ObjectiveHepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. The main risk factors associated with HCC include viral hepatitis (B and/or C), alcohol abuse, and metabolic dysfunction-associated steatotic liver disease (MASLD), which progressively advance to liver fibrosis, cirrhosis, and ultimately evolve into HCC. Surgical resection represents the most effective treatment for HCC, while recent advances in immunotherapy, including immune checkpoint inhibitors and adoptive cell therapies, have provided improved treatment prospects for patients with unresectable HCC. However, the complex metabolic heterogeneity of HCC limits the therapeutic efficacy. Metabolic intermediates acyl-CoA not only provide energy and substrates for numerous biochemical reactions but also serve as donors for protein lysine acylation, a major class of post-translational modification (PTM). Therefore, a deeper understanding of the molecular mechanisms underlying protein lysine acylation and hepatocarcinogenesis is urgently needed. MethodsThe levels of protein lysine acylation and silence information regulator 5 (SIRT5) expression levels in clinical HCC samples were analyzed by Western blot. Quantitative malonylome and succinylome of HCC samples were analyzed by antibody-based affinity enrichment coupled with tandem mass spectrometry. The proliferation of HCC cells was analyzed with Cell Counting Kit-8 (CCK-8) assays, the apoptosis was quantified by Annexin V-FITC/propidium iodide (PI) staining coupled with flow cytometry, and the ability of cells to migrate was assayed by Transwell assays. The enzymatic activity of glutathione S-transferase Mu 1 (GSTM1) was quantified. Transgenic mice with hepatic overexpression of SIRT5 were constructed using CRISPR-Cas9, and primary hepatocarcinogenesis was induced by administration of diethylnitrosamine. ResultsWestern blot analysis indicated that the expression level of SIRT5 was elevated in clinical samples from HCC patients, and the levels of lysine malonylation, glutarylation, and succinylation were significantly reduced in HCC tissues. Knockout of SIRT5 in MHCC-97H and MHCC-97L hepatoma cells suppressed cell proliferation, and increased the percentage of apoptotic cells significantly. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially malonylome and succinylome of HCC samples revealed significant enrichment in two major classes of biological processes: core energy metabolism (e.g., glycolysis/gluconeogenesis, tricarboxylic acid metabolic process, fatty acid beta oxidation) and detoxification and oxidative stress response (e.g., response to toxic substance, chemical carcinogenesis, reactive oxygen species (ROS)). SIRT5 removes malonylation from lysine residues in GSTM1 and restores its detoxification activity, which is crucial for the survival of hepatocytes under stressed conditions. More importantly, in vivo experiment indicated that hepatic-specific overexpression of SIRT5 in mice accelerated diethylnitrosamine-induced liver fibrosis and hepatocarcinogenesis, indicating the critical role of SIRT5 in HCC progression. ConclusionThis study highlights the previously unrecognized SIRT5-GSTM1 axis as a key regulator in hepatocarcinogenesis, and suggests a potential target for the treatment of patients with HCC.

Objective To verify the efficacy of a domestic human DNA quantification kit based on real-time fluorescence quantitative PCR in detecting the total human DNA concentration,male DNA concen-tration in mixed male/female DNA samples,the degree of DNA degradation and inhibitor tolerance.Methods Samples with different concentrations,different male/female ratios,different concentrations of inhibitors,and different degradation degrees were tested using the domestic human DNA quantification kit based on real-time fluorescence quantitative PCR.This kit was compared with a similar product on the market and was applied to the detection of DNA from real cases.Results This human DNA quan-tification kit can effectively detect human DNA as low as 0.001 65 ng/μL,and 6.25 pg/μL of male DNA in mixed samples with a male-to-female ratio of 1∶15 000.Even when the sample contains as high as 400 ng/μL of humic acid or 1 000 μmol/L of hemin alone,the DNA concentration can still be accurately detected.The degradation index can effectively characterize the degradation degree of the sample.This kit has been successfully applied in forensic practice.Conclusion This human DNA quan-tification kit is accurate and reliable in detection.It can accurately reflect the degradation of DNA and inhibitor tolerance.It has good performance in quantitative accuracy,determination of the male/female ratio in mixed samples,and inhibitor tolerance.It has application potential in forensic case examination.

Objective:To investigate whether neoadjuvant therapy can improve the prognosis of patients with pancreatic cancer.Methods:A retrospective case-control study analyzed data from the Surveillance, Epidemiology, and End Results (SEER) database on 12, 103 patients who underwent surgical treatment between January 1, 2010, and December 31, 2021. Patients were divided into the neoadjuvant therapy group ( n=3 276) and the upfront surgery group ( n=8 827) based on whether they received neoadjuvant treatment. The neoadjuvant therapy group included 2 342 patients receiving neoadjuvant chemotherapy and 934 patients receiving neoadjuvant chemoradiotherapy. The upfront surgery group consisted of 4 335 patients receiving adjuvant chemotherapy, 1 987 patients receiving adjuvant chemoradiotherapy, 63 patients receiving adjuvant radiotherapy, and 2 442 patients undergoing surgery alone. Propensity score matching was used to eliminate group differences and create a cohort with no statistical differences in other clinicopathological features except for the grouping variable. Variables such as age, gender, tumor location, race, population of residence, tumor diameter, household income, TNM stage, and information on radiotherapy and chemotherapy were used for 1∶1 case matching. T stage, N stage, and the use of radiotherapy or chemotherapy were matched exactly. After matching, 1 182 patients were included in each group: the neoadjuvant therapy group contained 1 155 patients receiving neoadjuvant chemoradiotherapy and 27 receiving neoadjuvant chemotherapy, while the upfront surgery group comprised 848 patients receiving adjuvant chemotherapy and 334 receiving adjuvant chemoradiotherapy. TNM staging was reported according to the 7th edition of the AJCC guidelines. The primary outcome was overall survival. Measurement data with skewed distributions were expressed as M( Q1, Q3), and intergroup comparisons were conducted using the Wilcoxon rank-sum test. Categorical data were compared using the chi-square test or the Fisher′s exact test. The Log-rank test and subgroup analyses to assess interactions between neoadjuvant therapy and subgroup in COX regression models were used to compare survival benefits across variables. Landmark analysis was performed to create segmented survival curves, studying the impact of neoadjuvant therapy on prognosis during different follow-up periods. Results:The neoadjuvant therapy group had a higher proportion of T 4 tumor involving celiac axis, superior mesenteric artery, and/or common hepatic artery compared to the upfront surgery group (14.7% vs 2.8%, P<0.001). Additionally, significant differences were observed between groups in terms of race, location, population of residence, age, tumor diameter, tumor stage, and adjuvant therapy regimen ( P<0.05). The median overall survival time in the neoadjuvant therapy group was 30 months, compared to 22 months in the upfront surgery group ( P<0.001). In the neoadjuvant therapy group, the median survival was 30 months for both neoadjuvant chemotherapy and chemoradiotherapy patients; in the upfront surgery group, it was 26 months for both adjuvant chemotherapy and chemoradiotherapy patients, 17 months for adjuvant radiotherapy patients, and 12 months for surgery-only patients. After propensity score matching, there were no differences in the distribution of clinical characteristics between groups ( P>0.05), and all patients in the matched cohort had received chemotherapy. The matched neoadjuvant therapy group had a longer median overall survival compared to the upfront surgery group (30 months vs 27 months, P<0.001). Subgroup interaction analysis revealed that T stage had a significant interaction with neoadjuvant therapy, both before (T 4 stage: HR=0.382, 95% CI: 0.319-0.458; T 2-T 3 stages: HR=0.696, 95% CI: 0.656-0.738; T 1 stage: HR=1.199, 95% CI: 0.867-1.657; interaction P<0.001) and after matching (T 4 stage: HR=0.581, 95% CI: 0.414-0.814; T 2-T 3 stages: HR=0.827, 95% CI: 0.734-0.931; T 1 stage: HR=1.320, 95% CI: 0.716-2.433; interaction P=0.043). Subgroup interaction analysis indicated that T 1 patients did not benefit from neoadjuvant therapy; survival curves plotted for matched T 1 patients showed no difference in survival between the neoadjuvant therapy group and the upfront surgery group ( P=0.323). Conversely, non-T 1 (T 2-T 4) stage patients showed significant survival benefits in both unmatched and matched cohorts ( P<0.001). Landmark analysis showing that the survival benefits occurred mainly in the early postoperative period of up to 3 years ( P<0.001), but there was no difference in overall survival between the neoadjuvant therapy group and the upfront surgery group of >3 years ( P>0.05). Patients with Arterial invasion (T 4 stage compared to T 1-T 3 stages) showed a similarly significant interaction with the benefit of neoadjuvant therapy in both the pre-matching cohort (interaction P<0.001) and the post-matching cohort (interaction P=0.037). Patients with T 4 stage disease in the neoadjuvant therapy group had longer overall survival compared to the upfront surgery group (median overall survival in pre-matching cohort: 30 months vs 13 months, P<0.001; median overall survival in post-matching cohort: 28 months vs 18 months, P=0.001). Among T 4 stage patients in the post-matching cohort, neoadjuvant therapy provided significant survival benefits during the early postoperative period of up to 3 years ( P=0.001). However, there was no difference in overall survival between the neoadjuvant therapy group and the direct surgery group beyond 3 years( P=0.729). Conclusions:The prognosis in the neoadjuvant therapy group was better than in the upfront surgery group. Propensity score matching and subgroup interaction analysis showed that non-T 1 and T 4 stage patients benefited more from neoadjuvant therapy, with benefits mainly seen in the early postoperative period (≤3 years).

Multiple myeloma(MM)is a hematologic malignancy originating from plasma cells in the bone marrow.In recent years,the use of novel drugs and hematopoietic stem cell transplantation(HSCT)has significantly improved the prognosis of MM patients.However,MM remains challenging to cure and is prone to relapse and resistance.For patients with relapsed/refractory multiple myeloma(RRMM),there is an urgent need to explore novel therapeutic approaches.Bispecific antibodies(BsAbs)are a promising class of immunotherapeutics that functions by simultaneously binding to tumor cell antigens and endogenous effector cells,thereby forming an immunological synapse.This interaction facilitates the activation of effector cells,leading to the targeted lysis of tumor cells.Numerous clinical studies have demonstrated the significant efficacy of BsAbs,either as monotherapy or in combination with other treatment regimens,in patients with RRMM.This article summarizes the mechanisms of action,ef-ficacy,and safety of BsAbs,and discusses optimal sequencing strategies in immunotherapy,aiming to provide new perspectives for the treatment of MM.

This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

Objective To establish and evaluate a nomogram prediction model for spontaneous peritonitis in HBV-related primary liver cancer.Methods A retrospective study was conducted on 1298 patients with HBV-related primary liver cancer hospitalized in the Kunming Third People's Hospital from January 2012 to December 2022.General data and serological indicators were collected,and patients were divided into infection group(n=262)and control group(n=1036)based on the occurrence of spontaneous peritonitis.Univariate and LASSO regression analyses were used to screen variables,followed by binary logistic regression to analyze the influencing factors of spontaneous peritonitis in HBV-related primary liver cancer patients,leading to the establishment of a nomogram prediction model.Finally,the Hosmer-lemeshow(H-L)goodness of fit test,receiver operating characteristic(ROC)curve,calibration curve,decision curve analysis(DCA)and clinical impact curve(CIC)were utilized to evaluate the fit degree,accuracy,calibration,and clinical practicability of the nomogram prediction model.Results Single factor analysis revealed significant differences between infection group and control group in portal vein cancer thrombus(PVTT),Child-Pugh grade,China Liver Cancer Staging(CNLC)stage,alcohol consumption history,smoking history,white blood cell count(WBC),neutrophil count(NE),hemoglobin(Hb),fibrinogen(FIB),abnormal prothrombin(PIVKA-Ⅱ),aspartate aminotransferase(AST),alanine aminotransferase(ALT),total protein(TP),prealbumin(PA),γ-glutamyltransferase(GGT),alkaline phosphatase(ALP),cholinesterase(CHE),total bile acid(TBA),total cholesterol(TC),low density lipoprotein(LDL),creatinine(Cr),HBV DNA,CD3+T cells count,CD4+T cells count,CD8+T cells count,CD4+T cells/CD8+T cells ratio,procalcitonin(PCT),serum amyloid A(SAA),interleukin-6(IL-6),high-sensitivity C-reactive protein(hs-CRP),alpha-fetoprotein(AFP),and IL-4(P＜0.05).LASSO regression analysis identified 5 variables:Child-Pugh grade,PVTT,WBC,CHE and hs-CRP.Binary logistic regression analysis indicated that Child-Pugh grade(Grade B:OR=5.780,95％CI 3.271-10.213,P＜0.001;Grade C:OR=14.818,95％CI 7.697-28.526,P＜0.001),PVTT(OR=2.893,95％CI 2.037-4.108,P＜0.001),WBC(OR=1.088,95％CI 1.031-1.148,P=0.002),and hs-CRP(OR=1.005,95％CI 1.001-1.010,P=0.026)were the independent risk factors of spontaneous peritonitis in HBV-related primary liver cancer patients.Using these 4 variables,a nomogram prediction model was constructed and evaluated.The P-value of the H-L goodness of fit test was 0.760.Moreover,the area under ROC curve(AUC)was 0.866,with a sensitivity of 0.870 and a specificity of 0.716.The average absolute error of the calibration curve is 0.022.DCA and CIC analyses demonstrated that the nomogram prediction model possessed some clinical utility.Conclusion The nomogram prediction model for spontaneous peritonitis in HBV-related primary liver cancer patients,constructed using Child-Pugh grade,PVTT,WBC and hs-CRP,exhibits a high fitting degree and accuracy,with the prediction probability highly consistent with the actual occurrence probability,and possesses certain clinical practicability.

T7 RNA polymerase (T7 RNAP) is one of the simplest known RNA polymerases. Its unique structural features make it a critical model for studying the mechanisms of RNA synthesis. This review systematically examines the static crystal structure of T7 RNAP, beginning with an in-depth examination of its characteristic “thumb”, “palm”, and “finger” domains, which form the classic “right-hand-like” architecture. By detailing these structural elements, this review establishes a foundation for understanding the overall organization of T7 RNAP. This review systematically maps the functional roles of secondary structural elements and their subdomains in transcriptional catalysis, progressively elucidating the fundamental relationships between structure and function. Further, the intrinsic flexibility of T7 RNAP and its applications in research are also discussed. Additionally, the review presents the structural diagrams of the enzyme at different stages of the transcription process, and through these diagrams, it provides a detailed description of the complete transcription process of T7 RNAP. By integrating structural dynamics and kinetics analyses, the review constructs a comprehensive framework that bridges static structure to dynamic processes. Despite its advantages, T7 RNAP has a notable limitation: it generates double-stranded RNA (dsRNA) as a byproduct. The presence of dsRNA not only compromises the purity of mRNA products but also elicits nonspecific immune responses, which pose significant challenges for biotechnological and therapeutic applications. The review provides a detailed exploration of the mechanisms underlying dsRNA formation during T7 RNAP catalysis, reviews current strategies to mitigate this issue, and highlights recent progress in the field. A key focus is the semi-rational design of T7 RNAP mutants engineered to minimize dsRNA generation and enhance catalytic performance. Beyond its role in transcription, T7 RNAP exhibits rapid development and extensive application in fields, including gene editing, biosensing, and mRNA vaccines. This review systematically examines the structure-function relationships of T7 RNAP, elucidates the mechanisms of dsRNA formation, and discusses engineering strategies to optimize its performance. It further explores the engineering optimization and functional expansion of T7 RNAP. Furthermore, this review also addresses the pressing issues that currently need resolution, discusses the major challenges in the practical application of T7 RNAP, and provides an outlook on potential future research directions. In summary, this review provides a comprehensive analysis of T7 RNAP, ranging from its structural architecture to cutting-edge applications. We systematically examine: (1) the characteristic right-hand domains (thumb, palm, fingers) that define its minimalistic structure; (2) the structure-function relationships underlying transcriptional catalysis; and (3) the dynamic transitions during the complete transcription cycle. While highlighting T7 RNAP’s versatility in gene editing, biosensing, and mRNA vaccine production, we critically address its major limitation—dsRNA byproduct formation—and evaluate engineering solutions including semi-rationally designed mutants. By synthesizing current knowledge and identifying key challenges, this work aims to provide novel insights for the development and application of T7 RNAP and to foster further thought and progress in related fields.

Mitochondria, functioning not only as the central hub of cellular energy metabolism but also as semi-autonomous organelles, orchestrate cellular fate decisions through their endogenous mitochondrial DNA (mtDNA), which encodes core components of the electron transport chain. Emerging research has identified microRNAs localized within mitochondria, termed mitochondria-located microRNAs (mitomiRs). Recent studies have revealed that mitomiRs are transcribed from nuclear DNA (nDNA), processed and matured in the cytoplasm, and subsequently transported into mitochondria. mitomiRs regulate mtDNA through diverse mechanisms, including modulation of mtDNA expression at the translational level and direct binding to mtDNA to influence transcription. Aberrant expression of mitomiRs leads to mitochondrial dysfunction and contributes to the pathogenesis of metabolic diseases. Restoring mitomiR expression to physiological levels using mitomiRs mimics or inhibitors has been shown to improve mitochondrial function and alleviate related diseases. Consequently, the regulatory mechanisms of mitomiRs have become a major focus in mitochondrial research. Given that mitomiRs are located in mitochondria, targeted delivery strategies designed for mtDNA can be adapted for the delivery of mitomiRs mimics or inhibitors. However, numerous intracellular and extracellular barriers remain, highlighting the need for more precise and efficient delivery systems in the future. The regulation of mtDNA expression mediated by mitomiRs not only expands our understanding of miRNA functions in post-transcriptional gene regulation but also provides promising molecular targets for the treatment of mitochondrial-related diseases. This review systematically summarizes recent research progress on mitomiRs in regulating mtDNA expression and discusses the underlying mechanisms of mitomiRs-mtDNA interactions. Additionally, it provides new perspectives on precision therapeutic strategies, with a particular emphasis on mitomiRs-based regulation of mitochondrial function in mitochondrial-related diseases.

Multiple myeloma(MM)is a hematologic malignancy originating from plasma cells in the bone marrow.In recent years,the use of novel drugs and hematopoietic stem cell transplantation(HSCT)has significantly improved the prognosis of MM patients.However,MM remains challenging to cure and is prone to relapse and resistance.For patients with relapsed/refractory multiple myeloma(RRMM),there is an urgent need to explore novel therapeutic approaches.Bispecific antibodies(BsAbs)are a promising class of immunotherapeutics that functions by simultaneously binding to tumor cell antigens and endogenous effector cells,thereby forming an immunological synapse.This interaction facilitates the activation of effector cells,leading to the targeted lysis of tumor cells.Numerous clinical studies have demonstrated the significant efficacy of BsAbs,either as monotherapy or in combination with other treatment regimens,in patients with RRMM.This article summarizes the mechanisms of action,ef-ficacy,and safety of BsAbs,and discusses optimal sequencing strategies in immunotherapy,aiming to provide new perspectives for the treatment of MM.

OBJECTIVE: To screen out the key metabolites related to diabetic foot (DF) by integrating genome-wide association studies (GWAS) and metabolome genome-wide association studies (mGWAS). METHODS: The literature databases such as PubMed and China national knowledge infrastructure(CNKI), as well as genomics databases such as PAN UKBB, FinnGen, and IEU Open GWAS were systematically retrieved from database estobilishment to November 2024 on DF-related single nucleotide polymorphisms and genome-wide association studies. DF-single nucleotide polymorphism-metabolite network was constructed by mGWAS package and mGWAS-Explorer platform. The causal relationship between key factors was evaluated by two-sample Mendelian randomization. The genetic correlation between DF and 575 metabolites (source:IEU Open GWAS) was evaluated by linkage disequilibrium score regression. In vitro experiments were conducted to induce injury of human umbilical vein endothelial cells with 30 mM glucose and intervene with 20 μM γ-tocopherol. Changes in cell migration, scratch healing and tube formation function were detected. RESULTS: Twenty-senen literatures on single nucleotide polymorphism literatures and 3 studies on GWAS were included. Genetic analysis results showed DF-related single nucleotide polymorphisms were enriched in vascular endothelial dysfunction-related pathways (such as fluid shear stress and atherosclerosis). The results of metabolic network analysis screened out 19 associated metabolites, among which 12 such as γ -tocopherol and pyruvate had significant genetic correlations with DF. Mendelian randomization suggested matrix metalloproteinase-9(MMP-9) might be a potential driver of DF (β=0.658, P=0.063 8), and the occurrence of DF could reduce the level of high-density lipoprotein (β=-0.002, P=0.015 2). The results of in vitro experiments confirmed that γ -tocopherol could improve endothelial dysfunction induced by high glucose, specifically manifested as an increase in the number of cell migrations, improvement in the scratch healing rate, and recovery of tubule formation ability (P<0.05). CONCLUSION DF has a genetic basis centered on vascular endothelial dysfunction, and its occurrence can lead to further metabolic disorders. The key single nucleotide polymorphism loci integrated provided molecular markers for the risk stratification of foot ulcers in diabetic patients. In addition, γ -tocopherol has demonstrated clinical application potential as a therapeutic drug for DF by significantly improving the function of vascular endothelial cells in a high-glucose environment.
Humans ; Diabetic Foot/drug therapy* ; Polymorphism, Single Nucleotide ; Genome-Wide Association Study ; Genomics ; Metabolomics ; Metabolome