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.

Nonobstructive azoospermia (NOA), one of the most severe types of male infertility, etiology often remains unclear in most cases. Therefore, this study aimed to detect four biallelic detrimental variants (0.5%) in the minichromosome maintenance domain containing 2 ( MCMDC2 ) genes in 768 NOA patients by whole-exome sequencing (WES). Hematoxylin and eosin (H&E) demonstrated that MCMDC2 deleterious variants caused meiotic arrest in three patients (c.1360G>T, c.1956G>T, and c.685C>T) and hypospermatogenesis in one patient (c.94G>T), as further confirmed through immunofluorescence (IF) staining. The single-cell RNA sequencing data indicated that MCMDC2 was substantially expressed during spermatogenesis. The variants were confirmed as deleterious and responsible for patient infertility through bioinformatics and in vitro experimental analyses. The results revealed four MCMDC2 variants related to NOA, which contributes to the current perception of the function of MCMDC2 in male fertility and presents new perspectives on the genetic etiology of NOA.
Humans ; Male ; Azoospermia/genetics* ; Meiosis/genetics* ; Spermatogenesis/genetics* ; Adult ; Exome Sequencing ; Microtubule-Associated Proteins/genetics* ; Alleles ; Infertility, Male/genetics*

Objective To investigate programmed death including necroptosis,apoptosis,autophagy,ferroptosis,and pyroptosis in bone marrow megakaryocytes of mice during sepsis and its impact on platelet production capacity and coagulation function in mice.Methods C57BL/6J mice were randomly divided into a sham operation group(sham group)and a sepsis model group(CLP group).Peripheral blood platelets and coagulation function were measured by abdominal aortic blood sampling at 24 h postoperatively in both sham and CLP groups.After the mice were sacrificed,long bones of both lower limbs were taken,and bone marrow megakaryocytes were extracted using megakaryocyte separation solution and immunomagnetic bead separation.Laser confocal microscopy was used to observe the activation of programmed death-related marker molecules in mouse bone marrow megakaryocytes.Flow cytometry was used to detect programmed death rate,platelet production phenotype,and platelet surface markers(CD41,CD42b,CD61)of megakaryocytes.Western blotting was used to detect the expression of programmed death-related proteins in megakaryocytes.Results Compared with the sham group,the CLP group showed significant decreases in the number of platelets during acute sepsis(24 h)(P<0.000 1),significant increases in platelet distri-bution width(PDW)and mean platelet volume(MPV)(P<0.01),significant prolonging of thrombin time(TT),prothrombin time(PT),and activated partial thromboplastin time(APTT)(P<0.000 1,P<0.001,P<0.01),and significant reduction in fibrinogen(Fib)(P<0.000 1).Compared with the Con/sham group,the LPS/CLP group exhibited significant increases in the platelet production phenotype of megakaryocyte,the number of PLP in the supernatant,and the expression levels of platelet surface markers(CD41,CD42b,CD61).The rates of megakaryocyte necroptosis/apoptosis,pyroptosis,and ferroptosis were significantly elevated at 24 h post-CLP surgery.Laser confo-cal microscopy showed significant activation of LC3,P-MLKL,Caspase-1,and Fe2+in megakaryocytes of mice after CLP surgery.Western blotting results revealed that the CLP group exhibited a significant increase in the activa-tion rate of necroptosis-related protein P-MLKL(P<0.001),a significant increase in the cleavage of pyroptosis-related proteins GSDMD and GSDMD-N(P<0.01,P<0.001,respectively),a significant increase in the expres-sion of ferroptosis-related protein ACSL4(P<0.01),and a significant decrease in the expression of GPX4(P<0.01)compared to the sham group.Additionally,the CLP group demonstrated significant increases in the expression of apoptosis-related protein Bax,the cleavage of autophagy-related protein LC3B-Ⅱ,and the expression of P62(P<0.05,P<0.001,P<0.001,respectively).Inhibition of apoptosis with programmed cell death inhibitors decreased platelet production function of megakaryocyte,while inhibition of necroptosis and pyroptosis had limited effects on platelet production function of megakaryocyte.Inhibition of ferroptosis and autophagy enhanced platelet production function of megakaryocyte.Conclusion Significant programmed death of megakaryocytes was observed during the acute phase of sepsis(24 h).Among those megakaryocytes,apoptosis is an important mechanism for the differentia-tion of platelet production phenotype and increased platelet production capacity of megakaryocyte.Overactive autophagy and ferroptosis in megakaryocytes lead to megakaryocyte dysfunction,which is an important mechanism for coagulation abnormalities in sepsis.

Objective To investigate programmed death including necroptosis,apoptosis,autophagy,ferroptosis,and pyroptosis in bone marrow megakaryocytes of mice during sepsis and its impact on platelet production capacity and coagulation function in mice.Methods C57BL/6J mice were randomly divided into a sham operation group(sham group)and a sepsis model group(CLP group).Peripheral blood platelets and coagulation function were measured by abdominal aortic blood sampling at 24 h postoperatively in both sham and CLP groups.After the mice were sacrificed,long bones of both lower limbs were taken,and bone marrow megakaryocytes were extracted using megakaryocyte separation solution and immunomagnetic bead separation.Laser confocal microscopy was used to observe the activation of programmed death-related marker molecules in mouse bone marrow megakaryocytes.Flow cytometry was used to detect programmed death rate,platelet production phenotype,and platelet surface markers(CD41,CD42b,CD61)of megakaryocytes.Western blotting was used to detect the expression of programmed death-related proteins in megakaryocytes.Results Compared with the sham group,the CLP group showed significant decreases in the number of platelets during acute sepsis(24 h)(P<0.000 1),significant increases in platelet distri-bution width(PDW)and mean platelet volume(MPV)(P<0.01),significant prolonging of thrombin time(TT),prothrombin time(PT),and activated partial thromboplastin time(APTT)(P<0.000 1,P<0.001,P<0.01),and significant reduction in fibrinogen(Fib)(P<0.000 1).Compared with the Con/sham group,the LPS/CLP group exhibited significant increases in the platelet production phenotype of megakaryocyte,the number of PLP in the supernatant,and the expression levels of platelet surface markers(CD41,CD42b,CD61).The rates of megakaryocyte necroptosis/apoptosis,pyroptosis,and ferroptosis were significantly elevated at 24 h post-CLP surgery.Laser confo-cal microscopy showed significant activation of LC3,P-MLKL,Caspase-1,and Fe2+in megakaryocytes of mice after CLP surgery.Western blotting results revealed that the CLP group exhibited a significant increase in the activa-tion rate of necroptosis-related protein P-MLKL(P<0.001),a significant increase in the cleavage of pyroptosis-related proteins GSDMD and GSDMD-N(P<0.01,P<0.001,respectively),a significant increase in the expres-sion of ferroptosis-related protein ACSL4(P<0.01),and a significant decrease in the expression of GPX4(P<0.01)compared to the sham group.Additionally,the CLP group demonstrated significant increases in the expression of apoptosis-related protein Bax,the cleavage of autophagy-related protein LC3B-Ⅱ,and the expression of P62(P<0.05,P<0.001,P<0.001,respectively).Inhibition of apoptosis with programmed cell death inhibitors decreased platelet production function of megakaryocyte,while inhibition of necroptosis and pyroptosis had limited effects on platelet production function of megakaryocyte.Inhibition of ferroptosis and autophagy enhanced platelet production function of megakaryocyte.Conclusion Significant programmed death of megakaryocytes was observed during the acute phase of sepsis(24 h).Among those megakaryocytes,apoptosis is an important mechanism for the differentia-tion of platelet production phenotype and increased platelet production capacity of megakaryocyte.Overactive autophagy and ferroptosis in megakaryocytes lead to megakaryocyte dysfunction,which is an important mechanism for coagulation abnormalities in sepsis.

AIM To compare the chemical constituents in traditional decoction and formula granule decoction of classical famous prescription Wendan Decoction.METHODS The HPLC fingerprints were established,after which the contents of adenosine,synephrine,liquiritin,naringin,hesperidin,6-gingerol and adenosine cyclophosphate were determined,cluster analysis,principal component analysis and multidimensional scaling analysis were adopted in the investigation of component differences,and the equivalent of formula granules was adjusted.RESULTS The similarities of HPLC fingerprints for 10 batches of traditional decoctions were higher than those of HPLC fingerprints for 9 batches of formula granule decoctions(P＜0.01).Adenosine,synephrine,liquiritin,hesperidin and cyclic adenosine monophosphate demonstrated higher contents in traditional decoctions than those in formula granule decoctions(P＜0.05),6-gingerol displayed lower content than that in the latter produced by manufacturers A,C(P＜0.05),which was higher than that in the latter produced by manufacturer B(P＜0.01).Various batches of traditional decoctions and formula granule decoctions could be obviously distinguished,adenosine,synephrine and hesperidin exhibited great influences on the classification of principal component analysis,and the quality of formula granule decoctions produced by manufacturer C was closer to that of traditional decoctions.After equivalent correction,the contents of various constituents in formula granule decoctions produced by manufacturers A,C showed no significant differences as compared with those in traditional decoction(P＞0.05).CONCLUSION The formula granules of Wendan Decoction from different manufacturers exist quality differences,so the preparation process and extraction process of this preparation should be optimized to improve quality,and equivalent ratio should be adjusted according to actual requirements to ensure its scientific and rational clinical application.

AIM To compare the chemical constituents in traditional decoction and formula granule decoction of classical famous prescription Wendan Decoction.METHODS The HPLC fingerprints were established,after which the contents of adenosine,synephrine,liquiritin,naringin,hesperidin,6-gingerol and adenosine cyclophosphate were determined,cluster analysis,principal component analysis and multidimensional scaling analysis were adopted in the investigation of component differences,and the equivalent of formula granules was adjusted.RESULTS The similarities of HPLC fingerprints for 10 batches of traditional decoctions were higher than those of HPLC fingerprints for 9 batches of formula granule decoctions(P＜0.01).Adenosine,synephrine,liquiritin,hesperidin and cyclic adenosine monophosphate demonstrated higher contents in traditional decoctions than those in formula granule decoctions(P＜0.05),6-gingerol displayed lower content than that in the latter produced by manufacturers A,C(P＜0.05),which was higher than that in the latter produced by manufacturer B(P＜0.01).Various batches of traditional decoctions and formula granule decoctions could be obviously distinguished,adenosine,synephrine and hesperidin exhibited great influences on the classification of principal component analysis,and the quality of formula granule decoctions produced by manufacturer C was closer to that of traditional decoctions.After equivalent correction,the contents of various constituents in formula granule decoctions produced by manufacturers A,C showed no significant differences as compared with those in traditional decoction(P＞0.05).CONCLUSION The formula granules of Wendan Decoction from different manufacturers exist quality differences,so the preparation process and extraction process of this preparation should be optimized to improve quality,and equivalent ratio should be adjusted according to actual requirements to ensure its scientific and rational clinical application.

Human physiological indicators have become an important standard for assessing health in modern society.Traditional detection methods often require a separate laboratory,complex operation process and long detection time,so it is urgent to develop portable,fast and accurate on-site detection technologies for bioanalysis.Point-of-care testing(POCT),which differs from traditional laboratory testing,can realize the rapid in situ detection of biomarkers without the complicated analytical process of the laboratory.Smartphones,which are an essential tool in our daily life,not only have independent operating systems and built-in storage functions,but also have high-definition cameras,which have great application potential in POCT visualization.The combination of various biosensing technologies and smartphones has developed into a new direction in the field of POCT.This review mainly introduced the research progress of smartphone-based visual biosensors in POCT in recent years,including colorimetric sensors,fluorescence sensors,chemiluminescence sensors and electrochemiluminescence sensors.Finally,the problems faced by smart-phone-based visual biosensors in the application of POCT were summarized,and their future development was prospected.

Temporomandibular joint (TMJ) diseases are common clinical conditions. The number of patients with TMJ diseases is large, and the etiology, epidemiology, disease spectrum, and treatment of the disease remain controversial and unknown. To understand and master the current situation of the occurrence, development and prevention of TMJ diseases, as well as to identify the patterns in etiology, incidence, drug sensitivity, and prognosis is crucial for alleviating patients′suffering.This will facilitate in-depth medical research, effective disease prevention measures, and the formulation of corresponding health policies. Cohort construction and research has an irreplaceable role in precise disease prevention and significant improvement in diagnosis and treatment levels. Large-scale cohort studies are needed to explore the relationship between potential risk factors and outcomes of TMJ diseases, and to observe disease prognoses through long-term follw-ups. The consensus aims to establish a standard conceptual frame work for a cohort study on patients with TMJ disease while providing ideas for cohort data standards to this condition. TMJ disease cohort data consists of both common data standards applicable to all specific disease cohorts as well as disease-specific data standards. Common data were available for each specific disease cohort. By integrating different cohort research resources, standard problems or study variables can be unified. Long-term follow-up can be performed using consistent definitions and criteria across different projects for better core data collection. It is hoped that this consensus will be facilitate the development cohort studies of TMJ diseases.

AIM To investigate the ameliorative effects of Schisandrol A(Sol A)in Suhuang antitussive capsule on post-infectious cough(PIC).METHODS The in vivo mouse PIC model was established by intratracheal instillation of lipopolysaccharide(LPS)combined with cigarette smoke exposure.The mice were randomly divided into the control group,the model group,the Suhuang antitussive capsule group(14 g/kg),the montelukast sodium positive control group(3 mg/kg),and low and high dose Sol A groups(10,30 mg/kg).The in vitro PIC model was established by stimulating human bronchial epithelial cells(BEAS-2B)with LPS.The cells were divided into the control group,the model group,the Suhuang antitussive capsule group(10 μg/mL)and low and high dose Sol A groups(3,10 μmol/L).HE and Masson staining were used to detect the pathological changes of the lung and bronchial tissues.ELISA was used to detect the levels of IL-1β,IL-6,TNF-α,ROS,MDA,SOD and GSH in the lung tissues.RT-qPCR was used to detect the IL-1β,IL-6 and TNF-α mRNA expressions in BEAS-2B cells.And Western blot was applied to detect the protein expressions of p-PI3K,p-Akt,NOX4,SIRT1,p-ERK,Fibronectin,E-cadherin,Vimentin and α-SMA in mouse lung tissue and BEAS-2B cells.RESULTS Compared with the model group,the groups intervened with Sol A or Suhuang antitussive capsule displayed prolonged cough latency(P＜0.01);reduced cough frequency(P＜0.01);relieved pulmonary inflammatory cell infiltration and collagen deposition in PIC mice;decreased pulmonary levels of IL-1β,IL-6,TNF-α,ROS,MDA and protein expressions of Fibronectin,Vimentin,α-SMA,p-ERK,p-PI3K,p-Akt,and NOX4(P＜0.05,P＜0.01);increased pulmonary levels of SOD and GSH and protein expressions of E-cadherin and SIRT1(P＜0.05,P＜0.01);decreased ROS level,IL-1β,IL-6,TNF-α mRNA expressions and p-ERK,p-PI3K,p-Akt,NOX4 protein expressions in vitro(P＜0.05,P＜0.01);and increased SIRT1 protein expression in vitro as well(P＜0.01).CONCLUSION Being the main antitussive component of Suhuang antitussive capsule upon the PIC model,Sol A inhibits the inflammation via SIRT1/ERK signaling pathway and relieve the oxidative stress via PI3K/Akt/NOX4 signaling pathway.

AIM To explore the effects of praeruptorin A from Suhuang antitussive capsules on cough variant asthma(CVA).METHODS The rats were randomly divided into the normal group,the model group,the dexamethasone group(0.5 mg/kg),the Suhuang antitussive capsules group(7 g/kg)and the low,medium and high dose praeruptorin A groups(15,30 and 60 mg/kg).The rat model of CVA was established by intraperitoneal injection of sensitizer(1 mg/mL ovalbumin and 10 mg/mL aluminum hydroxide)and aerosol inhalation of 1%ovalbumin followed by the corresponding dosing of drugs by gavage initiated on the 14th day.Another 14 days later,the rats had their pathological pulmonary changes observed by HE,Masson and PAS stainings;their number of inflammatory cells in bronchoalveolar lavage fluid(BALF)detected by hematology analyzer;and their levels of IL-4,IL-5,IL-13 and MUC5AC in BALF detected by ELISA.The RAW264.7 cell inflammatory model induced by lipopolysaccharide(LPS)was treated with 4,8,16 μmol/L praeruptorin A or 0.25 mg/mL Suhuang antitussive capsules,respectively.And the cells had their NO level detected by Griess method,and their ROS expression observed using fluorescence microscopy.The detections of the pulmonary and cellular mRNA expressions of IL-6,IL-1β,COX-2,iNOS and PPAR-γ by RT-qPCR;and the protein expressions of p-P65,P65,p-IκBα,IκBα,NLRP3,caspase-1(p20)and IL-1β by Western blot were conducted in both the cells and the rats.RESULTS The in vivo result showed that praeruptorin A reduced the cough frequency(P＜0.01);prolonged the cough latency(P＜0.05,P＜0.01);reduced the number of eosinophils and neutrophils in BALF(P＜0.05,P＜0.01);decreased the levels of IL-4,IL-5,IL-13 and MUC5AC in BALF and the pulmonary mRNA expressions of IL-6,IL-1β,COX-2 and iNOS(P＜0.05,P＜0.01);and decreased the phosphorylation of P65 and IκBα protein and NLRP3,caspase-1(p20)and IL-1β protein expressions(P＜0.05,P＜0.01)as well.The in vitro result showed that praeruptorin A inhibited the release of LPS-induced NO and reduce the ROS level(P＜0.01);decreased the mRNA expressions of IL-1β,COX-2 and iNOS(P＜0.05,P＜0.01);increased PPAR-γ mRNA expression(P＜0.05),and decreased the phosphorylation of P65 and IκBα protein and the expression of NLRP3 protein(P＜0.05,P＜0.01).CONCLUSION Praeruptorin A,one of the main antitussive components of Suhuang antitussive capsules,may improve CVA because of its anti-inflammatory and antitussive role by inhibiting the activation of NF-κB signaling pathway and reducing the expression of NLRP3 inflammatory corpuscles.