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.

Tacit knowledge of tongue inspection is content that affects diagnostic results but is difficult to be explicit. Its cognitive characteristics are rooted in xiang thinking and embodiment. It constructs multidimensional associations between tongue manifestations and disease mechanisms through imagery mapping， and it relies on the practitioner's bodily perception， clinical context， and long-term practice for continuous internalization. In response to the neglect of tacit transmission and individual cognitive processes in current traditional Chinese medicine （TCM） tacit knowledge research， an empirical research framework integrating cognitive neuroscience is proposed. Specifically， event-related potential （ERP） and eye tracking （ET） technologies can be used to capture the brain electrical activity and eye movement trajectories of experts and novices in different tongue diagnosis cognitive tasks. By integrating multimodal cognitive data such as behaviorism， ERP components， and ET indicators， the implicit cognitive advantages of experts in the perception， mapping， and decision-making stages of tongue diagnosis are presented， and the possibility of achieving tacit knowledge transformation through visual cognitive transfer training is explored. Exploring empirical pathways to uncover the cognitive mechanisms of tongue diagnosis tacit knowledge is of great significance for the inheritance of traditional Chinese medicine.

Objective To investigate the regulatory role and underlying mechanism of the long non-coding RNA (LncRNA) LINC01430 in the inhibitory effect of sanguinarine on non-small cell lung cancer (NSCLC). Methods Differentially expressed LncRNAs and coexpressed target genes in A549 and H1975 cells before and after sanguinarine treatment were identified through high-throughput lncRNA sequencing. LINC01430 expression and its prognostic value were analyzed by using the TCGA database and experimental validation. An LINC01430-overexpressing A549 cell model was established, and CCK-8, TUNEL, RT-qPCR, Western blot analysis, and dual-luciferase reporter assays were performed to evaluate the influence of LINC01430 overexpression on sanguinarine-mediated cell proliferation, apoptosis and NOX4/IL-6 expression. Results A total of 361 differentially expressed LncRNAs, among which LINC01430 was significantly downregulated (P<0.001), were identified after sanguinarine treatment. Pathway enrichment analysis revealed pathways associated with oxidative stress and inflammation, and the mRNA and protein levels of the target genes NOX4 and IL-6 were markedly reduced (P<0.05, P<0.01). LINC01430 exhibited high expression in NSCLC tissues and cell lines (P<0.05), with a trend toward poor prognosis (HR=1.15). The overexpression of LINC01430 significantly attenuated the inhibitory effect of sanguinarine on cell proliferation and the promoting effect on apoptosis in NSCLC cells (P<0.01) and partially reversed the downregulation of NOX4 and IL-6 induced by sanguinarine (P<0.05). LINC01430 directly activated IL-6 promoter transcriptional activity (P<0.05). Meanwhile, its effect on the NOX4 promoter showed an upward trend without statistical significance. Conclusion Sanguinarine may suppress NSCLC by downregulating LINC01430 to inhibit NOX4/IL-6 signaling. LINC01430 represents a promising target for NSCLC therapy.

Based on the theory of "spleen functions as wei （卫） qi"， this paper discusses the mechanism of "inflammation-metabolism-immunity" regulation in colorectal cancer cachexia and the therapeutic strategy. It is proposed that spleen deficiency with failure in transportation， leading to the maldistribution of wei （卫） qi and pathogenic constraint transforming into fire， is the source of inflammation. Spleen deficiency with failure in transportation， leading to the maldistribution of subtle essences and loss of nourishment to the physical body， is the root of metabolic depletion. Spleen deficiency with failure in transportation， leading to wei qi depletion and healthy qi decline， is the cause of immune impairment. Accordingly， a spleen-centered strategy of fortifying the spleen and restoring its defensive function is established. The specific therapeutic approaches include fortifying the spleen and boosting qi， restoring defensive function and securing the exterior to replenish the source of wei qi， with modified Sijunzi Decoction （四君子汤）， fortifying the spleen and removing toxin， resolving phlegm and stasis to restrain exuberant internal toxin and constrained fire， with modified Baitouweng Decoction （白头翁汤）， activating the spleen and harmonizing the stomach， promoting the transportation and distribution of food essence to correct nutritional and metabolic disorders， with modified Xiangsha Liujunzi Decoction （香砂六君子汤）， and fortifying the spleen and nourishing the kidney， replenishing primordial qi and consolidating the root to restore immune competence， with Buzhong Yiqi Decoction （补中益气汤） and Zuogui Beverage （左归饮） as the basic prescription.

Based on the theory of "spleen functions as wei （卫） qi"， this paper discusses the mechanism of "inflammation-metabolism-immunity" regulation in colorectal cancer cachexia and the therapeutic strategy. It is proposed that spleen deficiency with failure in transportation， leading to the maldistribution of wei （卫） qi and pathogenic constraint transforming into fire， is the source of inflammation. Spleen deficiency with failure in transportation， leading to the maldistribution of subtle essences and loss of nourishment to the physical body， is the root of metabolic depletion. Spleen deficiency with failure in transportation， leading to wei qi depletion and healthy qi decline， is the cause of immune impairment. Accordingly， a spleen-centered strategy of fortifying the spleen and restoring its defensive function is established. The specific therapeutic approaches include fortifying the spleen and boosting qi， restoring defensive function and securing the exterior to replenish the source of wei qi， with modified Sijunzi Decoction （四君子汤）， fortifying the spleen and removing toxin， resolving phlegm and stasis to restrain exuberant internal toxin and constrained fire， with modified Baitouweng Decoction （白头翁汤）， activating the spleen and harmonizing the stomach， promoting the transportation and distribution of food essence to correct nutritional and metabolic disorders， with modified Xiangsha Liujunzi Decoction （香砂六君子汤）， and fortifying the spleen and nourishing the kidney， replenishing primordial qi and consolidating the root to restore immune competence， with Buzhong Yiqi Decoction （补中益气汤） and Zuogui Beverage （左归饮） as the basic prescription.

GPR126, also known as ADGRG6, is one of the most deeply studied aGPCRs. Initially, GPR126 was thought to be a receptor associated with muscle development and was primarily expressed in the muscular and skeletal systems. With the deepening of research, it was found that GPR126 is expressed in multiple mammalian tissues and organs, and is involved in many biological processes such as embryonic development, nervous system development, and extracellular matrix interactions. Compared with other aGPCRs proteins, GPR126 has a longer N-terminal domain, which can bind to ligands one-to-one and one-to-many. Its N-terminus contains five domains, a CUB (complement C1r/C1s, Uegf, Bmp1) domain, a PTX (Pentraxin) domain, a SEA (Sperm protein, Enterokinase, and Agrin) domain, a hormone binding (HormR) domain, and a conserved GAIN domain. The GAIN domain has a self-shearing function, which is essential for the maturation, stability, transport and function of aGPCRs. Different SEA domains constitute different GPR126 isomers, which can regulate the activation and closure of downstream signaling pathways through conformational changes. GPR126 has a typical aGPCRs seven-transmembrane helical structure, which can be coupled to Gs and Gi, causing cAMP to up- or down-regulation, mediating transmembrane signaling and participating in the regulation of cell proliferation, differentiation and migration. GPR126 is activated in a tethered-stalk peptide agonism or orthosteric agonism, which is mainly manifested by self-proteolysis or conformational changes in the GAIN domain, which mediates the rapid activation or closure of downstream pathways by tethered agonists. In addition to the tethered short stem peptide activation mode, GPR126 also has another allosteric agonism or tunable agonism mode, which is specifically expressed as the GAIN domain does not have self-shearing function in the physiological state, NTF and CTF always maintain the binding state, and the NTF binds to the ligand to cause conformational changes of the receptor, which somehow transmits signals to the GAIN domain in a spatial structure. The GAIN domain can cause the 7TM domain to produce an activated or inhibited signal for signal transduction, For example, type IV collagen interacts with the CUB and PTX domains of GPR126 to activate GPR126 downstream signal transduction. GPR126 has homology of 51.6%-86.9% among different species, with 10 conserved regions between different species, which can be traced back to the oldest metazoans as well as unicellular animals.In terms of diseases, GPR126 dysfunction involves the pathological process of bone, myelin, embryo and other related diseases, and is also closely related to the occurrence and development of malignant tumors such as breast cancer and colon cancer. However, the biological function of GPR126 in various diseases and its potential as a therapeutic target still needs further research. This paper focuses on the structure, interspecies differences and conservatism, signal transduction and biological functions of GPR126, which provides ideas and references for future research on GPR126.

BACKGROUND:Osteoporotic fracture is the most serious complication of osteoporosis.Previous studies have demonstrated that gut microbiota has a regulatory effect on skeletal tissue and that gut microbiota has an important relationship with osteoporotic fracture,but the causal relationship between the two is unclear. OBJECTIVE:To explore the causal relationship between gut microbiota and osteoporotic fractures using Mendelian randomization method. METHODS:The genome-wide association study(GWAS)datasets of gut microbiota and osteoporotic fracture were obtained from the IEU Open GWAS database and the Finnish database R9,respectively.Using gut microbiota as the exposure factor and osteoporotic fracture as the outcome variable,Mendelian randomization analyses with random-effects inverse variance weighted,MR-Egger regression,weighted median,simple model,and weighted model methods were performed to assess whether there is a causal relationship between gut microbiota and osteoporotic fracture.Sensitivity analyses were performed to test the reliability and robustness of the results.Reverse Mendelian randomization analyses were performed to further validate the causal relationship identified in the forward Mendelian randomization analyses. RESULTS AND CONCLUSION:The results of this Mendelian randomization analysis indicated a causal relationship between gut microbiota and osteoporotic fracture.Elevated abundance of Actinomycetales[odds ratio(OR)=1.562,95%confidence interval(CI):1.027-2.375,P=0.037),Actinomycetaceae(OR=1.561,95%CI:1.027-2.374,P=0.037),Actinomyces(OR=1.544,95%CI:1.130-2.110,P=0.006),Butyricicoccus(OR=1.781,95%CI:1.194-2.657,P=0.005),Coprococcus 2(OR=1.550,95%CI:1.068-2.251,P=0.021),Family ⅩⅢ UCG-001(OR=1.473,95%CI:1.001-2.168,P=0.049),Methanobrevibacter(OR=1.274,95%CI:1.001-1.621,P=0.049),and Roseburia(OR=1.429,95%CI:1.015-2.013,P=0.041)would increase the risk of osteoporotic fractures in patients.Elevated abundance of Bacteroidia(OR=0.660,95%CI:0.455-0.959,P=0.029),Bacteroidales(OR=0.660,95%CI:0.455-0.959,P=0.029),Christensenellacea(OR=0.725,95%CI:0.529-0.995,P=0.047),Ruminococcaceae(OR=0.643,95%CI:0.443-0.933,P=0.020),Enterorhabdus(OR=0.558,95%CI:0.395-0.788,P=0.001),Eubacterium rectale group(OR=0.631,95%CI:0.435-0.916,P=0.016),Lachnospiraceae UCG008(OR=0.738,95%CI:0.546-0.998,P=0.048),and Ruminiclostridium 9(OR=0.492,95%CI:0.324-0.746,P=0.001)would reduce the risk of osteoporotic fractures in patients.We identified 16 gut microbiota associated with osteoporotic fracture by the Mendelian randomization method.That is,using gut microbiota as the exposure factor and osteoporotic fracture as the outcome variable,eight gut microbiota showed positive causal associations with osteoporotic fracture and another eight gut microbiota showed negative causal associations with osteoporotic fracture.The results of this study not only identify new biomarkers for the early prediction of osteoporotic fracture and potential therapeutic targets in clinical practice,but also provide an experimental basis and theoretical basis for the study of improving the occurrence and prognosis of osteoporotic fracture through gut microbiota in bone tissue engineering.

Objective To investigate the risk factors for pneumonia complicating infectious mononucleosis(IM)in adults and construct a nomogram prediction model.Methods A retrospective analysis was conducted on 198 IM patients admitted to the Second Affiliated Hospital of Air Force Medical University from January 2015 to December 2021.Patients were divided into pneumonia group(n=52)and non-pneumonia group(n=146)based on whether pulmonary infection occurred during hospitalization.The baseline data(age,gender,place of onset,etc.),clinical manifestations(maximum body temperature,lymph node enlargement,splenomegaly,etc.),and inflammatory indicators[white blood cell count(WBC),C-reactive protein(CRP),etc.]were compared between the two groups.Kaplan-Meier curves were plotted to analyze the key indicators affecting the hospital stay of IM patients.Multivariate logistic regression was used to analyze the independent risk factors for pneumonia complicating IM in adults and construct a nomogram prediction model based on the identified risk factors.The predictive efficacy of the model was evaluated using the receiver operating characteristic(ROC)curve and the consistency of the model was assessed using the calibration curve.The fit of the model was evaluated using the Hosmer-Lemeshow test.Additionally,the sensitivity,specificity,and accuracy of the model were assessed using confusion matrix.Results Compared with non-pneumonia group,the pneumonia group had a significantly higher proportion of patients from rural areas,with body mass index(BMI)≥24 kg/m2,smoking history,hepatomegaly,fever duration of≥7 d,as well as increased total hospitalization costs and average daily hospitalization costs,and prolonged hospital stay(P<0.05).The proportion of patients with a history of antibiotic use was lower in the pneumonia group(P<0.05).Kaplan-Meier survival analysis showed that patients from rural areas,with BMI≥24 kg/m2,smoking history,no prophylactic use of antibiotics,fever duration≥7 d,and hepatomegaly had significantly prolonged hospital stays(P<0.05).Multivariate logistic regression analysis revealed that living in a rural area(OR=4.089,P<0.05),hepatomegaly(OR=4.082,P<0.05),and elevated WBC(OR=1.205,P<0.05)were independent risk factors for pneumonia complicating IM in adults,while the prophylactic use of antibiotics(OR=0.142,P<0.05)was an independent protective factor.The area under the ROC curve of the constructed nomogram prediction model was 0.827(95%CI 0.762-0.892),and the slope of the calibration curve was close to 1,and the Hosmer-Lemeshow test showed χ2=5.299,P=0.725,indicating good consistency and fit of the prediction model.The results of the confusion matrix assessment showed that the sensitivity of the model was 0.669(0.624-0.773),the specificity was 0.827(0.724-0.930),and the accuracy was 0.732(0.665-0.793).Conclusion The nomogram prediction model based on place of onset,hepatomegaly,the prophylactic use of antibiotics and WBC has excellent fit and discrimination,providing an effective quantitative tool for prognosis assessment of IM.

BackgroundGraduate students frequently face life events， many of which may adversely affect their mental well-being. However， the interaction between life events and the development of depression， anxiety， and somatic symptoms remains unclear. ObjectiveTo explore the relationship between life events and the development of depressive， anxiety and somatic symptoms in graduate students， thereby informing prevention strategies for these conditions. MethodsA sample of 6 722 newly enrolled graduate students at a comprehensive university in Southwest China from September to November 2018 was selected. The assessment was conducted using the Adolescent Self-rating Life Events Checklist （ASLEC）， the 7-item Generalized Anxiety Disorder scale-7 item （GAD-7）， the Patient Health Questionnaire Depression Scale-9 item （PHQ-9）， and the Patient Health Questionnaire-15 （PHQ-15）. Network analysis was implemented by using the bootnet and qgraph packages in the R software （version 4.2.3）， with centrality indices calculated to identify core and bridge symptoms within the network. ResultsThe study encompassed a total of 6 171 graduate students， representing 91.80% of the target population. The prevalence rates of anxiety， depressive， and somatic symptoms among graduate students were 12.59% （777/6 171）， 16.63% （1 026/6 171）， and 27.66% （1 707/6 171）， respectively. Network analysis revealed that 'academic stress' was the core symptom with the highest strength and expected influence （both values=1.207）， while 'feeling down， depressed， or hopeless' was the bridge symptom with the highest bridge strength and bridge expected influence （both values=0.454）. There was no significant difference in global network strength and edge weight between women and men （P>0.05）. ConclusionAcademic stress， emerging as the core symptom， assumes a dominant position within the symptom network and exhibits strong interactions with other negative affective states. There was no gender difference in the network structure.