ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

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 This study reports on the obligatory parasitism of water mites Arrenurus sp. on Anopheles messeae at the Manzhouli Port, Inner Mongolia, China. Methods Duing July 2024, a survey on the mosquito diversity was conducted at the Manzhouli Port. Captured mosquitoes and their ectoparasites were identified to species level. Results A total of 1840 adult mosquitoes were collected, representing species from three genera: Culex(Cx. modestus, Cx. pipiens pallens), Aedes(Ae. dorsalis, Ae. flavidorsalis, Ae. flavescens), and Anopheles (An. messeae). Among all the mosqutioes specimens,3 out of 150 captured An. messeae were found to carry ectoparasitic mites, with number of 2,4,27 mites separately. Morphological and molecular identification reached the same result as water mites(Hydrachnidiae, Hydracrina). COI gene sequence showed 94% similarity with the closest species Arrenurus truncatellus. Conlusions Literature review suggests water mites are host-specific parasitism of mosquito species and herein with the first record of Arrenurus sp. parasiting on An. Messeae in the most high-latitude region globally.

Intraoperative cell salvage (IOCS) has been widely applied as an important blood conservation measure in surgical operations. However, there is currently a lack of clinical practice guidelines for the implementation of IOCS in patients with malignant tumors. This report aims to provide clinicians with recommendations on the use of IOCS in patients with malignant tumors based on the review and assessment of the existed evidence. Data were derived from databases such as PubMed, Embase, the Cochrane Library and Wanfang. The guideline development team formulated recommendations based on the quality of evidence, balance of benefits and harms, patient preferences, and health economic assessments. This study constructed seven major clinical questions. The main conclusions of this guideline are as follows: 1) Compared with no perioperative allogeneic blood transfusion (NPABT), perioperative allogeneic blood transfusion (PABT) leads to a more unfavorable prognosis in cancer patients (Recommended); 2) Compared with the transfusion of allogeneic blood or no transfusion, IOCS does not lead to a more unfavorable prognosis in cancer patients (Recommended); 3) The implementation of IOCS in cancer patients is economically feasible (Recommended); 4) Leukocyte depletion filters (LDF) should be used when implementing IOCS in cancer patients (Strongly Recommended); 5) Irradiation treatment of autologous blood to be reinfused can be used when implementing IOCS in cancer patients (Recommended); 6) A careful assessment of the condition of cancer patients (meeting indications and excluding contraindications) should be conducted before implementing IOCS (Strongly Recommended); 7) Informed consent from cancer patients should be obtained when implementing IOCS, with a thorough pre-assessment of the patient's condition and the likelihood of blood loss, adherence to standardized internally audited management procedures, meeting corresponding conditions, and obtaining corresponding qualifications (Recommended). In brief, current evidence indicates that IOCS can be implemented for some malignant tumor patients who need allogeneic blood transfusion after physician full evaluation, and LDF or irradiation should be used during the implementation process.

Objective: To conduct a scoping review on the types, construction methods and predictive performance of health literacy prediction models based on machine learning methods, so as to provide the reference for the improvement and application of such models. Methods: Publications on health literacy prediction models conducted using machine learning methods were retrieved from CNKI, Wanfang Data, VIP, PubMed and Web of Science from inception to May 1, 2024. The quality of literature was assessed using the Prediction Model Risk of Bias ASsessment Tool. Basic characteristics, modeling methods, data sources, missing value handling, predictors and predictive performance were reviewed. Results: A total of 524 publications were retrieved, and 22 publications between 2007 and 2024 were finally enrolled. Totally 48 health literacy prediction models were involved, and 25 had a high risk of bias (52.08%), with major issues focusing on missing value handling, predictor selection and model evaluation methods. Modeling methods included regression models, tree-based machine learning methods, support vector machines and neural network models. Predictors primarily encompassed factors at four aspects: individual, interpersonal, organizational and society/policy aspects, with age, educational level, economic status, health status and internet use appearing frequently. Internal validation was conducted in 14 publications, and external validation was conducted in 4 publications. Forty-two models reported the areas under the receiver operating characteristic curve, which ranged from 0.52 to 0.983, indicating good discrimination. Conclusion Health literacy prediction models based on machine learning methods perform well, but have deficiencies in risk of bias, data processing and validation.

Objective:To investigate the influencing factors of diarrhea during early enteral nutrition(EEN)therapy in severe patients with temporary ileostomy.Method:A total of 154 patients with temporary ileostomy who received EEN in the Department of General Surgery,Jinling Hospital from November 2019 to November 2023 were included in this study.All patients were divided into two groups:the diarrhea group(n=43)and the non-diarrhea group(n=111).The clinical data of the patients were retrospectively collected and analyzed,and univariate analysis was performed to compare the differences between groups.The indicators with significant differences were subjected to multivariate logistic regression analysis to determine the influencing factors of diarrhea during EEN therapy in severe patients with temporary ileostomy.Result:Among the 154 patients,43 developed diarrhea during EEN therapy,with an incidence of 27.9%.Multivariate logistic regression analysis showed that enteral nutrition(EN)infusion rate(OR=6.342,P=0.001,95%CI:2.055～19.572),antibiotics type(OR=8.342,P=0.013,95%CI:1.577～44.119),mechanical ventilation(OR=7.011,P=0.001,95%CI:2.272～21.629),EN formulation type(OR=6.497,P=0.001,95%CI:2.177～19.392),and diabetes(OR=3.321,P=0.036,95%CI:1.080～10.215)were closely associated with EN-related diarrhea in severe patients with temporary ileostomy.Conclusion:There was a high incidence of diarrhea in severe patients with temporary ileostomy who received EEN.EN infusion rate,antibiotics use,mechanical ventilation,EN formulation type and diabetes were the influencing factors for presence of EEN-related diarrhea in the patients.Our data could provide a reference for preventing EEN-related diarrhea in severe patients with temporary ileostomy after surgery.

Objective To establish a rapid,sensitive,and specific multiplex PCR detection method for the simultaneous detection of Cryptosporidium,Eimeria,and bovine parvovirus.Methods Specific primers targeting the SSU rRNA genes of Cryptosporidium and Eimeria,as well as the VP2 gene of bovine parvovirus were designed and the corresponding recombinant plasmid standards were constructed.To establish the multiplex PCR method,the reaction conditions were optimized using temperature gradient PCR and single-variable control methods.The sensitivity,specificity,reproducibility,and clinical application of the protocol were evaluated.Results The optimal annealing temperature was found to be 60.5℃,and the forward and reverse primer concentrations were determined to be 0.2 μmol/L for Eimeria,and 0.4 μmol/L for Cryptosporidium and bovine parvovirus.The assay demonstrated high sensitivity,with detection limits of 243,260,and 3 110 copies for the recombinant plasmid standards of Cryptosporidium,Eimeria,and bovine parvovirus,respectively.Specificity testing showed no cross-reactivity with ten common bovine pathogens,including Salmonella,bovine viral diarrhea virus,and bovine rotavirus.Consistent intra-and inter-batch results confirmed the strong reproducibility of the method.Clinical application to 81 diarrhea samples from various regions in the Ganzi Prefecture,Sichuan,revealed positivity rates of 18.52%(15/81)for Cryptosporidium,34.57%(28/81)for Eimeria,and 18.52%(15/81)forbovineparvovirus,withamixedinfectionrateof3.7%(3/81).Conclusions Themultiplex PCR method established in this study offers a reliable tool for differential diagnosis and epidemiological investigation of the three common diarrheal pathogens in yaks.

Objective To establish a sensitive,accurate and rapid detection method for 10 drugs and metabolites in hair with triple quadrupole tandem mass spectrometry,addressing the identification of drugs in real hair samples.Methods After cryogenic grinding and ultrasonic extraction,hair was separated by Restek Allure PFPP column(100 mm × 2.1 mm,5 μm).The mobile phase A was 20 mmol/L ammonium acetate,0.1％formic acid and 5％acetonitrile aqueous solution.The mobile phase B was acetonitrile.An electrospray ionization source was used for data acquisition in scheduled MRM mode.Results The method showed good linearity for all analytes within the validated range(R2＞0.995),with the limits of detection ranging from 0.5 to 6 pg/mg,the limits of quantification ranging from 2.5 to 10 pg/mg.Accuracy ranged from 89.1％to 114.6％,with intra-day precision ranging from 0.2％to 11.7％,inter-day precision ranging from 4.0％to 15.8％.the matrix effects ranging from 89.4％to 118.4％,the recoveries ranging from 63.6％～112.1％.Totally 20 cases were detected positive in 196 actual hair samples.Conclusion The time-scheduled scanning method exhibits high stability and sensitivity,enabling high-throughput detection,and is suitable for forensic toxicology laboratories to identify drugs in hair.

Objective To investigate the value of pre-treatment MRI features in predicting cachexia in hepatocellular carcinoma(HCC).Methods A retrospective analysis was conducted on 399 patients with hepatocellular carcinoma,recording their pre-treatment clinical and MRI data.All patients underwent MRI plain and enhanced scan,and their weight was followed up 6 months after the MRI examination.According to the diagnostic criteria for cachexia,patients were divided into cachexia group and non-cachexia group.They were randomly divided into the training set(n=279)and the validation set(n=120).Univariable and multivariable logistic regression analyses were used to screen variables associated with cachexia in hepatocellular carcinoma and to establish a predictive model.The receiver operating characteristic(ROC)curve was used to evaluate the predictive performance of different models.The DeLong test was used to compare the AUC values of different models,and the best-performing model was used to establish a predictive nomogram for cachexia in hepatocellular carcinoma.Results Multivariable logistic regression analysis showed that serum albumin<40 g/dL,serum alpha-fetoprotein>100 ng/mL,tumor diameter>5 cm,portal vein tumor thrombus,intratumoral arterial enhancement,and arterial phase peritumoral enhancement were independent predictors of cachexia in hepatocellular carcinoma.The clinical-imaging model showed the best predictive performance,with an AUC of 0.843 in the training set and 0.854 in the validation set.Conclusion The nomogram based on MRI features can predict cachexia in hepatocellular carcinoma 6 months earlier than clinical diagnosis,which has important clinical guidance significance.