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.

OpenSim is an open source, free motion simulation and gait analysis software, which can be used to dynamically simulate and analyze the complex motion of the human body, and is widely used in human biomechanical research. Since OpenSim can analyze multi-dimensional motion data such as muscle strength, joint torque, and muscle synergistic activation during human movement, it can be used to study the biomechanical mechanism of musculoskeletal imbalance diseases and various treatment methods in TCM orthopedics, and has a broad application prospect in the field of TCM orthopedics. By the analysis of the basic characteristics, elements, analysis process, and application prospects of OpenSim, it is concluded that OpenSim musculoskeletal model has a large application space in the field of traditional Chinese medicine orthopedic, which is helpful to explain the pathogenesis and mechanism of diseases, and promote the precision diagnosis and treatment of orthopedics diseases;the application of OpenSim musculoskeletal model can solve the problem that the previous research paid attention to the bone malalignment and not enough attention to the tendon, and provide a new method for the research of orthopedic diseases. At present, there are still problems in the promotion and application of OpenSim, such as large equipment requirements and high operation threshold. Therefore, multidisciplinary cooperation, clinical research, and data sharing are the basic research strategies in this field.
Humans ; Biomechanical Phenomena ; Orthopedics ; Traumatology ; Software ; Medicine, Chinese Traditional ; Musculoskeletal System ; Models, Biological

Objective:To explore the influence of human induced pluripotent stem cell (iPSC) derived skin organoid-conditioned culture medium (SO-CM) on the function of human dermal fibroblasts (Fbs) induced by high glucose, with the aim of providing treatment ideas for diabetic wounds.Methods:This study was an experimental research. Human iPSCs were induced into skin organoids. Human iPSC-derived skin organoids and human dermal Fbs were seeded into skin organoid culture medium (SOM) and cultured for three days, respectively. Then the cell culture supernatants were collected as SO-CM and Fb-conditioned culture medium (Fb-CM), respectively. The content of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), IL-18, C-C motif chemokine ligand 2 (CCL-2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), and VEGF-β in SOM, Fb-CM, and SO-CM was detected by enzyme-linked immunosorbent assay. Human Fbs of passage 8 and 9 induced by high glucose were divided into SOM group, Fb-CM group, and SO-CM group according to the random number table method, and were cultured with SOM, Fb-CM, and SO-CM all containing glucose at final molarity of 35 mmol/L, respectively. After 24 hours of culture, the Ki67 positive ratio of cells was calculated after immunofluorescence staining, and the cell absorbance value was detected by using cell counting kit-8, representing cell proliferation activity. The cell scratch test was performed to calculate the cell migration rate at 13 hours after scratching. After 48 hours of culture, the expression of reactive oxygen species in cells was detected by fluorescent probe method, and the rate of β-galactosidase-positive staining of cells was detected by β-galactosidase staining kit, representing cellular senescence. The sample size was three.Results:There was no statistically significant difference in the content of TNF-α, PDGF, and TGF-β among the three culture media ( P>0.05). Compared with that in SOM, the content of IL-10 and EGF in Fb-CM and SO-CM was significantly decreased ( P<0.05), while the content of CCL-2 in FB-CM and VEGF in SO-CM was significantly increased ( P<0.05). After 24 hours of culture, the Ki67 positive ratio ((45.2±6.0)% and (57.4±4.0)%) and absorbance values (124±5 and 158±12) of cells in the Fb-CM group and the SO-CM group were significantly higher than those in the SOM group ((29.6±2.1)% and 100±6, P<0.05), and the Ki67 positive ratio and absorbance value of cells in the SO-CM group were significantly higher than those in the Fb-CM group ( P<0.05). At 13 hours after scratching, the cell migration rates in the Fb-CM group and the SO-CM group were significantly higher than that in the SOM group ( P<0.05). After 48 hours of culture, the level of reactive oxygen species in the SO-CM group was significantly higher than that in the SOM group and the Fb-CM group (with both P values <0.05). After 48 hours of culture, there was no statistically significant difference in the rate of β-galactosidase-positive staining of cells among the three groups ( P>0.05). Conclusions:The SO-CM has high content of VEGF and can significantly promote the proliferation, migration, and expression of reactive oxygen species in human dermal Fbs induced by high glucose, but has no significant effect on cell senescence.

Objective:To explore the structural equation model to explore the levels of work-related musculoskeletal disorders (WMSDs) and various risk factors in the feet and ankle of China's occupational population, providing scientific basis for for preventing WMSDs in feet and ankles.Methods:Data of 73497 national occupational epidemiological cases were selected from June 2018 to December 2023 used the Chinese version of the Electronic Questionnaire on Musculoskeletal Disorders. The adverse ergonomic factors and their source classification standard and confirmatory factor analysis were used to investigate foot and ankle WMSDs and their related risk factors (including individual factors, work organization, work posture, work type, fatigue, etc.) in key occupational groups in China, and structural equation model hypothesis, fitting, verification, and path and intermediary effect analysis were carried out. The model fit evaluation indexes included Chi-square specific degrees of freedom ( χ2/ df), gauge fit index (NFI), Tucker Lewis index (TLI), goodness of Fit index (GFI), adjusted Goodness of Fit index (AGFI) and approximate root mean square error (RMSEA) . Results:A total of 73497 occupational workers were surveyed, with local muscle fatigue and WMSDs incidence rates in the feet and ankles being 17.17% and 12.06%, respectively. The fitting index of the adjusted structural equation model basically meets the standard (GFI=1, AGFI=1, RMESA=0.042, NFI=0.716, TLI=0.663). The top three factors affecting feet and ankle WMSDs are feet and ankle muscle fatigue, work type, and work organization, with standardized path coefficients of 0.221, 0.105, and 0.095, respectively. The top two factors affecting feet and ankle muscle fatigue are work organization and work type, with standardized path coefficients of 0.548 and 0.383, respectively. Feet and ankle muscle fatigue, work type, work organization, and work posture have a direct effect on feet and ankle WMSDs, with effect values of 0.221, 0.105, 0.095, and 0.077, respectively. The organization and type of work can also have indirect effects through feet and ankle muscle fatigue, with effect values of 0.121 and 0.084, respectively.Conclusion:Feet and ankle muscle fatigue has a direct impact on WMSDs, and plays a mediating role between ankle and ankle WMSDs caused by work organization and work type. Feet and ankle muscle fatigue is an important pathway leading to feet and ankle WMSDs. It is recommended that employers and managers detect job fatigue early and take corresponding prevention and intervention measures, which can play a key role in preventing feet and ankle WMSDs.

Objective:To explore the structural relationship between WMSDs in the upper limbs and various risk factors in the occupational population in China, based on a large sample epidemiological survey and structural equation analysis, and to establish a structural equation model, so as to lay a foundation for the prevention and control of such diseases.Methods:The Chinese version of the Musculoskeletal Disorders Electronic Questionnaire was used to conduct a nationwide survey on the prevalence of WMSDs in the upper extremity. Six factors related to WMSDs in the upper extremity were extracted by the classification standard of adverse ergonomic factors and their source and confirmatory factor analysis, including work organization, work type, upper extremity work posture, individual factors, upper extremity fatigue and upper extremity WMSDs. The structural equation analysis was carried out and the structural equation model was established.Results:The incidence of WMSDs and fatigue in the upper limbs was 24.44% and 43.76%, respectively. The adjusted structural equation model fitting indicators were generally up to the standard (GFI=1.000, AGFI=1.000, RMSEA=0.043, NFI=0.808, TLI=0.784) . The four exogenous latent variables of work organization, work type, upper limb work posture and individual factors were correlated. There was a strong positive correlation between job type and upper limb work posture ( r=0.865) , a moderate positive correlation between work organization and job type and upper limb work posture ( r=0.570, 0.490) , and a weak negative correlation between individual factors and the other three exogenous latent variables. Upper limb work posture and individual factors had direct effects on upper limb WMSDs, and the effect coefficients were 0.10 and 0.06, respectively. Upper limb fatigue played a mediating role between work organization, work type, upper limb work posture and upper limb WMSDs. The effect coefficient was 0.46, and the composition ratios of indirect effects were 100.0%, 100.0%, and 38.3%, respectively. The direct path effect of upper limb work posture, individual factors and upper limb WMSDs was weaker than the mediating path through upper limb fatigue. Conclusion:When carrying out the prevention and control of upper limbWMSDs, it is necessary to comprehensively consider the pathogenesis path of upper limb muscle fatigue and upper limb WMSDs caused by work organization, work type, and upper limb work posture, so as to provide theoretical reference for improving the prevention and control level of such diseases.

Objective:To analyze the clinical phenotypes and genetic characteristics of pediatric patients with neurofibromatosis type 1 (NF1).Methods:A cross-sectional study was adopted. Clinical and imaging data of 25 pediatric patients diagnosed as having NF1 in Department of Pediatric Neurology, Linyi People's Hospital Affiliated to Shandong Second Medical University from January 2024 to July 2025 were collected. Whole exome sequencing and Sanger sequencing were used to conduct genetic testing on the pediatric patients and his/her parents. Protein 3D modeling of the domestic and foreign unreported variations was conducted using SWISS-MODEL software.Results:Among the 25 pediatric patients with NF1, 14 were male (56%) and 11 were female (44%), with age ranging from 8 months to 18 years. All pediatric patients exhibited café-au-lait macules, and 7 (28%) presented with plexiform neurofibromas. Genetic test identified 4 types of NF1 variants: nonsense variant ( n=11, 44%), frameshift variant ( n=9, 36%), missense variant ( n=3, 12%), and splice-site variant ( n=2, 8%). Importantly, 5 novel NF1 variants were discovered, including c.3455T>A, c.3709dupG, c.2665_2684del, c.7092_7095delinsTA, and c.3260del. Three pediatric patients inherited NF1 variant from their parents, while the remaining 22 harbored de novo mutation. Conclusion:NF1 exhibits a broad clinical spectrum, primarily affecting the skin and nervous system; this study identifies 5 previously unreported variants, expanding the genetic profile of NF1.

Objective:To explore the influence of human induced pluripotent stem cell (iPSC) derived skin organoid-conditioned culture medium (SO-CM) on the function of human dermal fibroblasts (Fbs) induced by high glucose, with the aim of providing treatment ideas for diabetic wounds.Methods:This study was an experimental research. Human iPSCs were induced into skin organoids. Human iPSC-derived skin organoids and human dermal Fbs were seeded into skin organoid culture medium (SOM) and cultured for three days, respectively. Then the cell culture supernatants were collected as SO-CM and Fb-conditioned culture medium (Fb-CM), respectively. The content of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), IL-18, C-C motif chemokine ligand 2 (CCL-2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), and VEGF-β in SOM, Fb-CM, and SO-CM was detected by enzyme-linked immunosorbent assay. Human Fbs of passage 8 and 9 induced by high glucose were divided into SOM group, Fb-CM group, and SO-CM group according to the random number table method, and were cultured with SOM, Fb-CM, and SO-CM all containing glucose at final molarity of 35 mmol/L, respectively. After 24 hours of culture, the Ki67 positive ratio of cells was calculated after immunofluorescence staining, and the cell absorbance value was detected by using cell counting kit-8, representing cell proliferation activity. The cell scratch test was performed to calculate the cell migration rate at 13 hours after scratching. After 48 hours of culture, the expression of reactive oxygen species in cells was detected by fluorescent probe method, and the rate of β-galactosidase-positive staining of cells was detected by β-galactosidase staining kit, representing cellular senescence. The sample size was three.Results:There was no statistically significant difference in the content of TNF-α, PDGF, and TGF-β among the three culture media ( P>0.05). Compared with that in SOM, the content of IL-10 and EGF in Fb-CM and SO-CM was significantly decreased ( P<0.05), while the content of CCL-2 in FB-CM and VEGF in SO-CM was significantly increased ( P<0.05). After 24 hours of culture, the Ki67 positive ratio ((45.2±6.0)% and (57.4±4.0)%) and absorbance values (124±5 and 158±12) of cells in the Fb-CM group and the SO-CM group were significantly higher than those in the SOM group ((29.6±2.1)% and 100±6, P<0.05), and the Ki67 positive ratio and absorbance value of cells in the SO-CM group were significantly higher than those in the Fb-CM group ( P<0.05). At 13 hours after scratching, the cell migration rates in the Fb-CM group and the SO-CM group were significantly higher than that in the SOM group ( P<0.05). After 48 hours of culture, the level of reactive oxygen species in the SO-CM group was significantly higher than that in the SOM group and the Fb-CM group (with both P values <0.05). After 48 hours of culture, there was no statistically significant difference in the rate of β-galactosidase-positive staining of cells among the three groups ( P>0.05). Conclusions:The SO-CM has high content of VEGF and can significantly promote the proliferation, migration, and expression of reactive oxygen species in human dermal Fbs induced by high glucose, but has no significant effect on cell senescence.

Objective:To analyze the clinical phenotypes and genetic characteristics of pediatric patients with neurofibromatosis type 1 (NF1).Methods:A cross-sectional study was adopted. Clinical and imaging data of 25 pediatric patients diagnosed as having NF1 in Department of Pediatric Neurology, Linyi People's Hospital Affiliated to Shandong Second Medical University from January 2024 to July 2025 were collected. Whole exome sequencing and Sanger sequencing were used to conduct genetic testing on the pediatric patients and his/her parents. Protein 3D modeling of the domestic and foreign unreported variations was conducted using SWISS-MODEL software.Results:Among the 25 pediatric patients with NF1, 14 were male (56%) and 11 were female (44%), with age ranging from 8 months to 18 years. All pediatric patients exhibited café-au-lait macules, and 7 (28%) presented with plexiform neurofibromas. Genetic test identified 4 types of NF1 variants: nonsense variant ( n=11, 44%), frameshift variant ( n=9, 36%), missense variant ( n=3, 12%), and splice-site variant ( n=2, 8%). Importantly, 5 novel NF1 variants were discovered, including c.3455T>A, c.3709dupG, c.2665_2684del, c.7092_7095delinsTA, and c.3260del. Three pediatric patients inherited NF1 variant from their parents, while the remaining 22 harbored de novo mutation. Conclusion:NF1 exhibits a broad clinical spectrum, primarily affecting the skin and nervous system; this study identifies 5 previously unreported variants, expanding the genetic profile of NF1.

Objective:To explore the structural equation model to explore the levels of work-related musculoskeletal disorders (WMSDs) and various risk factors in the feet and ankle of China's occupational population, providing scientific basis for for preventing WMSDs in feet and ankles.Methods:Data of 73497 national occupational epidemiological cases were selected from June 2018 to December 2023 used the Chinese version of the Electronic Questionnaire on Musculoskeletal Disorders. The adverse ergonomic factors and their source classification standard and confirmatory factor analysis were used to investigate foot and ankle WMSDs and their related risk factors (including individual factors, work organization, work posture, work type, fatigue, etc.) in key occupational groups in China, and structural equation model hypothesis, fitting, verification, and path and intermediary effect analysis were carried out. The model fit evaluation indexes included Chi-square specific degrees of freedom ( χ2/ df), gauge fit index (NFI), Tucker Lewis index (TLI), goodness of Fit index (GFI), adjusted Goodness of Fit index (AGFI) and approximate root mean square error (RMSEA) . Results:A total of 73497 occupational workers were surveyed, with local muscle fatigue and WMSDs incidence rates in the feet and ankles being 17.17% and 12.06%, respectively. The fitting index of the adjusted structural equation model basically meets the standard (GFI=1, AGFI=1, RMESA=0.042, NFI=0.716, TLI=0.663). The top three factors affecting feet and ankle WMSDs are feet and ankle muscle fatigue, work type, and work organization, with standardized path coefficients of 0.221, 0.105, and 0.095, respectively. The top two factors affecting feet and ankle muscle fatigue are work organization and work type, with standardized path coefficients of 0.548 and 0.383, respectively. Feet and ankle muscle fatigue, work type, work organization, and work posture have a direct effect on feet and ankle WMSDs, with effect values of 0.221, 0.105, 0.095, and 0.077, respectively. The organization and type of work can also have indirect effects through feet and ankle muscle fatigue, with effect values of 0.121 and 0.084, respectively.Conclusion:Feet and ankle muscle fatigue has a direct impact on WMSDs, and plays a mediating role between ankle and ankle WMSDs caused by work organization and work type. Feet and ankle muscle fatigue is an important pathway leading to feet and ankle WMSDs. It is recommended that employers and managers detect job fatigue early and take corresponding prevention and intervention measures, which can play a key role in preventing feet and ankle WMSDs.

Objective:To explore the structural relationship between WMSDs in the upper limbs and various risk factors in the occupational population in China, based on a large sample epidemiological survey and structural equation analysis, and to establish a structural equation model, so as to lay a foundation for the prevention and control of such diseases.Methods:The Chinese version of the Musculoskeletal Disorders Electronic Questionnaire was used to conduct a nationwide survey on the prevalence of WMSDs in the upper extremity. Six factors related to WMSDs in the upper extremity were extracted by the classification standard of adverse ergonomic factors and their source and confirmatory factor analysis, including work organization, work type, upper extremity work posture, individual factors, upper extremity fatigue and upper extremity WMSDs. The structural equation analysis was carried out and the structural equation model was established.Results:The incidence of WMSDs and fatigue in the upper limbs was 24.44% and 43.76%, respectively. The adjusted structural equation model fitting indicators were generally up to the standard (GFI=1.000, AGFI=1.000, RMSEA=0.043, NFI=0.808, TLI=0.784) . The four exogenous latent variables of work organization, work type, upper limb work posture and individual factors were correlated. There was a strong positive correlation between job type and upper limb work posture ( r=0.865) , a moderate positive correlation between work organization and job type and upper limb work posture ( r=0.570, 0.490) , and a weak negative correlation between individual factors and the other three exogenous latent variables. Upper limb work posture and individual factors had direct effects on upper limb WMSDs, and the effect coefficients were 0.10 and 0.06, respectively. Upper limb fatigue played a mediating role between work organization, work type, upper limb work posture and upper limb WMSDs. The effect coefficient was 0.46, and the composition ratios of indirect effects were 100.0%, 100.0%, and 38.3%, respectively. The direct path effect of upper limb work posture, individual factors and upper limb WMSDs was weaker than the mediating path through upper limb fatigue. Conclusion:When carrying out the prevention and control of upper limbWMSDs, it is necessary to comprehensively consider the pathogenesis path of upper limb muscle fatigue and upper limb WMSDs caused by work organization, work type, and upper limb work posture, so as to provide theoretical reference for improving the prevention and control level of such diseases.