenoid, oridonin represents the major bioactive compound in the medicinal plant Isodon rubescens (Hemsl.) Hara. This compound exhibits a broad range of pharmacological activities, including potent anticancer effects against various tumor types, as well as antibacterial and anti-inflammatory properties. Although the pharmacological properties of oridonin have been extensively characterized, its exact tissue-level distribution in leaves has yet to be elucidated. In this study, histochemical staining, desorption electrospray ionization mass spectrometry imaging (DESI–MSI), and dichloromethane-targeted extraction coupled with high-performance liquid chromatography (HPLC) were employed to determine the cellular distribution of oridonin in I. rubescens leaves. Histochemical staining with Hydrochloric acid–vanillin (HCl–vanillin) revealed intense fluorescence signals exclusively in peltate glandular trichomes, with no detectable fluorescence in mesophyll cells. DESI–MSI analysis showed that the characteristic ion signal of oridonin (m/z = 387.15) exhibited a punctate distribution pattern closely matching the spatial arrangement of glandular trichomes. Quantitative HPLC analysis demonstrated that oridonin content in glandular trichome extracts accounted for 77.44% of that in whole-leaf extracts, whereas mesophyll extracts contained only 10.20%, suggesting that glandular trichomes serve as the primary storage site. Furthermore, bioactivity assays revealed that glandular trichome-enriched extracts exhibited significant antibacterial activity against Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus, and showed cytotoxic effects on A549 human lung adenocarcinoma cells, with activity levels positively correlated with oridonin content. These convergent lines of evidence provide evidence that glandular trichomes are the main accumulation and storage sites of oridonin in I. rubescens leaves, and that trichome-stored oridonin constitutes the primary material basis for the antibacterial and cytotoxic activities of this plant. This study provides a cellular-level basis for the quality evaluation and breeding of high-oridonin I. rubescens varieties.
Bazedoxifene (BAZ), a selective estrogen receptor modulator, has recently been demonstrated to inhibit the IL-6/STAT3 signaling pathway; however, its direct effects on cardiac fibroblasts and the underlying mechanisms remain unclear. Isoproterenol (ISO)-stimulated Sprague–Dawley neonatal rat cardiac fibroblasts (CFs) were employed as an in vitro model. CCK-8 assay, flow cytometry, Transwell migration assay, ELISA, qRT-PCR, and Western blot were applied to evaluate the effects of BAZ on CF activation, proliferation, migration, and collagen synthesis. Additionally, IL-6 overexpression via lentivirus (Lv-IL-6) was used to assess mediation by IL-6/STAT3 signaling. BAZ (5 μmol/L) significantly inhibited ISO-induced CF proliferation by inducing G0/G1 cell-cycle arrest; migration and upregulation of α-SMA and Collagen I/III were reduced; IL-6, TGF-β1, and hydroxyproline concentrations in the conditioned medium were decreased; STAT3 phosphorylation was significantly suppressed. Supplementation with Lv-IL-6 partially reversed these effects. The suppression of ISO-induced CF activation and fibrotic phenotype was associated with inhibition of IL-6 expression and blockade of IL-6/STAT3 signaling, suggesting the involvement of this pathway in the anti-fibrotic effects of BAZ. These findings provide in vitro evidence supporting BAZ as a candidate anti-myocardial fibrosis agent.
Background: The Chungtien schizothoracin (Ptychobarbus chungtienensis) is a threatened freshwater fish endemic to the Qinghai–Tibet Plateau and adjacent high-altitude regions of northwestern Yunnan, China. Although a complete mitochondrial genome of P. chungtienensis has been previously reported, direct comparison with a mitogenome generated using high-accuracy long-read sequencing can provide additional information on mitochondrial genome structure and sequence variation. This study aimed to assemble and annotate a complete mitogenome of P. chungtienensis using PacBio HiFi sequencing and to compare its mitogenomic characteristics with previously published Ptychobarbus mitogenomes. Methods: High-molecular-weight genomic DNA from a single specimen was sequenced using PacBio HiFi long-read technology. The mitochondrial genome was assembled using MitoHiFi, annotated using MitoFinder followed by manual curation, and compared with previously published Ptychobarbus mitogenomes. Phylogenetic relationships were evaluated using maximum-likelihood analysis with expanded taxon sampling, and selection pressure on the 13 mitochondrial protein-coding genes was assessed using dN/dS-based branch and branch-site models. Results: The assembled mitogenome is 16,583 bp in length and contains the typical 37 mitochondrial genes, including 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes, together with a control region and the origin of light-strand replication (OL). The overall A + T content was 54.97%. Direct comparison with the previously reported 16,970 bp mitogenome showed that the 387 bp length difference was concentrated in non-coding regions, particularly the control region and the tRNA-Thr–tRNA-Pro intergenic region. Phylogenetic analysis based on 22 complete mitogenomes placed the newly assembled P. chungtienensis sequence in a strongly supported mitochondrial clade with Schizothorax macropogon (bootstrap = 100%), whereas the previously reported P. chungtienensis sequence clustered with P. kaznakovi (bootstrap = 100%), indicating that the two P. chungtienensis records represent distinct mitochondrial lineages. The dN/dS values of all 13 mitochondrial protein-coding genes were below 1, and neither branch nor branch–site analyses detected significant evidence of lineage-specific positive selection. Conclusions: This long-read-based mitogenome provides a high-quality genomic resource for P. chungtienensis and reveals substantial mitochondrial sequence and lineage variation among available records. These results provide a basis for comparative mitogenomic and conservation genetic studies while also indicating that species-level phylogenetic relationships and high-altitude adaptation should not be inferred from mitochondrial data alone.
Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers worldwide, with limited treatment options and a high prevalence of oncogenic KRAS mutations. Piper betle contains numerous bioactive phytochemicals with reported anticancer activities, although its activity against pancreatic cancer remains incompletely characterized. This study evaluated the phytochemical composition of P. betle leaf extracts and the cell viability effects of ethanolic extracts against human KRAS G12D-mutant pancreatic cancer cell lines (PANC-1, AsPC-1, Panc 02.03, Panc 04.03, Panc 08.13 and Panc 10.05). Phytochemical screening, total phenolic content (TPC), total flavonoid content (TFC) and gas chromatography–mass spectrometry (GC-MS) analyses were performed to characterize the extracts. The ethanolic extracts exhibited higher extraction yield (17.26%), TPC (395.94 ± 0.010 mg GAE/g) and TFC (121.28 ± 0.009 mg QE/g) than aqueous extracts. GC-MS analysis tentatively identified phenylpropanoid compounds, including chavicol, chavibetol, and eugenol. Based on these findings, the ethanolic extract was selected for biological evaluation. Treatment significantly reduced cell viability in a concentration-dependent manner across all pancreatic cancer cell lines, with estimated IC50 values ranging from 21.54 to 54.91 µg/mL. These findings suggest that ethanolic P. betle leaf extract possesses promising biological activity and support further studies to identify its active constituents and elucidate their mechanisms of action.
Emerging SARS-CoV-2 variants highlight the need for orally active, low-toxicity antivirals. We designed seven para-substituted flavonoid hybrids (M1–M7) against the main protease (Mpro). In silico ADME filtering revealed zero Lipinski, Veber, or Ghose violations, a SwissADME bioavailability score of 0.55, and selected favorable predicted absorption and transporter endpoints relative to lopinavir, without implying measured pharmacokinetic superiority. ProTox-III indicated that amino and nitro substitution increased predicted genotoxicity liabilities, whereas cyano and methoxy substitution reduced selected endocrine-related signals. AutoDock Vina docking to Mpro (PDB 9C8Q; redocking RMSD 0.316 Å) ranked the nitro analogue M6 first among the designed compounds (−8.1 kcal mol−1), with contacts involving His41 and neighboring active-site residues. During the 100 ns GROMACS simulations, the protein backbone remained stable, whereas M6 adopted a late reoriented pose that was retained in the active-site region and supported by late-window per-residue energetic contributions. DFT calculations at the B3LYP/6-311G(d,p) level identified the narrowest HOMO-LUMO gap (3.44 eV) and highest electrophilicity (ω = 6.2 eV) for M6. Overall, M6 is prioritized as a computational lead requiring Mpro inhibition, antiviral, and cytotoxicity validation.
