ObjectiveThis study leverages structural data from antigen-antibody complexes of the influenza A virus neuraminidase (NA) protein to investigate the spatial recognition relationship between the antigenic epitopes and antibody paratopes. MethodsStructural data on NA protein antigen-antibody complexes were comprehensively collected from the SAbDab database, and processed to obtain the amino acid sequences and spatial distribution information on antigenic epitopes and corresponding antibody paratopes. Statistical analysis was conducted on the antibody sequences, frequency of use of genes, amino acid preferences, and the lengths of complementarity determining regions (CDR). Epitope hotspots for antibody binding were analyzed, and the spatial structural similarity of antibody paratopes was calculated and subjected to clustering, which allowed for a comprehensively exploration of the spatial recognition relationship between antigenic epitopes and antibodies. The specificity of antibodies targeting different antigenic epitope clusters was further validated through bio-layer interferometry (BLI) experiments. ResultsThe collected data revealed that the antigen-antibody complex structure data of influenza A virus NA protein in SAbDab database were mainly from H3N2, H7N9 and H1N1 subtypes. The hotspot regions of antigen epitopes were primarily located around the catalytic active site. The antibodies used for structural analysis were primarily derived from human and murine sources. Among murine antibodies, the most frequently used V-J gene combination was IGHV1-12*01/IGHJ2*01, while for human antibodies, the most common combination was IGHV1-69*01/IGHJ6*01. There were significant differences in the lengths and usage preferences of heavy chain CDR amino acids between antibodies that bind within the catalytic active site and those that bind to regions outside the catalytic active site. The results revealed that structurally similar antibodies could recognize the same epitopes, indicating a specific spatial recognition between antibody and antigen epitopes. Structural overlap in the binding regions was observed for antibodies with similar paratope structures, and the competitive binding of these antibodies to the epitope was confirmed through BLI experiments. ConclusionThe antigen epitopes of NA protein mainly ditributed around the catalytic active site and its surrounding loops. Spatial complementarity and electrostatic interactions play crucial roles in the recognition and binding of antibodies to antigenic epitopes in the catalytic region. There existed a spatial recognition relationship between antigens and antibodies that was independent of the uniqueness of antibody sequences, which means that antibodies with different sequences could potentially form similar local spatial structures and recognize the same epitopes.

Acupuncture, a therapeutic treatment defined as the insertion of needles into the body at specific points (ie, acupoints), has growing in popularity world-wide to treat various diseases effectively, especially acute and chronic pain. In parallel, interest in the physiological mechanisms underlying acupuncture analgesia, particularly the neural mechanisms have been increasing. Over the past decades, our understanding of how the central nervous system and peripheral nervous system process signals induced by acupuncture has developed rapidly by using electrophysiological methods. However, with the development of neuroscience, electrophysiology is being challenged by calcium imaging in view field, neuron population and visualization in vivo. Owing to the outstanding spatial resolution, the novel imaging approaches provide opportunities to enrich our knowledge about the neurophysiological mechanisms of acupuncture analgesia at subcellular, cellular, and circuit levels in combination with new labeling, genetic and circuit tracing techniques. Therefore, this review will introduce the principle and the method of calcium imaging applied to acupuncture research. We will also review the current findings in pain research using calcium imaging from in vitro to in vivo experiments and discuss the potential methodological considerations in studying acupuncture analgesia.
Calcium ; Acupuncture Therapy ; Acupuncture ; Acupuncture Analgesia/methods* ; Acupuncture Points ; Technology

Objective To establish a rapid screening method for 34 emerging contaminants in surface water by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UHPLC-Q-TOF-MS).Methods The pretreatment conditions of solid phase extraction(SPE)were op-timized by orthogonal experimental design and the surface water samples were concentrated and ex-tracted by Oasis? HLB and Oasis? MCX SPE columns in series.The extracts were separated by Kine-tex? EVO C18 column,with gradient elution of 0.1%formic acid aqueous solution and 0.1%formic acid methanol solution.Q-TOF-MS'fullscan'and'targeted MS/MS'modes were used to detect 34 emerging contaminants and to establish a database with 34 emerging contaminants precursor ion,prod-uct ion and retention times.Results The 34 emerging contaminants exhibited good linearity in the con-centration range respectively and the correlation coefficients(r)were higher than 0.97.The limit of de-tection was 0.2-10 ng/L and the recoveries were 81.2%-119.2%.The intra-day precision was 0.78%-18.70%.The method was applied to analyze multiple surface water samples and 6 emerging contaminants were detected,with a concentration range of 1.93-157.71 ng/L.Conclusion The method is simple and rapid for screening various emerging contaminants at the trace level in surface water.

Objective To study the construction of risk prediction model for postoperative recurrence of ad-vanced epithelial ovarian cancer based on serum human epididymis protein 4(HE4),platelet count/lymphocyte count ratio(PLR),relaxin(RLX),karyopherin α2(KPNA2).Methods 124 patients with advanced epithelial o-varian cancer diagnosed and treated in Suzhou Municipal Hospital(East District)from January 2016 to January 2019 were selected as the study objects,patients with advanced epithelial ovarian cancer were divided into re-currence group and the non-recurrence group based on whether they had recurred or not.The level of HE4 was detected by electrochemical luminescence immunoassay,PLR was calculated according to the blood routine re-sults,and RLX and KPNA2 levels were detected by enzyme-related immunosorbent assay.Multivariate Logis-tic regression analysis was used to analyze the influencing factors of postoperative recurrence in patients with advanced epithelial ovarian cancer,and establish a risk prediction model for postoperative recurrence of ad-vanced epithelial ovarian cancer.Receiver operating characteristic(ROC)curve was used to evaluate the pre-dictive efficacy of the model for postoperative recurrence of advanced epithelial ovarian cancer,and Hosmer-Lemeshow test was used to analyze the fitting of recurrence risk prediction model for patients with advanced epithelial ovarian cancer.Results There was a statistically significant difference in International Federation of Gynecology and Obstetrics(FIGO)staging and serum levels of carbohydrate antigen 125,HE4,PLR,RLX and KPNA2 between the recurrence group and the non-recurrence group(P＜0.05).FIGO staging Ⅳ of cancer and elevated serum HE4,PLR,RLX and KPNA2 were risk factors for postoperative recurrence in patients with advanced epithelial ovarian cancer(P＜0.05).ROC curve analysis showed that,the area under the curve of the recurrence risk prediction model for postoperative recurrence risk of advanced epithelial ovarian cancer was 0.859,which was significantly higher than that single indicator detected by HE4,PLR,RLX and KP-NA2.Hosmer-Lemeshow test showed that the recurrence risk prediction model of advanced epithelial ovarian cancer had a good fitting(x2=7.869,P=0.437).Conclusion The risk prediction model for postoperative re-currence of advanced epithelial ovarian cancer based on serum HE4,PLR,RLX,KPNA2 and FIGO staging of cancer has high predictive value for evaluating postoperative recurrence of advanced epithelial ovarian cancer,and deserves clinical attention.

In the field of dental aesthetics,digital aesthetic design plays a crucial role in helping dentists to predict treatment outcomes vis-ually,as well as in enhancing the consistency of knowledge and understanding of aesthetic goals between dentists and patients.It serves as the foundation for achieving ideal aesthetic effects.However,there is no clear standard for this digital process currently in China and abroad.Many dentists lack of systematic understanding of how to carry out digital aesthetic design for treatment.To establish standardized processes for dental aesthetic design and to improve the homogeneity of treatment outcomes,Chinese Society of Digital Dental Industry(CSD-DI)convened domestic experts in related field to compile this consensus.This article elaborates on the key aspects of digital aesthetic data collection,integration steps,and the digital aesthetic design process.It also formulates a decision tree for dental aesthetics at macro level and outlines corresponding workflows for various clinical scenarios,serving as a reference for clinicians.

BACKGROUND:In recent years,the incidence of hyperuricemia caused by purine metabolism disorders has been increasing,which can induce inflammatory responses and lead to renal injury. OBJECTIVE:To explore the role and mechanism of solute carrier family 2 member 12(SLC2A12)in hyperuricemia-related renal injury. METHODS:Renal tubular cells(HK2 cells)were divided into five groups:HK2 group,HK2+uric acid group,HK2+uric acid+NC group,HK2+uric acid+siSLC2A12 group,and HK2+uric acid+siSLC2A12+MK-2206 group.HK2 cells were treated with uric acid and transfected with siRNA SLC2A12,followed by MK-2206 treatment to inhibit AKT expression.Cell proliferation was detected by CCK-8 assay.Apoptosis was detected by TUNEL assay.qRT-PCR and western blot assay were used to detect fibrogenic factors as well as activation of the AKT/FOXO3a pathway.The concentrations of inflammatory cytokines were measured by enzyme-linked immunosorbent assay. RESULTS AND CONCLUSION:(1)Uric acid treatment inhibited cell proliferation and promoted cell apoptosis in the HK2+uric acid group compared with the HK2 group.The proliferative ability of cells in the HK2+uric acid+siSLC2A12 group was further decreased and apoptotic cells were further increased compared with the HK2 group.Compared with the HK2+uric acid+siSLC2A12 group,the HK2+uric acid+siSLC2A12+MK-2206 group showed an increase in cell proliferation and a decrease in apoptotic cells.(2)Compared with the HK2 group,the connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA)and transforming growth factor beta(TGF-β)expressions increased in the HK2+uric acid group;CTGF,α-SMA and TGF-β expression further increased in the HK2+uric acid+siSLC2A12 group.Compared with the HK2+uric acid+siSLC2A12 group,the CTGF,α-SMA and TGF-β expressions decreased.(3)Compared with the HK2 group,the expression of p-AKT,FOXO3a,and p-FOXO3a elevated in the HK2+uric acid group;the expression of p-AKT further increased,while the expression of FOXO3a and p-FOXO3a decreased in the HK2+uric acid+siSLC2A12 group.Compared with the HK2+uric acid+siSLC2A12 group,p-AKT expression decreased;FOXO3a and p-FOXO3a expression increased in the HK2+uric acid+siSLC2A12+MK-2206 group.(4)Compared with the HK2 group,interleukin-6,interleukin-1 β,and tumor necrosis factor α levels increased in the HK2+uric acid group;interleukin-6,interleukin-1 β,and tumor necrosis factor α levels further increased in the HK2+uric acid+siSLC2A12 group.Compared with the HK2+uric acid+siSLC2A12 group,interleukin-6,interleukin-1 β,and tumor necrosis factor α levels diminished in the HK2+uric acid+siSLC2A12+MK-2206 group.(5)These findings indicate that SLC2A12 may protect against hyperuricemia-induced renal injury by counteracting uric acid-induced tubular fibrosis and inflammation through activation of the FOXO3a pathway.

Background::Hepatic ischemia-reperfusion injury (HIRI) remains a common complication during liver transplantation (LT) in patients. As a key downstream effector of the Hippo pathway, Yes-associated protein (YAP) has been reported to be involved in various physiological and pathological processes. However, it remains elusive whether and how YAP may control autophagy activation during ischemia-reperfusion.Methods::Human liver tissues from patients who had undergone LT were obtained to evaluate the correlation between YAP and autophagy activation. Both an in vitro hepatocyte cell line and in vivo liver-specific YAP knockdown mice were used to establish the hepatic ischemia-reperfusion models to determine the role of YAP in the activation of autophagy and the mechanism of regulation. Results::Autophagy was activated in the post-perfusion liver grafts during LT in patients, and the expression of YAP positively correlated with the autophagic level of hepatocytes. Liver-specific knockdown of YAP inhibited hepatocytes autophagy upon hypoxia-reoxygenation and HIRI ( P <0.05). YAP deficiency aggravated HIRI by promoting the apoptosis of hepatocytes both in the in vitro and in vivo models ( P <0.05). Attenuated HIRI by overexpression of YAP was diminished after the inhibition of autophagy with 3-methyladenine. In addition, inhibiting autophagy activation by YAP knockdown exacerbated mitochondrial damage through increasing reactive oxygen species ( P <0.05). Moreover, the regulation of autophagy by YAP during HIRI was mediated by AP1 (c-Jun) N-terminal kinase (JNK) signaling through binding to the transcriptional enhanced associate domain (TEAD). Conclusions::YAP protects against HIRI by inducing autophagy via JNK signaling that suppresses the apoptosis of hepatocytes. Targeting Hippo (YAP)-JNK-autophagy axis may provide a novel strategy for the prevention and treatment of HIRI.

Objective To construct SpyCatcher-mi3 nanoparticle vaccine delivery vectors,evaluate their role in enhancing the immunogenicity of the ovalbumin CD8+T-cell epitope peptide,OVA257-264,and determine its protective effect in a model which mice were immunized and subcutaneously challenged with E.G7-OVA tumor cells.Methods SpyCatcher-mi3 proteins were expressed by E.coli and purified by affinity chromatography and anion exchange chromatography sequentially.OVA257-264-SpyTag peptide was obtained by synthesis.The OVA257-264-mi3 nanoparticles were produced by the SpyTag/SpyCatcher system.The toxicity of OVA257-264-mi3 was evaluated using hemolysis assay,CCK-8 assay and mouse experiment.A total of 42 female SPF-grade C57BL/6 mice(6～8 weeks old,18～20 g)were randomly divided into OVA257-264-mi3,OVA257-264,and control groups,with 14 mice in each group.Then the mice in each group were immunized on days 0,14 and 28.In 14 d after the last immunization,the amounts of spot-forming cells(SFCs,indicating IFN-γ secreting cells in splenic lymphocytes)were determined using ELISpot assay to evaluate their immunogenicity.After the immunized mice were subcutaneously implanted with E.G7-OVA tumor cells,the antitumor effect of the vaccine in prophylactic xenograft tumor model was evaluate by observing tumor volumes with a caliper and tumor growth with MRI.Results Both SpyCatcher-mi3 and OVA257-264-mi3 could be self-assembled to form homogeneous and stable nanoparticles,with an average particle size of about 43.8 and 91.3 nm,respectively.The OVA257-264-mi3 was safe for in vitro and in vivo toxicity evaluation.The number of IFN--y secreting cells per 1 × 106 splenic lymphocytes reached 253 in the OVA257-264-mi3 group of mice,significantly higher than that in the OVA257-264 group and the Control group(P＜0.05).The tumor volume of mice in the OVA257-264-mi3 group was about 151.1 mm3 on day 22,which was significantly smaller than that of the OVA257-264 group and the Control group(P＜0.05),and the survival rate during the observation period reached 60％,which was significantly higher than that of the OVA257-264 groups(P＜0.05).Conclusion Nanoparticle vaccine OVA257-264-mi3 is successfully constructed,and it shows enhancing effect on the immunogenicity of the antigen epitope peptide,and exerts protective effect on prophylactic xenograft tumor model,providing a theoretical basis for the research of tumor neoantigen vaccines.

Purpose::Ischemia and hypoxia are the main factors limiting limb replantation and transplantation. Static cold storage (SCS), a common preservation method for tissues and organs, can only prolong limb ischemia time to 4 - 6 h. The normothermic machine perfusion (NMP) is a promising method for the preservation of tissues and organs, which can extend the preservation time in vitro by providing continuous oxygen and nutrients. This study aimed to evaluate the difference in the efficacy of the 2 limb preservation methods. Methods::The 6 forelimbs from beagle dogs were divided into 2 groups. In the SCS group ( n = 3), the limbs were preserved in a sterile refrigerator at 4 °C for 24 h, and in the NMP group ( n = 3), the perfusate prepared with autologous blood was used for the oxygenated machine perfusion at physiological temperature for 24 h, and the solution was changed every 6 h. The effects of limb storage were evaluated by weight gain, perfusate biochemical analysis, enzyme-linked immunosorbent assay, and histological analysis. All statistical analyses and graphs were performed using GraphPad Prism 9.0 one-way or two-way analysis of variance. The p value of less than 0.05 was considered to indicate statistical significance. Results::In the NMP group, the weight gained percentage was 11.72% ± 4.06%; the hypoxia-inducible factor-1α contents showed no significant changes; the shape of muscle fibers was normal; the gap between muscle fibers slightly increased, showing the intercellular distance of (30.19 ± 2.83) μm; and the vascular α-smooth muscle actin (α-SMA) contents were lower than those in the normal blood vessels. The creatine kinase level in the perfusate of the NMP group increased from the beginning of perfusion, decreased after each perfusate change, and remained stable at the end of perfusion showing a peak level of 4097.6 U/L. The lactate dehydrogenase level of the NMP group increased near the end of perfusion and reached the peak level of 374.4 U/L. In the SCS group, the percentage of weight gain was 0.18% ± 0.10%, and the contents of hypoxia-inducible factor-1α increased gradually and reached the maximum level of (164.85 ± 20.75) pg/mL at the end of the experiment. The muscle fibers lost their normal shape and the gap between muscle fibers increased, showing an intercellular distance of (41.66 ± 5.38) μm. The contents of vascular α-SMA were much lower in the SCS group as compared to normal blood vessels.Conclusions::NMP caused lesser muscle damage and contained more vascular α-SMA as compared to SCS. This study demonstrated that NMP of the amputated limb with perfusate solution based on autologous blood could maintain the physiological activities of the limb for at least 24 h.

Objective To develop a health belief model(HBM)based adolescent alcohol-related cognition scale to measure adolescent alcohol-related cognition and test its reliability and validity.Methods The adolescents'alcohol-related cognitive scale was developed based on HBM model.By using purposive sampling,three general high schools in Qingpu District,Shanghai were selected.One-third of the classes from grades 10 and 11 in each school were randomly selected,and the students from these classes were surveyed as the research subjects.Exploratory factor analysis and confirmatory factor analysis were used to analyze its reliability(internal consistency reliability and combination reliability)and validity(structural validity,convergent validity,discriminative validity and criterion validity).Results A total of 970 questionnaires were collected,of which 948 were valid,with an effective rate of 97.7%.The adolescents'alcohol-related cognitive scale contained 22 items.Five common factors were extracted from exploratory factor analysis,including perceived susceptibility,perceived severity,perceived benefits,perceived obstacles,and self-efficacy.The cumulative variance contribution rate reached 83.89%.The results of confirmatory factor analysis confirmed the overall fit of the model.The average variance extracted value(AVE)of each dimension was greater than 0.5,and the convergent validity of the model was ideal.The AVE square root of each dimension of the scale was greater than its correlation coefficient,indicating good discrimination validity.Cronbach's α coefficient of the total volume table was 0.892,indicating good overall reliability.Conclusion The adolescents'alcohol-related cognitive scale developed in this study has good reliability and validity,which can be used to measure adolescents'alcohol-related perceptions.