Inteins are unique protein insertion sequences capable of self-excision, enabling the covalent ligation of flanking extein peptides via amide bond formation. This process proceeds spontaneously without requiring external enzymes, cofactors, or chemical reagents, granting inteins exceptional biocompatibility and traceless performance in protein engineering applications. Split inteins represent a specialized and versatile subclass whose splicing domains are encoded by two separate gene fragments rather than a single continuous open reading frame. These fragments, known as the N-terminal (IntN) and C-terminal (IntC) split inteins, associate through non-covalent interactions including hydrophobic forces, hydrogen bonds, and van der Waals forces to assemble into an active three-dimensional structure, which then drives efficient extein ligation and enables protein trans-splicing. Protein trans-splicing mediated by split inteins has become a cornerstone for traceless protein ligation owing to its high specificity and irreversibility, fundamentally reshaping strategies for protein modification, assembly, and functional regulation. Compared with traditional chemical ligation methods, split intein systems require no complex chemical derivatization of peptide fragments and can operate efficiently at micromolar concentrations under physiological conditions, thus avoiding structural and functional damage caused by organic reagents. In contrast to enzymatic ligation tools such as sortase, split inteins eliminate the need for additional enzymes or cofactors, simplifying reaction systems, reducing costs, and minimizing non-specific side products. These distinctive advantages render split inteins highly promising for applications in chemical biology, synthetic biology, and biopharmaceutical development. In recent years, deepened mechanistic understanding has established structure-guided rational design as the primary approach to overcoming key limitations of split inteins, including intrinsic aggregation propensity, strict extein sequence dependence, and limited splicing efficiency. Bioinformatic tools have been used to identify aggregation-prone regions in the IntN fragment, and site-directed mutagenesis of hydrophobic residues, relocation of split sites, or removal of misfolding-prone sequences has substantially reduced in vitro aggregation and improved soluble expression and assembly activity. Rational engineering of catalytic residues and adjacent flexible loops has relaxed strict amino acid preferences at extein junctions, enhancing sequence tolerance and reducing the risk of functional impairment in target proteins. Consensus design based on multiple sequence alignments has yielded ultra-fast splicing variants such as Cfa DnaE and Cat-TerL, which exhibit significantly accelerated kinetics and improved tolerance to denaturing conditions. Meanwhile, advances in structural biology have further clarified the conformational dynamics and catalytic mechanisms of splicing, supporting the precise design of high-performance intein modules. On this basis, electrostatic interaction tuning and metagenomic screening have yielded multiple mutually orthogonal split intein pairs, enabling selective multi-fragment protein ligation and providing new routes for the efficient synthesis of large multi-domain functional proteins. With these engineered split inteins offering continuously improved performance and expanded applicability, protein trans-splicing has been widely applied in numerous cutting-edge areas of protein research and biomedicine. In gene delivery, split intein-based systems overcome the packaging limit of adeno-associated viral vectors, enabling the accurate reconstitution of large therapeutic proteins and base editors in target cells, thereby enhancing the efficacy and scope of gene therapy for genetic diseases. In internal protein sequence editing, split inteins mediate precise sequence replacement and modification in flexible regions or loops of target proteins, without the need for complex multi-step ligation and protein refolding involved in traditional protein semisynthesis. In protein-protein interaction studies, intein-mediated splicing covalently captures transient and weak intracellular complexes, enabling sensitive, high-throughput interaction detection and drug screening. In synthetic biology, conditionally controllable splicing systems support the construction of diverse intracellular and cell-surface biological logic gates for the precise regulation of cellular behavior. In mechanistic biochemical research, split inteins enable photocatalytic proximity labeling and site-specific tagging, allowing the preparation of homogeneous protein samples carrying precise post-translational modifications such as ubiquitination and polyglutamylation for chromatin interactome analysis and epigenetic studies. Moreover, covalent trapping strategies using split inteins stabilize transient enzymatic intermediates, providing unprecedented insights into molecular mechanisms such as nucleosome ubiquitination that are difficult to elucidate using conventional methods. This review systematically summarizes key technological advances in split inteins over the past decade, highlighting engineering strategies, mechanistic insights, and the development of orthogonal components. It comprehensively surveys emerging applications at the frontiers of protein research, analyzes current core challenges, and proposes future directions, particularly emphasizing artificial intelligence-driven de novo design and novel splicing pathways to break existing technical bottlenecks. By enabling traceless, efficient, and versatile protein manipulation, split inteins continue to serve as indispensable tools that drive innovation in protein engineering and fundamental life science research.

Objective: To investigate the nutritional status and influencing factors among Tibetan and Mongolian children and adolescents aged 7-18 years in high-altitude regions, so as to provide evidence for early prevention and control of malnutrition in this population. Methods: From May to June 2022, a cluster sampling method was employed to recruit 1 019 Tibetan and Mongolian children and adolescents aged 7-18 years from two primary and secondary schools in Golmud City. Physical examinations, dietary frequency questionnaires, and physical activity assessments were conducted. Nutritional status was classified as obesity, combined overweight/obesity, underweight, or central obesity according to national standards including Screening for Overweight and Obesity among School-age Children and Adolescents, Screening Standard for Malnutrition of School-age Children and Adolescents, Blue Book on Obesity Prevention and Control in China. Chi-square tests, t-test and Logistic regression analyses were performed to identify factors associated with different nutritional statuses. Results: The detection rates of obesity, combined overweight/obesity, underweight, and central obesity were 8.0%, 18.1%, 5.2%, and 19.7%, respectively. The height of children and adolescents across all age groups was generally lower than the national standard values. Tibetan participants exhibited significantly lower height-for-age Z-scores (HAZ)(9-10, 13-17 years, Z =2.01, 2.78, 4.16, 3.38, 4.12, 3.63, 3.00) and BMI-for-age Z-scores (BAZ) compared to Mongolian participants ( Z =-2.95, -2.47, -2.31, -2.89, -2.14, -2.17)( P < 0.05 ). Multivariate Logistic regression revealed that Mongolian children and adolescents had higher risks of obesity ( OR =2.20) and combined overweight/obesity ( OR = 2.18 ) ( P <0.05). Additionally, insufficient moderate-to-vigorous physical activity (MVPA) was associated with an increased risk of central obesity ( OR =1.48, P <0.05), compared with children and adolescents who meet the standard of MVPA. Conclusions The rates of overweight and obesity among Tibetan and Mongolian children and adolescents in Golmud City are higher, influenced by multiple factors. Nutrition interventions and physical activity strategies tailored to ethnic characteristics should be implemented, with emphasis on promoting MVPA to improve nutritional outcomes in this population.

BACKGROUND: Chronic kidney disease (CKD) is associated with common pathophysiological processes, such as inflammation and fibrosis, in both the heart and the kidney. However, the underlying molecular mechanisms that drive these processes are not yet fully understood. Therefore, this study focused on the molecular mechanism of heart and kidney injury in CKD. METHODS: We generated an microRNA (miR)-26a knockout (KO) mouse model to investigate the role of miR-26a in angiotensin (Ang)-II-induced cardiac and renal injury. We performed Ang-II modeling in wild type (WT) mice and miR-26a KO mice, with six mice in each group. In addition, Ang-II-treated AC16 cells and HK2 cells were used as in vitro models of cardiac and renal injury in the context of CKD. Histological staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), and Western blotting were applied to study the regulation of miR-26a on Ang-II-induced cardiac and renal injury. Immunofluorescence reporter assays were used to detect downstream genes of miR-26a, and immunoprecipitation was employed to identify the interacting protein of LIM and senescent cell antigen-like domain 1 (LIMS1). We also used an adeno-associated virus (AAV) to supplement LIMS1 and explored the specific regulatory mechanism of miR-26a on Ang-II-induced cardiac and renal injury. Dunnett's multiple comparison and t -test were used to analyze the data. RESULTS: Compared with the control mice, miR-26a expression was significantly downregulated in both the kidney and the heart after Ang-II infusion. Our study identified LIMS1 as a novel target gene of miR-26a in both heart and kidney tissues. Downregulation of miR-26a activated the LIMS1/integrin-linked kinase (ILK) signaling pathway in the heart and kidney, which represents a common molecular mechanism underlying inflammation and fibrosis in heart and kidney tissues during CKD. Furthermore, knockout of miR-26a worsened inflammation and fibrosis in the heart and kidney by inhibiting the LIMS1/ILK signaling pathway; on the contrary, supplementation with exogenous miR-26a reversed all these changes. CONCLUSIONS Our findings suggest that miR-26a could be a promising therapeutic target for the treatment of cardiorenal injury in CKD. This is attributed to its ability to regulate the LIMS1/ILK signaling pathway, which represents a common molecular mechanism in both heart and kidney tissues.
Animals ; MicroRNAs/metabolism* ; Angiotensin II/toxicity* ; Mice ; Renal Insufficiency, Chronic/chemically induced* ; Mice, Knockout ; Disease Models, Animal ; Male ; Signal Transduction/genetics* ; LIM Domain Proteins/genetics* ; Mice, Inbred C57BL ; Cell Line ; Humans

Objective To explore the protective effects and mechanism of Shenqi Qiangjing granules containing serum on hydrogen peroxide(H2O2)induced oxidative damage and ferroptosis in mouse spermatogonia(GC-1 spg).Methods Thir-ty SPF grade healthy male SD rats were selected and randomly divided into blank group,levocarnitine group,and Shenqi Qiangjing granules group.After 7 days of intragastric administration,drug-containing serum was collected from each group.Using mouse sper-matogonia as a cell model,they were randomly divided into the normal control group,the model control group,blank serum group,levocarnitine containing serum group,and Shenqi Qiangjing granules containing serum group.Except for the normal control group,the other groups used hydrogen peroxide at a concentration of 600 μmol·L-1 to induce injury to mouse spermatogonia for 4 hours,and then established oxidative stress injury models,after 24 hours of medication intervention in each group.The survival rate of cells was detected using CCK-8 method;The levels of intracellular reactive oxygen species(ROS),malondialdehyde(MDA),catalase(CAT),glutathione(GSH),and superoxide dismutase(SOD)were detected by ELISA;The intracellular iron level was detected by iron ion colorimetry;The activities of Caspase-3 and Caspase-9 were detected by colorimetry;The mRNA levels of glu-tathione peroxidase 4(GPX4)and ACSL4 were determined by qRT-PCR.Results Compared with the normal control group,the cell proliferation activity of the model control group decreased significantly,the levels of ROS,MDA and Fe3+were significantly increased,while the activities of CAT,GSH and SOD were significantly decreased in the model control group,however,Caspase-3 and Caspase-9 activities were significantly increased,the results showed significant difference(P＜0.05).Compare with the model control group,Shenqi Qiangjing granules containing serum could significantly increase the cell proliferation activity,decrease the levels of ROS,MDA and Fe3+,increase the activities of CAT,GSH and SOD,and decrease the activities of Caspase-3 and Caspase-9,the results showed significant difference(P＜0.05).The qRT PCR results showed that compared with the model control group,the expression of GPX4 mRNA was upregulated and ACSL4 mRNA was downregulated in blank serum group containing Shenqi Qiangjing granules,and the differences were statistically significant(P＜0.05).Conclusions Shenqi Qiangjing granules have significant protective effects on hydrogen peroxide-induced oxidative stress injury and ferroptosis of spermatogonia in mice.The mechanism may be related to the decrease of Caspase-3 and Caspase-9 activities and the inhibition of oxidative stress injury and ferroptosis.

Objective To explore the protective effects and mechanism of Shenqi Qiangjing granules containing serum on hydrogen peroxide(H2O2)induced oxidative damage and ferroptosis in mouse spermatogonia(GC-1 spg).Methods Thir-ty SPF grade healthy male SD rats were selected and randomly divided into blank group,levocarnitine group,and Shenqi Qiangjing granules group.After 7 days of intragastric administration,drug-containing serum was collected from each group.Using mouse sper-matogonia as a cell model,they were randomly divided into the normal control group,the model control group,blank serum group,levocarnitine containing serum group,and Shenqi Qiangjing granules containing serum group.Except for the normal control group,the other groups used hydrogen peroxide at a concentration of 600 μmol·L-1 to induce injury to mouse spermatogonia for 4 hours,and then established oxidative stress injury models,after 24 hours of medication intervention in each group.The survival rate of cells was detected using CCK-8 method;The levels of intracellular reactive oxygen species(ROS),malondialdehyde(MDA),catalase(CAT),glutathione(GSH),and superoxide dismutase(SOD)were detected by ELISA;The intracellular iron level was detected by iron ion colorimetry;The activities of Caspase-3 and Caspase-9 were detected by colorimetry;The mRNA levels of glu-tathione peroxidase 4(GPX4)and ACSL4 were determined by qRT-PCR.Results Compared with the normal control group,the cell proliferation activity of the model control group decreased significantly,the levels of ROS,MDA and Fe3+were significantly increased,while the activities of CAT,GSH and SOD were significantly decreased in the model control group,however,Caspase-3 and Caspase-9 activities were significantly increased,the results showed significant difference(P＜0.05).Compare with the model control group,Shenqi Qiangjing granules containing serum could significantly increase the cell proliferation activity,decrease the levels of ROS,MDA and Fe3+,increase the activities of CAT,GSH and SOD,and decrease the activities of Caspase-3 and Caspase-9,the results showed significant difference(P＜0.05).The qRT PCR results showed that compared with the model control group,the expression of GPX4 mRNA was upregulated and ACSL4 mRNA was downregulated in blank serum group containing Shenqi Qiangjing granules,and the differences were statistically significant(P＜0.05).Conclusions Shenqi Qiangjing granules have significant protective effects on hydrogen peroxide-induced oxidative stress injury and ferroptosis of spermatogonia in mice.The mechanism may be related to the decrease of Caspase-3 and Caspase-9 activities and the inhibition of oxidative stress injury and ferroptosis.

Objective To investigate the clinical value of intracoronary injection of recombinant human prourokinase for injection in patients with acute ST-segment elevation myocardial infarction(STEMI)undergoing emergency percutaneous coronary intervention(PCI).Methods Retrospective analysis was used to collect STEMI patients who underwent emergency PCI in department of cardiology from January to December 2019.According to the targeted injection of drugs through a guided catheter by intraoperative patients before stent implantation,the patients were divided into a tirofiban group(70 cases)and a recombinant human urokinase group(70 cases).TIMI blood flow grade,ST segment fall value(STR)of infarct-related artery,plasma D-dimer,left ventricular ejection fraction(LVEF)and coronary microcirculation dysfunction were compared between the two groups.At the same time,the general information of the patients,major adverse cardiovascular events(MACE)and bleeding were recorded.Results Compared with the tirofiban group,the TIMI blood flow level,LVEF,plasma D-dimer,and the proportion of the patients with STR＞70％in the recombinant human prourokinase group were significantly increased,while the incidence of slow flow/no reflow and MACE were significantly reduced,and the differences between the two groups were statistically significant(P＜0.05).The incidence of bleeding events in the recombinant human prourokinase group showed a downward trend,but there was no significant difference between the two groups(P＞0.05).Conclusion Intracoronary injection of recombinant human prourokinase through the guiding catheter can significantly improve myocardial perfusion and prognosis in patients with STEMI.It also reduces adverse cardiovascular events and has a high safety margin,which is worth promoting in the clinic.

Gouty arthritis is a condition resulting from the dysregulation of purine metabolism in the human body. It is characterized by redness, swelling, recurrent episodes of hot pain, and joint stiffness. Currently, Western medicine primarily relies on uric acid-lowering, anti-inflammatory, and analgesic drugs, but these medications often have significant side effects. In contrast, external therapies in traditional Chinese medicine are considered simpler, safer, and less likely to produce adverse reactions, thus making them more appealing to patients. This article summarizes the various external treatment methods used in traditional Chinese medicine to alleviate the clinical symptoms of gouty arthritis, offering a novel approach for clinical management.

Objective: To explore the related factors of negative conversion time (NCT) of nucleic acid in children with COVID-19. Methods: A retrospective cohort study was conducted. A total of 225 children who were diagnosed with COVID-19 and admitted to Changxing Branch of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from April 3^rd to May 31^st 2022 were enrolled in the study. The infection age, gender, viral load, basic disease, clinical symptoms and information of accompanying caregivers were retrospectively analyzed. According to age, the children were divided into<3 years of age group and 3-<18 years of age group. According to the viral nucleic acid test results, the children were divided into positive accompanying caregiver group and negative accompanying caregiver group. Comparisons between groups were performed using Mann-Whitney U test or Chi-square test. Multivariate Logistic regression analysis was used to analyze the related factors of NCT of nucleic acid in children with COVID-19. Results: Among the 225 patients (120 boys and 105 girls) of age 2.8 (1.3, 6.2) years, 119 children <3 years and 106 children 3-<18 years of age, 19 cases were diagnosed with moderate COVID-19, and the other 206 cases were diagnosed with mild COVID-19. There were 141 patients in the positive accompanying caregiver group and 84 patients in the negative accompanying caregiver group.Patients 3-<18 years of age had a shorter NCT (5 (3, 7) vs.7 (4, 9) d, Z=-4.17, P<0.001) compared with patients <3 years of age. Patients in the negative accompanying caregiver group had a shorter NCT (5 (3, 7) vs.6 (4, 9) d,Z=-2.89,P=0.004) compared with patients in the positive accompanying caregiver group. Multivariate Logistic regression analysis showed that anorexia was associated with NCT of nucleic acid (OR=3.74,95%CI 1.69-8.31, P=0.001). Conclusion: Accompanying caregiver with positive nucleic acid test may prolong NCT of nucleic acid, and decreased appetite may be associated with prolonged NCT of nucleic acid in children with COVID-19.
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Male ; Young Adult ; China/epidemiology* ; COVID-19/genetics* ; Nucleic Acids ; Retrospective Studies

Objective:To explore the molecular mechanism of Danggui Niantong Decoction in the treatment of gouty arthritis (GA) based on network pharmacology and molecular docking.Methods:By selecting for the active components and targets of Danggui Niantong Decoction with TCMSP, and retrieving the GeneCards, OMIM, PharmGKB and DrugBank databases to obtain GA related targets. The potential targets of Danggui Niantong Decoction in the treatment of GA were obtained by the intersection of mappings. The regulation network of Chinese medicine compound and protein-protein interaction network of Danggui Niantong Decoction were constructed by Cytoscape software, and the targets of Danggui Niantong Decoction in the treatment of GA were analyzed by GO and KEGG enrichment by David Database. Finally, molecular docking was performed by using Autodock software.Results:There are 198 active components that could treat GA in Danggui Niantong Decoction. The key active components are Quercetin and Kaempferol. There are 46 key targets, the core targets are NFE2L2, HMOX1, PPARA, PTGS2, IL1β, CXCL8. GO enrichment suggests that the key genes are primarily involved in many biological processes such as Inflammatory response regulation, response to oxidative stress, Fatty acid metabolism process, steroid metabolism, lipopolysaccharide response and reactive oxygen species metabolism. KEGG pathway indicates that Danggui Niantong Decoction mainly acted on IL-17 signal pathway, HIF-1 signal pathway, TNF signal pathway and AGE-RAGE signal pathway. Molecular docking shows that the active components of Danggui Niantong Decoction and action target of GA can combine toghether with high efficiency, and the structure is stable.Conclusion:Danggui Niantong Decoction has multi-component, multi pathway and multi-protein characteristics. Danggui Niantong Decoction can treat GA by regulating immune inflammatory reaction and oxidative stress reaction.