Background/Aims: Liver transplantation (LT) following total hepatectomy is a life-saving treatment for hepatocellular carcinoma (HCC). The HCC recurrence after LT hinders the effectiveness of the procedure. The objective of this study is to develop a pre-operative risk stratification model based on a liquid biopsy. Methods: We conducted a comprehensive multi-omics study of 260 HCC patients from three centers, including clinical data, low-coverage whole-genome sequencing of cell-free DNA (cfDNA) from plasma, as well as whole-exome, single-nucleus RNA, and spatial transcriptomics from matched tumor and non-tumor tissues. Results: We identified cfDNA-derived copy number alteration (CNA) signatures associated with post-transplant recurrence. By integrating cfDNA-derived CNA profiles with single-cell transcriptomic data, we traced recurrence-associated cfDNA to a distinct subpopulation of malignant cells within the primary tumor. These cells were embedded in a pro-metastatic microenvironment of specialized endothelial subtypes and cancer-associated fibroblasts. Notably, most recurrence-associated lesions were detectable in cfDNA prior to liver transplantation (LT). Building on these insights, we developed the ZJU Criteria based on CNA fragments and tumor markers, a pre-LT risk prediction tool that integrates conventional clinical factors with cfDNA-derived CNA signatures, and validated it using internal and independent external cohorts. Conclusion Our findings suggest that post-transplant recurrence commonly originates from advanced subclones that emerge late during tumor evolution. The ZJU Criteria provides an accurate, non-invasive strategy that significantly improves pre-LT risk stratification and clinical decision-making for patients with HCC.

ObjectiveTo establish a highly specific and sensitive triplex TaqMan quantitative PCR method for the detection of mouse hepatitis virus (MHV), mouse parvovirus (MPV), and reovirus type 3 (Reo-3) in laboratory mice. MethodsRecombinant plasmid standards were constructed using conserved genomic regions of the three target viruses. Specific primers and probes were designed, and a triplex TaqMan quantitative PCR (qPCR) system was optimized through preliminary experiments. Sensitivity was evaluated using 10-fold serial dilutions (10¹-10⁷ copies/μL). Intra- and inter-assay repeatability were assessed via reproducibility experiments. ResultsA strong linear correlation (R ²>0.99) was observed between copy number and Ct value at template concentrations of 10²-10⁷ copies/μL. Intra-assay and inter-assay coefficients of variation (CV) were both below 5%. No cross-reactivity was observed with mouse cytomegalovirus (MCMV), vesicular stomatitis virus (VSV), Sendai virus (SeV), or pneumonia virus of mice (PVM). ConclusionThe triplex TaqMan quantitative PCR method established in this study has high sensitivity, good repeatability, and strong specificity. It enables the rapid, specific, and sensitive detection of MHV, MPV, and Reo-3, allows the simultaneous detection of multiple pathogens in a single tube, and can be applied to the detection of clinical samples from laboratory animals and to epidemiological investigations, thereby providing effective scientific and technological support for the prevention and control of viral infection and the interruption of transmission.

Objective To explore the molecular mechanism of Jianfu mixture in the treatment of erectile dysfunction （ED） by network pharmacology and molecular docking techniques, and validate its core targets and mechanisms through in vitro experiments. Methods The active components and corresponding molecular targets of Jianfu mixture were searched by searching TCMSP and Batman-TCM databases, and the disease targets of ED were searched by using GeneCards database. Find the intersection of drug ingredient target and disease target. The interaction between intersected targets was described and analyzed by String database, and the analysis results were visualized by Cytoscape software to determine the core target and the corresponding active components. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for intersection targets; the core target within the intersection were found through MCODE plug-in on Cytoscape software and molecular docking was performed with the corresponding active ingredients. An endothelial dysfunction model was established by transfecting HUVECs with si-eNOS. Intervene with different concentrations of the Jianfu mixture for the model cells for 24 h. QPCR was used to detect mRNA expression of core targets （MAPK1, MAPK3, JUN, ESR1, MAPK8）; Western blot was used to analyze protein expression （eNOS, JUN, p-JUN, MAPK, p-MAPK） and phosphorylation levels. Results 144 effective active components and 168 active components target-disease targe intersection of Jianfu mixture were obtained. GO analysis revealed 200 5 biological processes, 151 molecular functions, and 63 cellular components. KEGG analysis yielded 181 pathways. 5 core targets including MAPK1, MAPK3, JUN, ESR1 and MAPK8 were screened out. The active components such as β-sitosterol, kaempferol, astapterocarpan had good binding affinity with the core target. In vitro experiments confirmed successful construction of the endothelial dysfunction model （eNOS expression significantly decreased after si-eNOS transfection）. Jianfu mixture dose-dependently inhibited mRNA expression of MAPK1, MAPK3, JUN, ESR1, and MAPK8. Additionally, it reduced phosphorylation levels of JUN and MAPK, indicating inhibition of the JNK/c-Jun and ERK/MAPK signaling pathways to improve endothelial function. Conclusion Jianfu mixture treats ED by suppressing abnormal activation of multi-target signaling pathways （MAPK/JUN/ESR1）, reducing endothelial apoptosis, and promoting NO synthesis. This mechanism aligns with the traditional Chinese medicine principle of “activating blood circulation, resolving stasis, tonifying Qi, and strengthening cardiovascular function.” The study provided molecular-level evidence for the therapeutic efficacy of Jianfu mixture in ED management.

Objective To explore the molecular mechanism of Jianfu mixture in the treatment of erectile dysfunction （ED） by network pharmacology and molecular docking techniques, and validate its core targets and mechanisms through in vitro experiments. Methods The active components and corresponding molecular targets of Jianfu mixture were searched by searching TCMSP and Batman-TCM databases, and the disease targets of ED were searched by using GeneCards database. Find the intersection of drug ingredient target and disease target. The interaction between intersected targets was described and analyzed by String database, and the analysis results were visualized by Cytoscape software to determine the core target and the corresponding active components. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for intersection targets; the core target within the intersection were found through MCODE plug-in on Cytoscape software and molecular docking was performed with the corresponding active ingredients. An endothelial dysfunction model was established by transfecting HUVECs with si-eNOS. Intervene with different concentrations of the Jianfu mixture for the model cells for 24 h. QPCR was used to detect mRNA expression of core targets （MAPK1, MAPK3, JUN, ESR1, MAPK8）; Western blot was used to analyze protein expression （eNOS, JUN, p-JUN, MAPK, p-MAPK） and phosphorylation levels. Results 144 effective active components and 168 active components target-disease targe intersection of Jianfu mixture were obtained. GO analysis revealed 200 5 biological processes, 151 molecular functions, and 63 cellular components. KEGG analysis yielded 181 pathways. 5 core targets including MAPK1, MAPK3, JUN, ESR1 and MAPK8 were screened out. The active components such as β-sitosterol, kaempferol, astapterocarpan had good binding affinity with the core target. In vitro experiments confirmed successful construction of the endothelial dysfunction model （eNOS expression significantly decreased after si-eNOS transfection）. Jianfu mixture dose-dependently inhibited mRNA expression of MAPK1, MAPK3, JUN, ESR1, and MAPK8. Additionally, it reduced phosphorylation levels of JUN and MAPK, indicating inhibition of the JNK/c-Jun and ERK/MAPK signaling pathways to improve endothelial function. Conclusion Jianfu mixture treats ED by suppressing abnormal activation of multi-target signaling pathways （MAPK/JUN/ESR1）, reducing endothelial apoptosis, and promoting NO synthesis. This mechanism aligns with the traditional Chinese medicine principle of “activating blood circulation, resolving stasis, tonifying Qi, and strengthening cardiovascular function.” The study provided molecular-level evidence for the therapeutic efficacy of Jianfu mixture in ED management.

ObjectiveTo address the shortage of high-caliber multidisciplinary professionals in China's rehabilitation engineering field and the prominent issue of "separation between medicine and engineering" in existing curricula, this study introduces the World Health Organization rehabilitation competency framework (RCF) to construct a systematic curriculum system for rehabilitation engineering. MethodsGuided by the four core competency domains of RCF (practice, professionalism, learning and development, management and leadership) and their associated supporting knowledge, the rehabilitation engineering curriculum was restructured into a modular framework, piloted and validated within the Biomedical Engineering (Intelligent Robotics) program at University of Health and Rehabilitation Sciences. ResultsA four-tiered curriculum system comprising General Education-Disciplinary Platform-Professional Core-Diverse Electives with a total of 160 credits was established, achieving precise mapping between competency objectives and course content. The students in the pilot cohort demonstrated outstanding academic performance (50% ranked in the top ten of their major), they also gained a deeper understanding and mastery of interdisciplinary knowledge integrating medicine and engineering. ConclusionThe RCF-based curriculum system effectively promotes the organic integration of medical and engineering knowledge, providing an internationally aligned competency-oriented paradigm for rehabilitation engineering talent cultivation, which holds significant value for broader application.

Multidrug-resistant (MDR) bacterial infections have emerged as a serious challenge of global public health crisis. The overuse and misuse of conventional antibiotics have dramatically accelerated the emergence, evolution and worldwide spread of drug-resistant bacterial strains, necessitating urgent exploration of novel antibacterial strategies. Bacteriophages serve as natural bacterial predators offering distinct advantages including high host specificity, autonomous self-replication capabilities and cost-effective large-scale production. However, wild-type phages present significant clinical limitations due to their narrow host ranges, susceptibility to rapid immune clearance and poor penetration of bacterial biofilms, which severely restrict their therapeutic applications. The convergence of synthetic biology, nanotechnology and advanced gene editing technologies has accelerated the development of engineered bacteriophage platforms, providing programmable, scalable and clinically translatable pathways to overcome these inherent biological constraints. Here, we systematically delineate four fundamental strategies for engineered bacteriophage development. Chemical modification utilizes reactive functional groups such as amino, carboxyl and thiol moieties on capsid proteins through esterification, amidation or click chemistry reactions to achieve precise drug conjugation and surface functionalization. In vivo editing encompasses ultraviolet or chemical mutagenesis for random mutation induction, homologous recombination for targeted genetic alterations, recombineering methodologies including electroporation-mediated bacteriophage recombination engineering, and CRISPR-Cas systems for precise genome editing to enable exact genetic reconstruction and host range reprogramming. In vitro synthesis leverages genome engineering platforms where intact phage genomes are transferred into yeast or host bacteria to facilitate highly efficient homologous recombination, enabling large DNA fragment assembly and cross-gene host range expansion without bacterial toxicity constraints. Directed evolution combines artificial selection through mutation library screening with rational design approaches involving chimeric receptor binding protein construction or site-specific mutagenesis, effectively balancing the discovery of unknown adaptive pathways with targeted host specificity modification. Moreover, we comprehensively discuss therapeutic applications across diverse clinical scenarios. Engineered bacteriophage effectively disrupt bacterial biofilms through sophisticated functionalized delivery platforms including nanozyme-conjugated phages, phage-liposome nanoconjugates and bio-responsive hydrogels, demonstrating significantly enhanced bactericidal efficiency compared to unmodified free phages. These bioengineered vectors attenuate bacterial virulence and resensitize pathogens to antibiotics by delivering CRISPR-Cas systems or base editors to disrupt critical virulence factors such as pili, capsule synthesis machineries and quorum sensing systems, or by inactivating antibiotic resistance determinants including beta-lactamase genes. As an intelligent nanomedicine delivery platform, engineered bacteriophage enable precise pathogen elimination an through photocatalytic reactive oxygen species generation, immunomodulatory interventions, or controlled release of antibacterial drugs. Furthermore, oral administration of engineered bacteriophage facilitates microbiota modulation, which selectively eliminate intestinal pathogens while preserve beneficial commensal microbiota, thereby restoring microbial community balance and preventing complications associated with dysbiosis. Finally, we critically analyze persistent challenges including host strain matching complexity, evolution of bacterial resistance mechanisms, pharmacokinetic optimization requirements, optimal administration route selection, large-scale production quality control standards and clinical dosing determination protocols. Through multidisciplinary integration of synthetic biology, infectious disease medicine and immunology, future translational medicine studies of bacteriophage should establish comprehensive technical platforms encompassing rapid phage screening, intelligent rational design, rigorous in vivo evaluation and standardized clinical validation processes, ultimately advancing engineered bacteriophage from laboratory innovations to clinically approved therapeutics for effectively combating MDR bacterial infections.

Background: Diabetic peripheral neuropathy (DPN) is a common chronic complication of diabetes mellitus that significantly impairs patients’ quality of life. Traditional Chinese medicine (TCM), as a major component of complementary and alternative medicine, has accumulated numerous effective formulas for the clinical management of DPN. However, systematic approaches for optimizing TCM formulas remain limited. Objective: To establish a pathway-oriented approach for TCM formula optimization and to evaluate the efficacy and mechanisms of the optimized formula in DPN. Methods: We developed 2 formula optimization algorithms that defined pathway-oriented herbal correlation (HC) and herbal contribution (HO) to screen and construct a new herbal formula, Qihongtongbi (QHTB), for treating Qi deficiency and blood stasis syndrome complicated with DPN. An animal experiment was subsequently conducted to evaluate the therapeutic effectiveness of QHTB. A total of 32 specific-pathogen-free male ob/ob mice (18–20 g) were randomly divided into 4 groups: model, Mudan Granules (MDG), QHTB low- and high-dose groups (n = 8). Ten male C57BL/6J mice (18–20 g) served as the control group. Metabolomics analysis was further employed to elucidate the biological mechanisms underlying the effects of QHTB. Results: Based on our previous study on TCM medication patterns for treating DPN, 22 candidate herbs were selected for formula optimization. The top 5 candidate herbs (total HC = 25.24 × 10 ) exhibited HC values comparable to those of MDG (total HC = 26.1 × 10 ). These herbs were Carthamus tinctorius L. (HC = 5.40 × 10 ), Astragalus mongholicus Bunge (HC = 5.23 × 10 ), Salvia miltiorrhiza Bunge (HC = 5.15 × 10 ), Commiphora myrrha (T. Nees) Engl. (HC = 5.02 × 10 ), and Glycyrrhiza glabra L. (HC = 4.44 × 10 ). HO analysis showed that the cumulative HO of these 5 herbs exceeded 50%. In vivo experiments demonstrated that, compared with the model group, QHTB significantly lowered blood glucose (P < 0.05), enhanced nerve conduction velocity (P < 0.01), improved nociceptive hypersensitivity (P < 0.01), and ameliorated sciatic nerve morphology, with efficacy comparable to MDG. Serum and fecal metabolomics further revealed that QHTB exerted multipathway regulatory effects, among which nicotinate and nicotinamide metabolism represented a key mechanism. Conclusion: In summary, this study provides a methodological reference for the systematic optimization of TCM formulas.

Triclosan (TCS) and triclocarban (TCC) are widely used synthetic broad-spectrum antibacterial agents that can enter the human body through the skin, gastrointestinal tract, and other pathways. More and more studies have found that exposure to TCS and TCC can affect human health, but currently, review reports on the health effects of human exposure to TCS and TCC are limited. Therefore, this study reviewed population studies on the relationship between TCS and TCC exposure and health effects by searching the PubMed database, summarized the associated health outcomes, and elucidated the biological mechanisms. A total of 56 studies were retrieved, among which cross-sectional studies (25 studies, 44.64%) and cohort studies (25 studies, 44.64%) accounted for a relatively large proportion, while case-control studies (6 studies, 10.72%) were relatively few. Studies on TCS exposure (48 studies, 85.71%) were far more prevalent than those on TCC exposure (2 studies, 3.57%). The remaining 6 studies involved both TCS and TCC exposure. The research results revealed that TCS exposure was associated with male and female abnormal reproductive functions, fetal growth restriction, abnormal behavior development in children, obesity, gestational diabetes mellitus (GDM), and immune-related diseases. Although the results of different studies show significant differences, they have indicated that exposure to TCS is a potential risk factor for these health problems. Due to the limited number of studies, the evidence for the relationship between TCC exposure and most of the aforementioned health effects is insufficient. Population studies and in vitro and in vivo studies have shown that exposure to TCS and TCC can interfere with the microbial homeostasis, the endocrine system, oxidative stress and immune function of the body, which are potential mechanisms causing adverse health effects. In the future, large-scale prospective cohort studies, as well as in vivo and in vitro studies, are still needed to further clarify the associations between TCS and TCC exposure and health effects, and to deeply explore its mechanism of action. These efforts will provide references for clarifying the human health hazards of TCS and TCC exposure and formulating targeted prevention and control strategies.

OBJECTIVE To establish the UPLC fingerprint and the method for multi-component content determination in Jingangteng capsules， and to evaluate its quality by combining chemical pattern recognition analysis. METHODS An UPLC method was established. Separation was performed on a Zorbax SB-C 18 Rapid Resolution HD column， with acetonitrile-0.1% formic acid as the mobile phase for gradient elution.Using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines （2012 edition）， UPLC fi ngerprints were established for 10 batches of Jingangteng capsules， and similarity was evaluated. SPSS 22.0 and SIMCA 14.1 software were used to perform hierarchial-cluster analysis and orthogonal partial least squares discriminant analysis （OPLS-DA）， respectively. The same UPLC method was employed to determine the contents of chlorogenic acid， 3，5-dihydroxy-2-methylbenzoic acid-3- O -glucoside （M1）， caffeic acid， astilbin， oxyresveratrol， quercitrin and resveratrol in the 10 batches of samples. RESULTS A total of 17 common peaks were identified in UPLC fingerprints of the 10 batches of samples， of which 7 were identified as chlorogenic acid， M1， caffeic acid， astilbin， oxyresveratrol， quercitrin， and resveratrol. The similarities of 10 batches of samples ranged from 0.820 to 0.985. The results of hierarchial-cluster analysis showed that 10 batches of samples were grouped into four categories： S1-S4 formed one group， S5 and S6 formed another， S7， S8 and S10 formed a third， and S9 formed a fourth， consistent with the OPLS-DA results； the variable importance projection values for peaks 7， 10， 2， 16 （resveratrol）， 13 （oxyresveratrol）， 11， 6 （caffeic acid）， 5 （M1） and 15 （quercitrin） were ＞1. Quantitative analysis results showed that the contents of chlorogenic acid， M1， caffeic acid， astilbin， oxyresveratrol， quercitrin， and resveratrol were 1.650 8-4.213 7， 0.636 2-2.161 7， 0.031 0-0.086 5， 0.239 1-1.069 3， 0.211 9-1.104 0， 0.488 8-2.399 2， and 0.164 0-0.699 8 mg/g， respectively. CONCLUSIONS UPLC fingerprint and content determination methods established in this study are simple to operate， accurate， reliable and reproducible； when combined with chemical pattern recognition analysis， they can be used to evaluate the quality of Jingangteng capsules. Nine components， such as resveratrol， oxyresveratrol， caffeic acid， M1 and quercitrin， may serve as markers of quality variation.

ObjectiveTo investigate the role of Ras-GTPase-activating protein SH3 domain-binding protein 2 （G3BP2） in regulating the activation， proliferation， and migration of hepatic stellate cells （HSCs）. MethodsThe mouse HSCs （JS-1 cell line） were treated with 5 μg/L transforming growth factor-beta 1（TGF-β1） for 24 hours to establish an HSC activation and proliferation model. A G3BP2 knockdown system was constructed using siRNA interference technology. The experiment was divided into four groups： Control， TGF-β1 treatment， TGF-β1+si-NC， and TGF-β1+ G3BP2-siRNA. The expression levels of key fibrosis indicators， including type I collagen （Collagen I）， α-smooth muscle actin （α-SMA）， and G3BP2， were detected by Western blot and RT-qPCR. Cell proliferation activity was assessed using the CCK-8 proliferation assay kit and EdU fluorescence labeling technology. Cell migration ability was analyzed by scratch wound healing assay and Transwell migration assay. The formation level of stress granules was quantified by immunofluorescence microscopy to investigate the effects of G3BP2 on stress granule formation in activated HSCs. ResultsStimulation with TGF-β1 upregulated the expression of G3BP2 in JS-1 cells （RT-qPCR： P0.000 1； Western blot： P0.000 1）， while a downward trend in its expression was observed in the G3BP2‑silenced group （RT-qPCR： P0.01； Western blot： P0.000 1）. Compared with the control group， the TGF-β1 group exhibited increased protein expression levels of α-SMA and Collagen I （RT-qPCR： both P0.01； Western blot： P0.01 and P0.05， respectively）， concomitant with an increased number of stress granules and enhanced cell proliferation and migration capacity （all P0.001）. The experimental results demonstrated that G3BP2 knockout effectively reversed the aforementioned phenotypes， with the G3BP2-silenced group showing reduced expression of fibrotic markers （all P0.01）， decreased stress granule formation （P0.01）， and reduced cell proliferation and migration capacity （all P0.05）， compared to the negative control group. ConclusionG3BP2 enhances the activation， proliferation， and migration of HSCs by promoting the formation of stress granules， thereby accelerating the pathological progression of liver fibrosis. This suggests that stress granules may serve as important regulators in controlling the activation， proliferation， and migration of HSCs.