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”.

To understand the infection status of main intestinal protozoa in BALB/c mice and pro-vide a basis for further control of intestinal protozoa infection.Five hundred and forty BALB/c mice provided by four domestic suppliers of BALB/c mice were detected for intestinal protozoa,in which 140 from supplier A,130 from supplier B,135 from supplier C,and 135 from supplier D,re-spectively.Fresh faecal samples were collected from each mouse separately to extract the genome and amplified by nested PCR based on primers for the 18S rRNA gene sequences of Pent-atrichomonas hominis(P.hominis)and Cryptosporidium tyzzeri(C.tyzzeri),and the 16S-like rRNA gene sequence of Tritrichomonas muris(T.muris)and sequenced.The results showed that the total intestinal protozoan infection rate was 7.1％(10/140)in 140 mice faecal samples provided by supplier A.Among them,the positivity rate of T.muris was 7.1％(10/140),C.tyzzeri was 2.1％(3/140),and P.hominis was 7.1％(10/140),the co-infection rate of two intestinal protozoa was 7.1％(10 mice:T.muris+P.hominis),and three intestinal protozoa was 2.1％(3 mice:T.muris+P.hominis+C.tyzzeri).The total intestinal protozoan infection rate in 135 mice faecal samples provided by supplier C was 7.4％,in which,7.4％(10/135)was positive for T.muris.There are no intestinal protozoa to be detected in 130 mice faecal samples from supplier B and 135 mice faecal samples from supplier D.The homology analysis showed that the homology of ampli-fied sequence of T.muris,P.hominis and C.tyzzeri was 98.52％,98.27％and 99.87％compared with published sequence of GenBank No:AY886846.1,GenBank No:AF156964.1 and GenBank No:KJ000486.1,which was clustered as an independent branch by phylogenetic analysis respec-tively.In conclusion,there are intestinal protozoan infection in BALB/c mice in some animal sup-pliers.The co-infections of more than 3 parasites such as T.muris,P.hominis and C.tyzzeri has been found.It will provide a basis for control of intestinal protozoa infection in BALB/c mice in the future.

This study was aimed at investigating the effects of demethylase fat mass and obesity-associated protein(FTO)and methyltransferase methyltransferase like protein 3(METTL3),key molecules in N6-methyladenosine(m6A)modification,on the replication and proliferation of Japanese encephalitis virus(JEV).Recombinant lentiviruses were generated by packaging the FTO and green fluorescent protein into lentiviral vectors.Neuro2a cells,a mouse neuroblastoma cell line,were infected with the lentivirus,and stable FTO-expressing cell lines were obtained through puromycin selection.Successful overexpression of FTO was confirmed through fluorescence microscopy,real-time quantitative PCR,and western blot analysis.When Neuro2a cells overexpressing FTO were infected with JEV,the overexpression of FTO decreased JEV replication in the cells,and increased the expression of interferon(IFN)and related molecules.Additionally,treatment of JEV-infected Neuro2a cells with the METTL3-specific inhibitor STM2457 resulted in a dose-dependent decrease in JEV replication and viral protein expression.These findings suggested that lowering m6A methylation levels inhibits JEV replication,thus shedding light on the regulatory role of methylation modification in JEV replication.

Objective:To investigate the clinicopathological features and differential diagnosis of male genital system lympho-ma(MGSL).Methods:We retrospectively analyzed the clinicopathological and immunophenotypic features and prognosis of 80 ca-ses of MGSL.Results:The onset age of the MGSL patients ranged from 4 to 85(median 62)years old.All the cases showed non-specificity of the imaging features and clinical manifestations.MGSL was located mainly in the testis(n=66),followed by the pros-tate(n=7),epididymis(n=3),scrotum(n=3)and penile glans(n=1).Diffused large B cell lymphoma(DLBCL)was the most common pathological type(n=62),next came extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue(MALT)(n=7)and other rare types(n=12).During the 1-112-month follow-up of 10 of the 19 patients,1 died at 1 month af-ter diagnosed with prostatic B-lymphoblastic lymphoma(B-LBL)and another 1 died at 50 months after diagnosed with testicular DLBCL.Conclusion:MGSL is rare clinically,mainly of the DLBCL type pathologically,lacking specificity in clinical symptoms and imaging manifestation.The definite diagnosis of the malignancy depends on histopathology combined with related molecular exami-nation and immunohistochemical labeling,and R-CHOP chemotherapy is the first choice for its treatment.

Objective To explore the expression of Nudix hydrolase 5(NUDT5)protein in breast cancer tissues and its correlation with ultrasound signs and clinicopathological characteristics.Methods A total of 108 patients with breast cancer admitted to our hospital from October 2019 to April 2022 were selected as the research objects.Ultrasound examination was performed before operation to observe the ultrasound signs of patients.Immunohistochemical method was used to detect the expressions of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER-2)in breast cancer tissues,and the expression levels of NUDT5 protein in breast cancer tissues and adjacent tissues,and the relationships of NUDT5 protein expression with ultrasound signs and clinicopathological characteristics of patients were analyzed.Results Among 108 patients,there were 62 cases with ER positive,60 cases with PR positive,and 28 cases with HER-2 positive.The positive expression rate of NUDT5 protein in breast cancer tissues was higher than that in adjacent tissues(P<0.05).The positive expression rate of NUDT5 protein was higher in breast cancer tissues with microcalcification,blood flow imaging grade(grade 2 to 3)and lymph node metastasis(P<0.05).The positive expression of NUDT5 protein was correlated with histological grade,ER and HER-2(P<0.05).The expression of NUDT5 protein in breast cancer tissue was positively correlated with ER negative and HER-2 positive(rs=0.463,0.398,P<0.05).Conclusion The positive expression rate of NUDT5 protein in breast cancer tissues is higher than that in adjacent tissues,and its expression has a certain correlation with microcalcification,blood flow imaging grade(grade 2 to 3),lymph node metastasis in ultrasound signs and histological grade,ER,and HER-2 in clinicopathological characteristics.

Objective:To evaluate the efficacy and safety of telitacicept in the treatment of systemic lupus erythematosus (SLE) .Methods:The clinical data of 25 SLE patients who received standard therapy combined with telitacicept at the Department of Dermatology, Xiangya Second Hospital, Central South University, from 2021 to 2024 were retrospectively collected. Baseline demographic and clinical characteristics were analyzed. Changes in skin lesions, joint pain symptoms, complete blood count, and biochemical parameters at 4, 12, and 24 weeks of treatment were compared with baseline (week 0). The Wilcoxon signed-rank test was used to compare complement C3 and C4 levels before and after treatment, and univariate logistic regression analysis was performed to explore factors influencing the efficacy of telitacicept.Results:Among the 25 SLE patients, 3 were male (12.0%) and 22 were female (88.0%). Based on the SLE Disease Activity Index (SLEDAI) -2000 scores, 8 patients were mild, 13 were moderate, and 4 were severe. Of the 11 SLE patients with rashes before treatment, 6 achieved complete remission at 12 weeks. Among the 7 patients with joint pain before treatment, 4 experienced symptom resolution at 24 weeks. The proportion of patients with leukopenia at baseline and at 4, 12, and 24 weeks was 10/25 (40.0%), 0/24 (0), 1/22 (4.5%), and 2/19 (10.5%), respectively. The proportion of patients with thrombocytopenia was 6/25 (24.0%), 3/24 (12.5%), 1/22 (4.5%), and 1/19 (5.3%), respectively, and the proportion of patients with anemia was 7/25 (28.0%), 3/24 (12.5%), 1/22 (4.5%), and 1/19 (5.3%), respectively. At baseline, 11 out of 25 patients (44.0%) had proteinuria. At 12 weeks, the urinary protein quantification level (0.4 [0, 0.6] g/L) was significantly lower than at baseline (0.9 [0.8, 1.2] g/L). The SLE responder index-4 (SRI4) response rates at 4, 12, and 24 weeks were 14/18, 15/17, and 12/14, respectively. Complement C3 and C4 levels were significantly higher at 4, 12, and 24 weeks compared to baseline (all P < 0.001). Univariate logistic regression analysis showed that age, disease duration, glucocorticoid dosage, baseline complement C4 levels, antinuclear antibody titer, and SLEDAI-2K score did not significantly affect the efficacy of telitacicept (SRI4 response rate at 12 weeks) (all P > 0.05). No serious adverse reactions related to telitacicept were observed in patients. Conclusions:Telitacicept improved skin lesions, complement C3 and C4 levels, and anti-double-stranded DNA antibody levels in SLE patients. No association was found between the efficacy of telitacicept and baseline SLEDAI-2K scores, antinuclear antibody titers, or complement C4 levels, suggesting that telitacicept is an effective and safe treatment for SLE patients.

Programmed cell death protein 1(PD-1)and its ligand,programmed cell death 1 ligand 1(PD-L1),represent a pair of prototypical immune checkpoint that plays a critical role in tumor immune evasion.However,the development of targeted therapeutics against these two proteins is limited by their low levels and high glycosylation modifications in eukaryotic cells.In this study,we designed two func-tional but truncated variants of PD-1(PD-133-150)and PD-L1(PD-L1 19-239);and subcloned them into eukaryotic expression vectors using golden gate assembly technology.Using these vectors,we achieved high level yields of these two proteins in transiently-transfected HEK-293T cells.After one-step affinity purification,the yields of PD-133-150 and PD-L119 239 proteins reached 5 mg and 3 mg per liter of cell cul-ture medium,with over 95％purity.Using biolayer interferometry and flow cytometry analysis,we deter-mined the binding kinetics,equilibrium constants,and the cellular binding activities of these proteins.Compared with the PD-1 extracellular domain expressed in insect or E.coli cells,the PD-133-150 purified from HEK-293T cells has a 24-fold and a 50-fold increase in its binding affinity to PD-L1.In addition,the dissociation rate of the binding decreased to less than l/400th of the original rate.Thus,we speculate that N-glycosylation could modulate the PD-1/PD-L1 interactions.Together,we established an effective eukaryotic expression and purification platform for functional characterization of PD-133150/PD-L119239 in-teractions,thereby providing high-quality molecular tools for PD-1/PD-L1 antibody screening and im-mune checkpoint research.

Jiaxing city is one of the pilot cities for the Long-term Care Insurance(LTCI)policy in Zhejiang Province.This study examines the current implementation status of Jiaxing's LTCI policy in terms of financing(including financing targets,channels,methods,and standards)and benefit payments(including payment forms,contents,standards,and levels).It summarizes the experiences and challenges encountered during the pilot process.Currently,Jiaxing's LTCI policy has undergone continuous improvement in financing and benefit payment mechanisms.The financing targets now cover both urban and rural residents,and the coverage has expanded from individuals with severe disabilities to include those with severe dementia.The benefit content has been refined,with the introduction of a detailed service project directory and service pricing.For the future,it is recommended to expand the coverage to individuals with moderate disabilities,based on the fund's capacity and sustainability,and to differentiate benefit standards according to the severity of disability.Furthermore,it is suggested to strengthen the responsibilities of employers and individuals,diversify financing channels,and gradually explore an independent financing mechanism for LTCI.A combination of fixed-amount and proportional financing methods should be considered,alongside factors such as intergenerational equity and population aging trends,to establish a scientific and dynamic premium adjustment mechanism.

Diabetic nephropathy(DN)is a serious complication of diabetes mellitus and a leading cause of end-stage renal diseases.In DN patients,key pathological mechanisms include proteinuria,glomerulo-sclerosis,and fibrosis,largely driven by poor glycemic control and oxidative stress caused by prolonged hyperglycemia.This stress damages renal podocytes and triggers inflammatory mesenchymal infiltration of renal tubular cells,exacerbating the progression of proteinuria and fibrosis.Human umbilical cord-de-rived mesenchymal stem cells(hUC-MSCs)offer promising potential for treating DN due to their strong anti-oxidative properties.In this study,we developed a DN mouse model and treated the mouse via tail vein injections of hUC-MSCs(1×106 cells/mouse).The results indicated that hUC-MSCs significantly lowered fasting blood glucose levels(22.5±3.0 vs 14.7±1.1,P＜0.01)and improved glucose toler-ance,as shown by intraperitoneal glucose tolerance test(IPGTT)results(P＜0.05).Additionally,the renal function improved in hUC-MSCs-treated mice,with marked reductions in oxidative stress markers,including blood urea nitrogen(BUN),urinary creatinine(Ucr),urinary protein(PRO),superoxide dismutase(SOD),and malondialdehyde(MDA)(P＜0.05).Histological analyses through hematoxy-lin-eosin(H&E),Periodic Acid-Schiff(PAS),and Sirius red staining demonstrated alleviation of glo-merular mesangial hyperplasia,glomerular hypertrophy,and tubular inflammation.Furthermore,hUC-MSCs treatment downregulated the expression of oxidative stress-related proteins,such as NADPH oxi-dase 4(NOX4)and thioredoxin-interacting protein(TXNIP),and reduced reactive oxygen species(ROS)production(P＜0.05).Meanwhile,human renal cortical proximal tubule epithelial cells(HK-2 cells)were selected for validation in vitro experiments using high glucose treatment followed by super-natants of hUC-MSCs(MSC-CM),and Western blotting showed that the expression of both NOX4 and TXNIP was inhibited(P＜0.05)and ROS expression was reduced.In conclusion,hUC-MSC treatment effectively lowered blood glucose levels and improved renal function in DN mice,likely through the sup-pression of NOX4 expression and TXNIP-mediated oxidative stress.