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.

AIM:To compare the effects and effi-cacy of methoxyamine and norepinephrine on sub-lingual microcirculation in patients with high-alti-tude septic shock.METHODS:A total of forty-six patients with septic shock from the Xining area of Qinghai Province were randomized into two groups:the M-hydroxylamine group(Group M,n=22)and the norepinephrine group(Group N,n=24).Baseline data as well as the incidence of acute kid-ney injury(AKI),distal limb necrosis/ischemia,and 28-day survival rates were documented.Sublingual microcirculation parameters were monitored and recorded at four time points:before treatment(T0),24 hours post-treatment(T1),48 hours post-treatment(T2),and 72 hours post-treatment(T3)using side-flow dark-field(SDF)imaging.The pa-rameters included total vascular density(TVD),per-fused vascular density(PVD),perfused vessel ratio(PPV),microvascular flow index(MFI),and hetero-geneity index(HI).Additionally,changes in heart rate(HR),mean arterial pressure(MAP),blood urea nitrogen(BUN),serum creatinine(Cr),and lac-tate(Lac)levels were also recorded.RESULTS:There were no significant differences between the two groups in terms of baseline data,incidence of AKI,distal limb necrosis/ischemia,and 28-day sur-vival rate.Additionally,there were no significant differences in HR,MAP,and Lac indices between the two groups at any time point.However,within-group comparisons showed that HR,MAP,and Lac indices improved over the course of treatment,with statistically significant differences.The BUN level at the T3 time point was significantly lower in the mehydroxyamine group compared to the nor-epinephrine group(P<0.05).Similarly,Cr levels were significantly lower at both T2 and T3 time points(P<0.05).There were no significant differenc-es in sublingual microcirculation indices between the two groups at any time point.However,TVD,PVD,PPV,MIF,and HI in both groups improved over the course of treatment,with statistically sig-nificant differences.CONCLUSION:Both methoxy-amine and norepinephrine can enhance both the macrocirculation and sublingual microcirculation in patients with septic shock at high altitudes.Addi-tionally,methoxyamine leads to a faster recovery of creatinine levels within 72 hours.

Osteoporosis is a prevalent skeletal condition characterized by reduced bone mass and strength, leading to increased fragility. Buqi-Tongluo (BQTL) decoction, a traditional Chinese medicine (TCM) prescription, has yet to be fully evaluated for its potential in treating bone diseases such as osteoporosis. To investigate the mechanism by which BQTL decoction inhibits osteoclast differentiation in vitro and validate these findings through in vivo experiments. We employed MTS assays to assess the potential proliferative or toxic effects of BQTL on bone marrow macrophages (BMMs) at various concentrations. TRAcP experiments were conducted to examine BQTL's impact on osteoclast differentiation. RT-PCR and Western blot analyses were utilized to evaluate the relative expression levels of osteoclast-specific genes and proteins under BQTL stimulation. Finally, in vivo experiments were performed using an osteoporosis model to further validate the in vitro findings. This study revealed that BQTL suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and osteoclast resorption activity in vitro in a dose-dependent manner without observable cytotoxicity. The inhibitory effects of BQTL on osteoclast formation and function were attributed to the downregulation of NFATc1 and c-fos activity, primarily through attenuation of the MAPK, NF-κB, and Calcineurin signaling pathways. BQTL's inhibitory capacity was further examined in vivo using an ovariectomized (OVX) rat model, demonstrating a strong protective effect against bone loss. BQTL may serve as an effective therapeutic TCM for the treatment of postmenopausal osteoporosis and the alleviation of bone loss induced by estrogen deficiency and related conditions.
Animals ; NFATC Transcription Factors/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Ovariectomy ; Osteoclasts/metabolism* ; Female ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; NF-kappa B/genetics* ; Osteoporosis/genetics* ; Signal Transduction/drug effects* ; Bone Resorption/genetics* ; Cell Differentiation/drug effects* ; Humans ; RANK Ligand/metabolism* ; Mitogen-Activated Protein Kinases/genetics* ; Transcription Factors

Objective:To explore the feasibility of applying deep learning image reconstruction (DLIR) in low-radiation dose liver dual-energy CT to further improve image quality, diagnostic confidence of lesion, and accuracy of iodine concentration (IC) measurement.Methods:This prospective cohort study enrolled 60 patients scheduled for enhanced liver CT at the First Affiliated Hospital of Anhui Medical University from June 2023 to January 2024. The participants were randomly assigned into the standard dose group and low radiation dose group with 30 cases in each using randomized block method. The standard radiation dose group underwent standard-radiation dose 120 kVp scans during the venous phase, while the low radiation dose group underwent low radiation dose scans with a rapid kVp-switching spectral scanning mode at 80 kVp and 140 kVp. The effective radiation dose (ED) was calculated for both groups. The standard radiation dose group was reconstructed using adaptive statistical iterative reconstruction-V (ASIR-V) algorithm 40% (AR40 120 kVp). The low radiation dose group using high-intensity DLIR (DLIR-H) to reconstructed 40 keV and 50 keV virtual monoenergetic images (VMI) (DH-VMI 40 keV, DH-VMI 50 keV). The image quality of the above three groups was objectively evaluated through the measurement of image noise and calculation of contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) for the liver and portal vein; and the image quality was subjectively scored for image noise, contrast, lesion conspicuity, and diagnostic confidence. In the low radiation dose group, DLIR-H and ASIR-V40% reconstructed iodine maps were used to measure the liver and portal vein of IC values, standard deviations (SD), and coefficients of variation (CV). One-way analysis of variance or Kruskal-Wallis H test was used to compare the differences of subjective and objective image quality among the three groups, and paired t-test was used to compare the differences in measurement indexes between DLIR-H and ASIR-V40% reconstructed iodine maps. Results:The ED in the low radiation dose group [(2.2±0.5) mSv] was reduced by 56.8% compared to the conventional radiation dose group [(5.4±1.4) mSv]. Objective evaluations demonstrated that DH-VMI 40 keV had higher image noise, CNR, and SNR for liver and portal veins compared to AR40 120 kVp ( P<0.001). DH-VMI 50 keV had lower image noise ( P=0.200), with higher CNR and SNR for the liver and portal vein compared to AR40 120 kVp( P<0.001). In subjective evaluation, there was no statistically significant difference in image noise scores between DH-VMI 40 keV and AR40 120 kVp ( P>0.05), while the image noise score for DH-VMI 50 keV was lower than that of AR40 120 kVp ( P<0.05). Both DH-VMI 40 keV and DH-VMI 50 keV had higher scores for contrast, lesion conspicuity, and diagnostic confidence compared to those of AR40 120 kVp ( P<0.05). In the low radiation dose group, there was no statistically significant difference in IC values for the liver and portal vein between the ASIR-V40% and DLIR-H algorithm reconstructed iodine maps ( P>0.05). The SD and CV of liver and portal vein in the DLIR-H reconstructed iodine maps were lower than those in the ASIR-V40% reconstructed iodine maps ( P<0.001). Conclusions:DLIR can effectively reduce the image noise of low-energy (40, 50 keV) VMI, enhance lesion conspicuity and diagnostic confidence, and improve measurement accuracy without affecting IC values.

Objective:To analyze the factors related to spontaneous re-eruption after intruded injury in permanent anterior teeth in children and adolescents.Methods:Clinical data from 5-to 17-year-old pa-tients who sustained intrusive luxation of permanent anterior teeth and treated in the Department of Pedia-tric Dentistry of Peking University School and Hospital of Stomatology from June 2015 to August 2024 were reviewed.Information of age,gender,degree of intrusion,direction of intrusion,tooth develop-ment,concomitant injuries,luxation and post-osteoclastic eruption of the adjacent teeth were recorded.The patients were divided into two groups based on whether they showed spontaneous re-eruption during advised observation after intrusion.Univariate and multifactor analysis were performed using Logistic re-gression.Results:Data from 170 teeth in 139 patients whose age ranging from 5.3-16.3 years[mean age(9.0±2.1)years]were examined.A gender disparity was observed among the patients,with 84 be-ing male and 55 being female.Among the 170 teeth,112 were categorized as successfully spontaneous re-eruption during advised observation after intrusion,while 58 were not.In terms of the degree of intru-sion,45 teeth(26.47％)had intrusion less than 3 mm,102 teeth(60.00％)experienced intrusion be-tween 3-7 mm,and 23 teeth(13.53％)were faced with intrusion exceeding 7 mm.As for the direction of intrusion,117 teeth(68.82％)were straight intrusion while mesial-distal and buccal-lingual intrusion respectively accounting for 17(10.00％)and 23(13.53％).Multivariate Logistic regression analysis showed that mesial-distal intrusion(OR=0.167,95％CI:0.031-0.9048,P=0.038),intrusion of＞7 mm(OR=0.065,95％CI:0.014-0.299,P＜0.001)and luxation of adjacent teeth(OR=0.369,95％CI:0.144-0.944,P=0.037)were independent risk factors for spontaneous re-eruption of trau-matically intruded permanent anterior teeth in children and adolescents during advised observation after intrusion,while intrusion of＜3 mm(OR=9.860,95％CI:2.430-40.009,P=0.001)and post-osteoclastic eruption of adjacent teeth(OR=4.712,95％CI:1.528-14.531,P=0.007)were inde-pendent protective factors.The possibility of spontaneous re-eruption in permanent anterior teeth during advised observation after intrusion was decreased by 61.1％with the increase of root development using Cvek's classification(OR=0.611,95％CI:0.408-0.914,P=0.017).Age(OR=1.077,95％CI:0.763-1.521,P=0.673)and laceration of gingival(OR=0.865,95％CI:0.290-2.578,P=0.794)didn't significantly affect the spontaneous re-eruption during advised observation after intrusion.Conclusion:In this study,mesial-distal intrusion,intrusion of＞7 mm and luxation of adjacent teeth were independent risk factors for spontaneous re-eruption of traumatically intruded permanent anterior teeth in children and adolescents during advised observation,while intrusion of＜3 mm and post-osteoclastic eruption of adjacent teeth were served as independent protective factors.

Objective:To analyze the epidemic trend of influenza-like illness（ILI）cases，etiological detection results，and the evolution of the hemagglutinin（HA）gene of the predominant strains in Hanzhong City，Shaanxi Province during the 2018-2024 influenza seasons.Methods:ILI sentinel surveillance data and network laboratory test results during the 2018-2024 influenza seasons in Hanzhong City，Shaanxi Province were collected for descriptive analysis. The HA gene sequences of 25 predominant strains were obtained through whole-genome deep sequencing method，and then compared with the vaccine strains recommended by the World Health Organization in the same period to analyze the evolution of the virus.Results:A total of 37 770 cases of ILI were reported in Hanzhong City during the 2018-2024 influenza seasons，and the proportion of total ILI cases（ILI%）was 2.87%（37 770/1 316 009）. The epidemic trend of ILI showed an obvious epidemic peak in winter and spring（from December of the current year to March of the following year）. The specimens with the highest positive rate were of type A（H3N2）（39.12%，365/933），and the predominant epidemic strains in each influenza season alternated among A（H1N1）pdm09（in the 2018-2019 and 2022-2023 influenza seasons），A（H3N2）（in the 2019-2020 and 2023-2024 influenza seasons）and B（Victoria）（in the 2021-2022 influenza season）. The phylogenetic relationship gradually became more distant over time across different influenza seasons. Among them，the epidemic strains of A（H1N1）pdm09 belonged to the 6B.1 clade，and the evolution mainly occurred in the Sa and Sb regions of the HA epitope. Meanwhile，the epidemic strains of A（H3N2）belonged to the 3C clade，and the evolution mainly took place in the A，B and C regions of the HA epitope. The strains of the B（Victoria）lineage belonged to the V1a.3a.2 clade，and the evolution mainly occurred in the 120-loop，150-loop，and 190-helix regions of the HA epitope.Conclusions:The influenza epidemic in Hanzhong City has obvious seasonality，and the amino acids of the epidemic strains have shown a certain degree of variation over the years. In future prevention and control work，influenza surveillance should be continuously strengthened，and the change trend of the predominant circulating strains should be closely monitored.

BACKGROUND:A novel aggregation induced luminescence fluorescence probe based on the mechanism of intramolecular motility restriction can be used for the detection of disease markers,tumor diagnosis,and bacterial imaging recognition.OBJECTIVE:To prepare a near-infrared Ⅱ nanoprobe called FA-DSPE-PEG-AIE@NPs based on aggregation-induced luminescence,and to explore its potential of targeted fluorescence imaging and photothermal therapy for prostate cancer.METHODS:Lecithin,polyethylene glycol phospholipids,folate polyethylene glycol phospholipids,and aggregation induced luminescent molecule 2TT-oC26B were used as raw materials.The folate-targeted nanoprobe FA-DSPE-PEG-AIE@NPs were prepared by nanoprecipitation method,and basic characterization of the nanoprobe was detected.PC3 human prostate cancer cells and human umbilical vein endothelial cells were selected as experimental objects.The cytotoxicity and phototoxicity of FA-DSPE-PEG-AIE@NPs were detected.PC3 human prostate cancer cells were selected as the experimental objects.Flow cytometry and calcein/propidium iodide staining were used to assess the efficacy of photothermal therapy.PC3 human prostate cancer cells were injected subcutaneously into the abdomen of BALB/C nude mice to establish a tumor model,and nanoprobes FA-DSPE-PEG-AIE@NPs were injected into the tail vein.The mice were immediately subjected to near-infraced Ⅱ fluorescence imaging.12 hours later,the tumor was irradiated by laser for 5 minutes,and the photothermal treatment effect was observed within 14 days.RESULTS AND CONCLUSION:(1)The nanoprobes FA-DSPE-PEG-AIE@NPs with a mean diameter of(171.0±0.3)nm showed a well-defined spherical morphology.The nanoprobe had a wide absorption spectrum and tail emission extending to the near-infrared Ⅱ which emitted a bright near-infrared Ⅱ fluorescence signal under laser irradiation.(2)The nanoprobes FA-DSPE-PEG-AIE@NPs had low cytotoxicity and high phototoxicity.The results of flow cytometry and calcein/propidium iodide staining showed that nanoprobes FA-DSPE-PEG-AIE@NPs had an obvious photothermal killing effect on human prostate cancer cells.(3)The nanoprobes FA-DSPE-PEG-AIE@NPs successfully achieved near-infrared Ⅱ fluorescence imaging of mouse blood vessels and the maximum enrichment time of the tumor was 12 hours.The vessel widths of the hind leg and single blood vessels of abdomen were estimated to be 0.63 mm and 0.42 mm.The tumor volume of mice was significantly smaller after 14 days of treatment.(4)The results show that nanoprobes FA-DSPE-PEG-AIE@NPs can achieve near-infrared Ⅱ fluorescence imaging and photothermal therapy of prostate cancer effectively,which may provide a new method for early diagnosis and combined treatment of prostate cancer.

Aim To investigate the protective effect of liraglutide(LIRA)on acetaminophen(APAP)-in-duced hepatotoxicity at the in vivo level and to reveal the underlying mechanism.Methods Forty SPF grade male C57BL/6J mice were randomly divided into the Control,LIRA(200 μg·kg-1),APAP(500 mg·kg-1),LIRA+APAP,LIRA+APAP+3-methylade-nine(3-MA,30 mg·kg-1)groups,with eight mice in each group.The mice were administered for three con-secutive days,and the materials were taken after 24 h.The general condition and body weight of mice in each group were recorded,and liver morphology was ob-served.Serum ALT and AST levels,as well as SOD ac-tivity,MDA,and GSH content in liver homogenates,were measured using biochemical assay kits.The levels of inflammatory cytokines IL-6,TNF-α,and IL-1β in serum were detected by ELISA.Liver pathological changes were assessed by HE staining,while mitochon-drial and autophagosome structures in liver tissues were observed using transmission electron microscopy.The number of PCNA-positive cells in liver tissues was e-valuated using immunohistochemical staining.The pro-tein expression levels of LC3Ⅱ,p62,Bax,Bcl-2,PC-NA,and CyclinD1 in liver tissues were determined by Western blot.Results LIRA pretreatment can im-prove the general condition of mice with acetamino-phen-induced liver injury(AILI),reduce serum ALT and AST levels,and effectively ameliorate the appear-ance and morphology of the liver as well as the patho-logical damage to liver tissue.Simultaneously,the lev-els of inflammatory cytokines IL-6,TNF-α,and IL-1βare significantly decreased;SOD activity and GSH con-tent are significantly increased,while MDA content is significantly reduced.Transmission electron microsco-py observations reveal the presence of numerous auto-phagosomes in the cytoplasm of liver tissue.Immuno-histochemical staining results indicate a significant in-crease in the number of PCNA-positive cells.Further-more,the expression of LC3Ⅱ,Bcl-2,PCNA,and Cy-clinD1 proteins in liver tissue is significantly upregulat-ed,while the expression of p62 and Bax proteins is significantly downregulated.However,after interven-tion with the autophagy inhibitor 3-MA,the aforemen-tioned protective effects of LIRA are significantly.Conclusions LIRA pretreatment can significantly im-prove liver injury in AILI mice.Its protective mecha-nism may be related to enhancing autophagy in hepato-cytes,thereby reducing oxidative stress,inflammatory response and apoptosis in liver of AILI mice.

Guidelines for Digital Ancient Books of TCM Indexing(T/CIATCM 119-2024)is based on the theoretical knowledge,disciplinary methods,and practical applications of TCM classical cataloging.Taking digital ancient books of TCM as the object,it systematically reveals the content of TCM knowledge,which is an essential indexing processing standard for building an intelligent retrieval system for TCM ancient books,and can provide support for the deep development and innovative utilization of TCM knowledge.It can not only promote the co-construction and sharing of ancient book resources in the TCM industry,but also promote the standardization construction and application of TCM information.This standard specifies the principles,methods,and examples of free indexing of digital ancient books of TCM based on their original content.It is applicable to the indexing and processing of digital ancient books of TCM for TCM professional libraries and related institutions,and to the data processing and construction of various types of TCM ancient book databases.

AIM:To compare the effects and effi-cacy of methoxyamine and norepinephrine on sub-lingual microcirculation in patients with high-alti-tude septic shock.METHODS:A total of forty-six patients with septic shock from the Xining area of Qinghai Province were randomized into two groups:the M-hydroxylamine group(Group M,n=22)and the norepinephrine group(Group N,n=24).Baseline data as well as the incidence of acute kid-ney injury(AKI),distal limb necrosis/ischemia,and 28-day survival rates were documented.Sublingual microcirculation parameters were monitored and recorded at four time points:before treatment(T0),24 hours post-treatment(T1),48 hours post-treatment(T2),and 72 hours post-treatment(T3)using side-flow dark-field(SDF)imaging.The pa-rameters included total vascular density(TVD),per-fused vascular density(PVD),perfused vessel ratio(PPV),microvascular flow index(MFI),and hetero-geneity index(HI).Additionally,changes in heart rate(HR),mean arterial pressure(MAP),blood urea nitrogen(BUN),serum creatinine(Cr),and lac-tate(Lac)levels were also recorded.RESULTS:There were no significant differences between the two groups in terms of baseline data,incidence of AKI,distal limb necrosis/ischemia,and 28-day sur-vival rate.Additionally,there were no significant differences in HR,MAP,and Lac indices between the two groups at any time point.However,within-group comparisons showed that HR,MAP,and Lac indices improved over the course of treatment,with statistically significant differences.The BUN level at the T3 time point was significantly lower in the mehydroxyamine group compared to the nor-epinephrine group(P<0.05).Similarly,Cr levels were significantly lower at both T2 and T3 time points(P<0.05).There were no significant differenc-es in sublingual microcirculation indices between the two groups at any time point.However,TVD,PVD,PPV,MIF,and HI in both groups improved over the course of treatment,with statistically sig-nificant differences.CONCLUSION:Both methoxy-amine and norepinephrine can enhance both the macrocirculation and sublingual microcirculation in patients with septic shock at high altitudes.Addi-tionally,methoxyamine leads to a faster recovery of creatinine levels within 72 hours.