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.

Glycoengineering was carried out in the mammalian cell line CHO for the production of pro-tein-based drugs.Firstly,the genome sequence of the Rosa26 locus of CHO cells was determined,the gRNA sequences were designed,and the landing pad was integrated into the Rosa26 locus of CHO cells by CRISPR/Cas9 technology.Three targeting vectors co-expressed by glycosyltransferases,which are β-1,4 galactosyltransferase(B4GALT1),α-2,6-sialyltransferase 1(ST6GAL1)and N-acetaminoglycosyl-transferase Ⅲ(GnT Ⅲ),were constructed by overlapping PCR and seamless ligation technology,and the three glycosyltransferase genes were integrated into the CHO Rosa26 locus by Cre enzyme-mediated cassette exchange technology.PCR confirmed that three glycosyltransferases had been successfully site-directed integrated into the Rosa26 site.The mRNA expression levels of the three glycosyltransferases were more than 50 000-fold by qRT-PCR,and the protein expression levels of the three glycosyltrans-ferases were more than 4-fold via western blotting(P＜0.001).A CHO-engineered cell line with three glycosyltransferases integrated into Rosa26 site was successfully constructed.

Glycoengineering was carried out in the mammalian cell line CHO for the production of pro-tein-based drugs.Firstly,the genome sequence of the Rosa26 locus of CHO cells was determined,the gRNA sequences were designed,and the landing pad was integrated into the Rosa26 locus of CHO cells by CRISPR/Cas9 technology.Three targeting vectors co-expressed by glycosyltransferases,which are β-1,4 galactosyltransferase(B4GALT1),α-2,6-sialyltransferase 1(ST6GAL1)and N-acetaminoglycosyl-transferase Ⅲ(GnT Ⅲ),were constructed by overlapping PCR and seamless ligation technology,and the three glycosyltransferase genes were integrated into the CHO Rosa26 locus by Cre enzyme-mediated cassette exchange technology.PCR confirmed that three glycosyltransferases had been successfully site-directed integrated into the Rosa26 site.The mRNA expression levels of the three glycosyltransferases were more than 50 000-fold by qRT-PCR,and the protein expression levels of the three glycosyltrans-ferases were more than 4-fold via western blotting(P＜0.001).A CHO-engineered cell line with three glycosyltransferases integrated into Rosa26 site was successfully constructed.

This study aims to explore the mechanism of Zhuanggu Jianxi Decoction in reducing synovial tissue inflammation in human knee osteoarthritis(KOA) via the liver X receptors(LXRs)/nuclear factor(NF)-κB signaling pathway. The synovial tissue samples were collected from 5 healthy volunteers and 30 KOA synovitis patients and cultured in vitro. The samples from the heathy volunteers were set as the normal group, and those from KOA synovitis patients were randomized into synovitis, Zhuanggu Jianxi Decoction, LXRα inhibitor, and N-CoR inhibitor groups. The synovitis tissue samples of the 5 groups were treated with 10% blank serum, 10% blank serum, 10% drug-containing serum, 10% drug-containing serum+LXRα inhibitor, and 10% drug-containing serum+N-CoR inhibitor, respectively, for 7 days. After intervention, the synovial tissue samples of each group were collected and stained with hematoxylin-eosin for observation and scoring of the pathological changes. The expression intensity of the fibroblast-specific marker α-smooth musle actin(α-SMA) in the synovial tissue was observed by immunofluorescence staining. The levels of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), matrix metalloproteinase-3(MMP-3), and matrix metalloproteinase-13(MMP-13) in the supernatant of synovium homogenate were determined by ELISA. The mRNA and protein levels of LXRα, N-CoR, P50, and P65 in the synovial tissue were determined by RT-qPCR and Western blot, respectively. The results showed that compared with the normal group, the synovitis group showcased obvious synovial lining cell proliferation, inflammatory cell infiltration, synovial cell disarrangement, increased histopathological score(P<0.05), enhanced α-SMA fluorescence intensity and increased number of synovial fibroblasts(P<0.05), elevated levels of IL-1β, TNF-α, MMP-3, and MMP-13 in the synovial tissue(P<0.05), down-regulated mRNA and protein levels of LXRα and N-CoR, and up-regulated mRNA and protein levels of P50 and P65(P<0.05). Compared with the synovitis group, the Zhuanggu Jianxi Decoction group showed alleviated pathological changes, declined histopathological score of the synovial tissue(P<0.05), decreased α-SMA fluorescence intensity and number of synovial fibroblasts(P<0.05), lowered levels of IL-1β, TNF-α, MMP-3, and MMP-13(P<0.05), up-regulated mRNA and protein levels of LXRα and N-CoR, and down-regulated mRNA and protein levels of P50 and P65(P<0.05) in the synovial tissue. Compared with the Zhuanggu Jianxi Decoction group, the LXRα inhibitor group and N-CoR inhibitor group showed aggravated pathological changes, risen histopathological score of the synovial tissue(P<0.05), enhanced α-SMA fluorescence intensity and increased number of synovial fibroblasts(P<0.05), elevated levels of IL-1β, TNF-α, MMP-3, and MMP-13(P<0.05), down-regulated mRNA and protein levels of LXRα and N-CoR, and up-regulated mRNA and protein levels of P50 and P65(P<0.05). The results above indicated that Zhuanggu Jianxi Decoction could alleviate the synovial tissue inflammation in KOA patients by upregulating the mRNA and protein levels of LXRα and N-CoR in the LXRs/NF-κB pathway to downregulate the mRNA and protein levels of P50 and P65 and reduce the activity of the NF-κB pathway in the synovial tissue.
Humans ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; Male ; Liver X Receptors/immunology* ; Middle Aged ; NF-kappa B/metabolism* ; Female ; Synovial Membrane/metabolism* ; Aged ; Adult

OBJECTIVE: To observe the protective effect and mechanism of hydroxyl safflower yellow A (HSYA) from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with oxygen-glucose deprivation reperfusion (OGD/R) to simulate the ischemia reperfusion model, and cell counting kit-8 was used to detect the protective effect of different concentrations (1.25-160 µ mol/L) of HSYA on HUVECs after OGD/R. HSYA 80 µ mol/L was used for follow-up experiments. The contents of inflammatory cytokines interleukin (IL)-18, IL-1 β, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α) and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay. The protein expressions of toll-like receptor, NOD-like receptor containing pyrin domain 3 (NLRP3), gasdermin D (GSDMD) and GSDMD-N-terminal domain (GSDMD-N) before and after administration were detected by Western blot. NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt (CRID3 sodium salt, also known as MCC950) and agonist were added, and the changes of NLRP3, cysteine-aspartic acid protease 1 (Caspase-1), GSDMD and GSDMD-N protein expressions were detected by Western blot. RESULTS: HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18, IL-1 β, MCP-1, TNF-α and IL-6 (P<0.01 or P<0.05). At the same time, by inhibiting NLRP3/Caspase-1/GSDMD pathway, HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3, Caspase-1, GSDMD and GSDMD-N proteins (P<0.01). CONCLUSIONS The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis.
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Chalcone/analogs & derivatives* ; Quinones/pharmacology* ; Pyroptosis/drug effects* ; Caspase 1/metabolism* ; Glucose ; Phosphate-Binding Proteins/metabolism* ; Signal Transduction/drug effects* ; Intracellular Signaling Peptides and Proteins/metabolism* ; Oxygen/metabolism* ; Cytokines/metabolism* ; Gasdermins

This study aims to investigate the effect of salvianolic acid B (Sal B), the active ingredient of Salvia miltiorrhiza, on H9C2 cardiomyocytes injured by oxygen and glucose deprivation/reperfusion (OGD/R) through regulating mitochondrial fission and fusion. The process of myocardial ischemia-reperfusion injury was simulated by establishing OGD/R model. The cell proliferation and cytotoxicity detection kit (cell counting kit-8, CCK-8) was used to detect cell viability; the kit method was used to detect intracellular reactive oxygen species (ROS), total glutathione (t-GSH), nitric oxide (NO) content, protein expression levels of mitochondrial fission and fusion, apoptosis-related detection by Western blot. Mitochondrial permeability transition pore (MPTP) detection kit and Hoechst 33342 fluorescence was used to observe the opening level of MPTP, and molecular docking technology was used to determine the molecular target of Sal B. The results showed that relative to control group, OGD/R injury reduced cell viability, increased the content of ROS, decreased the content of t-GSH and NO. Furthermore, OGD/R injury increased the protein expression levels of dynamin-related protein 1 (Drp1), mitofusions 2 (Mfn2), Bcl-2 associated X protein (Bax) and cysteinyl aspartate specific proteinase 3 (caspase 3), and decreased the protein expression levels of Mfn1, increased MPTP opening level. Compared with the OGD/R group, it was observed that Sal B had a protective effect at concentrations ranging from 6.25 to 100 μmol·L^-1. Sal B decreased the content of ROS, increased the content of t-GSH and NO, and Western blot showed that Sal B decreased the protein expression levels of Drp1, Mfn2, Bax and caspase 3, increased the protein expression level of Mfn1, and decreased the opening level of MPTP. In summary, Sal B may inhibit the opening of MPTP, reduce cell apoptosis and reduce OGD/R damage in H9C2 cells by regulating the balance of oxidation and anti-oxidation, mitochondrial fission and fusion, thereby providing a scientific basis for the use of Sal B in the treatment of myocardial ischemia reperfusion injury.

This study explored the effects of Buyang Huanwu Decoction(BYHWD) on platelet activation and differential gene expression after acute myocardial infarction(AMI). SD rats were randomly divided into a sham-operated group, a model group, a positive drug(aspirin) group, and a BYHWD group. Pre-treatment was conducted for 14 days with a daily oral dose of 1.6 g·kg~(-1) BYHWD and 0.1 g·kg~(-1) aspirin. The AMI model was established using the high ligation of the left anterior descending coronary artery method. The detection indicators included myocardial infarct size, heart function, myocardial tissue pathology, peripheral blood flow perfusion, platelet aggregation rate, platelet membrane glycoprotein CD62p expression, platelet transcriptomics, and differential gene expression. The results showed that compared with the sham-operated group, the model group showed reduced ejection fraction and cardiac output, decreased peripheral blood flow, and increased platelet aggregation rate and CD62p expression, and activated platelets. At the same time, TXB_2 content increased and 6-keto-PGF1α content decreased in serum. Compared with the model group, BYHWD increased ejection fraction and cardiac output, improved blood circulation in the foot and tail regions and cardiomyocytes arrangement, reduced myocardial infarct size and inflammatory infiltration, down-regulated platelet aggregation rate and CD62p expression, reduced serum TXB_2 content, and increased 6-keto-PGF1α content. Platelet transcriptome sequencing results revealed that BYHWD regulated mTOR-autophagy pathway-related genes in platelets. The differential gene expression levels were detected using real-time quantitative PCR. BYHWD up-regulated mTOR, down-regulated autophagy-related FUNDC1 and PINK genes, and up-regulated p62 gene expression. The results demonstrated that BYHWD could regulate platelet activation, improve blood circulation, and protect ischemic myocardium in AMI rats, and its mechanism is related to the regulation of the mTOR-autophagy pathway in platelets.
Rats ; Animals ; Rats, Sprague-Dawley ; Drugs, Chinese Herbal/therapeutic use* ; Myocardial Infarction/genetics* ; Myocardium/metabolism* ; Aspirin/therapeutic use* ; TOR Serine-Threonine Kinases/metabolism* ; Membrane Proteins/metabolism* ; Mitochondrial Proteins

OBJECTIVE: To explore the carrier rate, genotype and phenotype of α-thalassemia fusion gene in Huadu district of Guangzhou, Guangdong province of China, and provide data reference for the prevention and control of thalassemia. METHODS: A total of 10 769 samples who were screened for thalassemia in Maternal and Child Health Hospital of Huadu District from July 2019 to November 2020 were analyzed retrospectively. Blood cell analysis and hemoglobin (Hb) electrophoresis were performed. Thalassemia genes were analyzed by gap-PCR and PCR-reverse dot blot hybridization (PCR-RDB). RESULTS: A total of 9 cases with α-thalassemia fusion gene were detected in 10 769 samples (0.08%). There were 7 cases with fusion gene heterozygote, 1 case with compound of α-thalassemia fusion gene and Hb G-Honolulu, 1 case with compound of α-thalassemia fusion gene and Hb QS. The MCV results of 4 samples of blood cell analysis were within the reference range, the Hb A2 value of 1 case was decreased, and there were no other abnormalities found. CONCLUSION The α-thalassemia fusion gene is common in Huadu district of Guangzhou, and heterozygotes are more common, and current screening methods easily lead to misdiagnosis.
Humans ; alpha-Thalassemia/genetics* ; Retrospective Studies ; beta-Thalassemia/genetics* ; Genotype ; Phenotype ; Heterozygote ; China ; Mutation

Objective: To investigate the clinicopathologic and molecular genetic characteristics, diagnosis, differential diagnosis, treatment and prognosis of histiocyte-rich rhabdomyoblastic tumor (HRRMT). Methods: The clinical data of two cases of HRRMT diagnosed in Fujian Provincial Hospital and Fujian University of Traditional Chinese Medicine Affiliated People's Hospital from 2020 to 2021 were collected. Histopathology and immunohistochemical (IHC) staining were used to assess morphological changes; the genetic changes were analyzed with next-generation sequencing. The relevant literature was reviewed. Results: Both cases showed well-defined solid nodules and soft masses. Microscopically, the tumors had a fibrous pseudocapsule with lymphocytic aggregation, and locally invaded the surrounding skeletal muscle tissue, and the tumor cells were fusiform to epithelioid with an intensive foamy histiocytic infiltrate. No necrosis or mitosis was observed. Immunophenotyping showed the tumor cells were positive for desmin, either one or both skeletal muscle markers (myogenin or MyoD1), and negative for h-caldesmon, ALK and SMA. The Ki-67 index was<5%. Using next-generation sequencing, one case was found to harbour KRAS (G12D) and MSH3 (Q470^*) mutations. Conclusions: HRRMT is a newly described skeletal muscle tumor with uncertain malignant potential. Its diagnosis and differential diagnosis depend on morphologic and IHC staining. No specific molecular genetics changes have been identified so far.
Biomarkers, Tumor/analysis* ; Diagnosis, Differential ; Histiocytes/pathology* ; Humans ; Molecular Biology ; Muscle Neoplasms/pathology* ; Prognosis