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”.

Post-orgasmic illness syndrome (POIS) is a rare condition characterized by the rapid onset of extreme fatigue, flu-like symptoms, difficulty concentrating, depression, nasal congestion, rhinorrhea, itchy eyes, and other physical and psychological discomforts following ejaculation. This report presents the outcomes of two patients with POIS who underwent a two-year course of autologous seminal plasma subcutaneous cluster immunotherapy. Treatment efficacy was assessed using methods such as the symptom Visual Analogue Scale (VAS), the Union Physio-Psycho-Social Assessment Questionnaire (UPPSAQ)-70, and the Short Form 36 Health Survey (SF-36). The results suggest that autologous seminal plasma subcutaneous cluster immunother-apy may be a safe and effective therapeutic approach for POIS.

AIM To evaluate the quality consistency of Tongmai Granules,Tongmai Tablets,Tongmai Capsules and Tongmai Oral Liquid.METHODS The HPLC fingerprints were established,after which the contents of danshensu,protocatechuic aldehyde,3'-hydroxy puerarin,puerarin,puerarin apioside,daidzin,ferulic acid,salvianolic acid B and salvianolic acid A were determined,and cluster analysis and principal component analysis were adopted in the quality analysis from the perspective of daily intake.RESULTS There were 21 common peaks in the fingerprints for 39 batches of samples with the similarities of 0.765-0.997.Various batches of samples were clustered into 5 categories,2 principal components demonstrated the accumulative variance contribution rate of 83.53％ .The daily intakes of various constituents in different dosage forms exhibited obvious differences,especially for that of salvianolic acid B,which were low in tablets and capsules,and their heterogeneities existed among the same dosage forms.CONCLUSION This simple and accurate method can provide a reference for the quality evaluation of Tongmai preparations from different manufacturers.

This study was aimed at exploring novel auxiliary diagnostic biomarkers for brucellosis and their potential miR-NA-mRNA regulatory networks.High-throughput sequencing was used to compare miRNA expression differences in serum ex-osomes between patients with brucellosis and healthy controls.Subsequently,RT-qPCR was used to validate the expression of significantly upregulated exosomal miRNAs.The diagnostic value of these miRNAs was assessed with ROC curves,and bioin-formatics analyses were performed to investigate the potential roles of the miRNAs in brucellosis infection.The ROC curve a-nalysis indicated that the area under the curve for exosomal hsa-miR-11400(P＜0.05),hsa-miR-199a-5p(P＜0.05),and hsa-miR-148a-5p(P＜0.05)was 0.79,0.81,and 0.74,respectively.A total of 465 differentially expressed miRNAs and their tar-get genes were predicted,including 25 immune-related target genes,most of which were closely associated with cancer-related proteoglycans,NF-kappa B signaling pathways,and IL-17 signaling pathways.The constructed differentially expressed gene network indicated that the immune genes PLXNA2,IL17RA,PRKCA,CD22,ACVR1B,and CBL might be regulated by hsa-miR-199a-5p and hsa-miR-148a-5p.These findings suggest that exosomal miRNAs might serve as auxiliary diagnostic indicators for brucellosis.Our exosomal miRNA-mRNA regulatory network provides new insights into the pathogenesis and treatment of brucellosis.

This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

Aim To investigate the effects of sodium lactate(NALA)on right heart failure induced by monocrotaline(MCT)-induced pulmonary arterial hy-pertension in rats and to reveal the underlying mecha-nisms.Methods Forty male Sprague-Dawley(SD)rats were randomly allocated into four groups,with ten rats in each group,namely,MCT group,NALA group,and NALA+MCT group;the MCT and NALA+MCT groups were administered a single intraperito-neal injection of MCT at 60 mg·kg-1 to induce pul-monary hypertension,and one week later,the NALA and NALA+MCT groups received intraperitoneal in-jections of NALA at 0.1 g·kg-1(once a day,for 5 weeks),while the CON and MCT groups received e-qual volumes of physiological saline(once a day,for 5 weeks);right heart function was assessed using echo-cardiography,right ventricular and pulmonary artery remodeling were evaluated via histopathological sec-tions,and the expression levels of ANP,BNP,and in-flammatory factors were measured by ELISA,along with assessments of oxidative stress levels,Western blot detection of the expression levels of proteins in the TLR4/NF-κB signaling pathway.Results Compared to the CON group,the MCT group exhibited increased RVSP and RVHI,decreased right heart function,in-creased collagen fiber deposition,and elevated oxida-tive stress and inflammatory factor expression,and the expression levels of proteins in the TLR4/NF-κB signa-ling pathway increased(P＜0.05);compared to the MCT group,the NALA+MCT group showed reduced RVSP and RVHI,improved right heart function,atten-uated pulmonary vascular remodeling,decreased ex-pression of ANP,BNP,inflammatory factors,and H2O2,along with increased antioxidant enzyme expres-sion,and the expression levels of proteins in the TLR4/NF-κB signaling pathway decreased(P＜0.05).Conclusion NALA can inhibit right ventric-ular remodeling in rats with pulmonary hypertension,and the underlying mechanism may involve the allevia-tion of inflammatory responses and oxidative stress through the inhibition of the TLR4/NF-κB signaling pathway.

Objective:Based on outpatient and emergency visit data,this study employs a staged VaDE model to uncover the heterogeneity structure of residents'healthcare-seeking behaviors and reveal cross-district mobility patterns.Methods:Outpatient and emergency visit records of permanent residents in District A,Shanghai,in 2024 were used.Each individual visit served as the analytical unit.A staged VaDE approach was applied for nonlinear dimensionality reduction and clustering,followed by feature analysis with cross-district visit rates.Results:The proposed model outperformed K-Means and VAE+K-Means in clustering performance,identifying seven typical healthcare-seeking patterns with significant differences in cross-district rates,demographic characteristics,care structures,and disease profiles.Cluster 1 comprised elderly patients with severe conditions and high cross-district rates;Cluster 2,young and middle-aged cross-district commuters;Cluster 3,mild-case or maternity-oriented visits;Cluster 4,infrequent visits by middle-aged and elderly patients;and Clusters 5～7,local chronic disease or low-burden visits.Conclusion:The staged VaDE model demonstrates strong capability in characterizing behavioral heterogeneity and identifying key cross-district mobility types,providing evidence for refined healthcare service management and regional coordination.

Kv1.3,a voltage-gated potassium channel,is highly expressed in T lymphocytes,the nervous system,and vascular smooth muscle cells.It plays a critical role in membrane excitability and electrical signal transduction,serving as an important target for studying T-cell function and providing a promising direction for developing therapeutics against autoimmune and inflammatory diseases.Therefore,the de-velopment of specific inhibitors of Kv1.3 channel has emerged as a novel therapeutic strategy for these disorders.In this study,we isolated and purified a novel Kv1.3-inhibitory peptide toxin,LmKTx13,from the venom of the scorpion Lychas mucronatus using reversed-phase high-performance liquid chroma-tography(RP-HPLC).LmKTx13 consists of 38 amino acid residues,including six cysteines that form three disulfide bonds.Whole-cell patch-clamp recordings revealed that LmKTx13 potently inhibited Kv1.3 with an IC50 of 7.92±3.0 nmol/L.Selectivity analysis showed that 2 μmol/L LmKTx13 also in-hibited Kv1.2 and Kv1.7,but exhibited no significant effects on other potassium channel subtypes or voltage-gated sodium channels.Further investigation into the mechanism demonstrated that LmKTx13 acts as a pore-blocking inhibitor of Kv1.3.By analyzing the effects of LmKTx13 on Kv1.3 channel gating ki-netics and performing sequence alignment of the pore regions of Kv1.3 and Kv1.5,we constructed site-directed mutants and identified the pore region of Kv1.3 as the critical binding site for LmKTx13.Key residues involved in the interaction included T425,G427,and H451.In summary,we discovered a no-vel pore-blocking Kv1.3 inhibitor,LmKTx13,from L.mucronatus venom,which exhibits high affinity and selectivity for Kv1.3.These findings highlight its potential as a potential lead molecule for developing Kv1.3-targeted therapeutics.

Hepatocellular carcinoma(HCC),which is essentially primary liver cancer,is closely related to CD8+T cell immune infiltration and immune suppression.We constructed a CD8+T cells related risk score model to pre-dict the prognosis of HCC patients and provided therapeutic guidance based on the risk score.Using integrated bulk RNA sequencing(RNA-seq)and single-cell RNA sequencing(scRNA-seq)datasets,we identified stable CD8+T cell signatures.Based on these signatures,a 3-gene risk score model,comprised of KLRB1,RGS2,and TN-FRSF1B was constructed.The risk score model was well validated through an independent external validation co-hort.We divided patients into high-risk and low-risk groups according to the risk score and compared the differ-ences in immune microenvironment between these two groups.Compared with low-risk patients,high-risk patients have higher M2-type macrophage content(P＜0.0001)and lower CD8+T cells infiltration(P＜0.0001).High-risk patients predict worse response to immunotherapy treatment than low-risk patients(P＜0.01).Drug sensitivity a-nalysis shows that PI3K-β inhibitor AZD6482 and TGFβRII inhibitor SB505124 may be suitable therapies for high-risk patients,while the IGF-1R inhibitor BMS-754807 or the novel pyrimidine-based anti-tumor metabolic drug Gemcitabine could be potential therapeutic choices for low-risk patients.Moreover,expression of these 3-gene mod-el was verified by immunohistochemistry.In summary,the establishment and validation of a CD8+T cell-derived risk model can more accurately predict the prognosis of HCC patients and guide the construction of personalized treatment plans.