ObjectiveTo explore the central neuroregulation mechanism of heat-sensitive moxibustion for knee osteoarthritis on pain relief. MethodsThirty patients who did not have experience of Deqi （得气） during heat-sensitive moxibustion treatment were assigned to the "non-Deqi group"， while another 30 patients who had experience of Deqi were assigned to the "Deqi group". Both groups received moxibustion at the left Heding （EX-LE2） acupoint. In the Deqi group， after the patients experienced sensation of Deqi at the acupoint， moxibustion was applied at approximately 3 cm from the skin for 10 minutes； in the non-Deqi group， moxibustion was also applied at approximately 3 cm from the skin for 10 minutes. Both groups received treatment once daily for 10 consecutive days. Knee joint pain was assessed before and after treatment using the visual analog scale （VAS）. Resting-state functional magnetic resonance imaging （rs-fMRI） scans were performed on all participants before the first treatment session and after the final session on the 10th day. The fractional amplitude of low-frequency fluctuations （fALFF） maps before and after treatment were processed using the SPM12 module by MATLAB. ResultsAfter treatment， VAS scores in both groups were significantly lower than before treatment （P＜0.05 or P＜0.01）， with the Deqi group showing significantly lower VAS scores than the non-Deqi group （P＜0.01）. Compared to before treatment， the Deqi group exhibited significant activation in the prefrontal cortex （t = 6.28）， white matter （t = 6.36）， and left temporal lobe （t = 9.33）， while significant inhibition was observed in the occipital lobe （t = -9.86） and right cerebrum （t = -4.54， P＜0.01）； in the non-Deqi group， significant changes after treatment were observed in the left occipital lobe （t = -6.42）， left medial frontal gyrus （t = -4.35）， left middle frontal gyrus （t = -4.74）， right superior frontal gyrus （t = -4.82）， right superior temporal gyrus （t = -6.61）， and right cerebellar posterior lobe （t = -8.64）， all of which were in inhibited states （P＜0.01）. Compared to the non-Deqi group， the Deqi group exhibited significant activation after treatment in the external nucleus （t = 5.77）， white matter （t = 3.58）， right cerebrum （t = 5.84）， left cerebellum （t = 5.35）， and left cerebrum （t = 4.32）， while significant inhibition was observed in the prefrontal cortex （t = -4.16）， occipital lobe （t = -4.87）， and precentral gyrus （t = -4.46， P＜0.01）. ConclusionsHeat-sensitive moxibustion provides better analgesic effects for knee osteoarthritis under state of Deqi. Its central neuroregulation mechanism may be related to the involvement of the frontal lobe， temporal lobe， occipital lobe， external nucleus， white matter， right cerebrum， left cerebellum， left cerebrum， and precentral gyrus in modulating pain signals.

Natural components serve the survival instincts of cells that are obtained through long-term evolution, while they often fail to meet the demands of engineered cells for efficiently performing biological functions in special industrial environments. Enzymes, as biological catalysts, play a key role in biosynthetic pathways, significantly enhancing the rate and selectivity of biochemical reactions. However, the catalytic efficiency, stability, substrate specificity, and tolerance of natural enzymes often fall short of industrial production requirements. Therefore, exploring and modifying enzymes to suit specific biomanufacturing processes has become crucial. In recent years, artificial intelligence (AI) has played an increasingly important role in the discovery, evaluation, engineering, and de novo design of proteins. AI can accelerate the discovery and optimization of proteins by analyzing large amounts of bioinformatics data and predicting protein functions and characteristics by machine learning and deep learning algorithms. Moreover, AI can assist researchers in designing new protein structures by simulating and predicting their performance under different conditions, providing guidance for protein design. This paper reviews the latest research advances in protein discovery, evaluation, engineering, and de novo design for biomanufacturing and explores the hot topics, challenges, and emerging technical methods in this field, aiming to provide guidance and inspiration for researchers in related fields.
Protein Engineering/methods* ; Artificial Intelligence ; Proteins/genetics* ; Computational Biology ; Machine Learning ; Data Mining ; Algorithms ; Deep Learning

Objective To explore the effect and preliminary mechanism of the plant-derived immunostimulatory molecule,ophiopogonin,on enhancing the immune response of a subunit vaccine with the receptor-binding domain(RBD)of coronavirus spike protein as the antigen.Methods CCK-8 assay was used to determine the cytotoxicity of ophiopogonin D'(OPD')on bone marrow-derived dendritic cells(BMDCs).Female Balb/c mice were randomly divided into RBD,RBD/OPD',RBD/Alum,and control groups.The immunization dose was 5 μg of antigen per mouse and 100 μg of adjuvant per mouse,and immunization was carried out according to the intramuscular injection immunization procedure on days 0,21,and 42.The titers of specific IgG and its subtype antibodies were detected by ELISA.The cytokine levels in the supernatant of splenocytes were detected using ELISA.The number of splenocytes secreting IFN-γ was detected by ELISpot.Laser confocal microscopy was employed to observe the uptake of antigen by BMDCs.The phagocytic ability of BMDCs for antigen was quantitatively analyzed by flow cytometry.The mechanism of its enhanced immune effect was preliminarily explored using transcriptomics technology combined with bioinformatics research.Results When the concentration of OPD'was less than 5 μg/mL,the survival rate of BMDCs was 100%.After a single intramuscular injection in mice,except for a slight decrease in body weight,the other biochemical indicators were within corresponding normal ranges.After intramuscular injection immunization of the vaccine,the titers of serum-specific IgG,IgG1,and IgG2a in the RBD/OPD'group were significantly higher than those in the RBD group(P<0.05).Compared with the RBD group,the RBD/OPD'group induced a high-level Th1 cell immune response of IL-1β,TNF-α,and IFN-γ(P<0.01)and had more lymphocytes secreting IFN-γ(P<0.001).Laser confocal microscopy displayed that BMDCs took up more antigens after OPD'treatment,which was further confirmed with flow cytometry in quantitative analysis on antigen uptake rate(P<0.01).Transcriptomics results indicated that there was more significant enrichment of the PPAR signaling pathway in the RBD/OPD'group than the RBD group,suggesting that OPD'may activate the PPAR signaling pathway to exert its adjuvant effect.Conclusion OPD'effectively enhances the immune response of the RBD subunit vaccine,and its action mechanism may be related to the activation of the PPAR signaling pathway.

Objective To establish a mouse model of infection with the minimum lethal dose of Vibrio vulnificus(V.vulnificus)and to evaluate the protective efficacy of inactivated whole-cell(IWC)vaccine using this model.Methods A mouse model of lethal-dose infection was established by intraperitoneal injection of different doses of V.vulnificus.Bacterial colonization in the organs was detected with tissue homogenate plating,and pathological changes in the organs were observed after tissue section staining.Flow cytometry was used to detect immune cell responses after liver tissues were digested into single-cell suspension.IWC vaccine of V.vulnificus was prepared,and the mice were immunized through different routes to observe the protective efficacy of the vaccine.Results A mouse model of infection with the minimum lethal dose at 1×106 CFU of V.vulnificus was successfully established.After infection,the bacteria were mainly colonized in the liver of mice and caused severe pathological damages.Compared with the uninfected mice,the proportion of neutrophils in the liver was significantly increased in the infected mice,whereas the proportions of B cells and T cells were correspondingly decreased(P<0.05).A single intramuscular or intraperitoneal injection of the IWC vaccine could protect the mice effectively against lethal infection of V.vulnificus in 7 d later(P<0.01),although the level of serum IgG having no significant increase.Conclusion A mouse model of lethal-dose infection with V.vulnificus is successfully established,with histopathological characteristics.The IWC vaccine of V.vulnificus rapidly mediates immune protection in this model probably independent of IgG.

Objective To prepare tubeimoside Ⅲ nanoemulsion(TBMⅢ-NE)and evaluate its adjuvant effect in vaccines.Methods TBMⅢ-NE was prepared using low-energy emulsification.Dynamic light scattering was used to characterize the particle size and polydispersity index of the obtained TBMⅢ-NE,and transmission electron microscopy(TEM)was employed to observe the morphology.CCK-8 assay was utilized to determine the cytotoxicity of TBMⅢ-NE on bone marrow-derived dendritic cells(BMDCs).The in vitro safety of TBMⅢ-NE was evaluated using a hemolysis assay.The ability of TBMⅢ-NE to promote the phagocytosis of antigens by DC2.4 cells was observed using confocal laser microscopy.After co-incubation of TBMⅢ-NE with BMDCs,the expression levels of CD40,CD86,MHC-Ⅰ,and CCR7 on the surface of BMDCs were detected using flow cytometry,and the levels of cytokines in the supernatant of BMDCs were measured using enzyme-linked immunosorbent assay(ELISA).After female BALB/c mice were immunized with the SARS-CoV-2 antigen RBD in combination with TBMⅢ-NE,ELISA was conducted to determine the serum levels of specific IgG,IgG2a,and IgG1 antibodies.The number of specific IFN-γ-secreting cells in mouse splenocytes was detected using enzyme-linked immunospot(ELISpot)assay.Results The prepared blank nanoemulsion(BNE)and TBMⅢ-NE were in a particle size of 25.46 and 25.89 nm,and a polydispersity index of 0.214 and 0.125,respectively.TEM displayed that TBMⅢ-NE was in uniform sphere and well dispersed.When the TBMⅢ-NE adjuvant was diluted by 400-fold,the survival rate of BMDCs was approximately 86%.Compared with free TBMⅢ,the hemolytic toxicity of TBMⅢ-NE was significantly reduced(P<0.01).TBMⅢ-NE promoted the phagocytosis of antigens by DC2.4 cells and significantly increased the expression of CCR7 on the surface of BMDCs(P<0.05),indicating its potential to promote more dendritic cells to effectively migrate to lymph nodes.TBMⅢ-NE also promoted the expression of IL-6 and IL-1β in the supernatant of BMDCs(P<0.05).When combined with RBD,TBMⅢ-NE significantly increased the levels of specific IgG,IgG2a,and IgG1 antibodies in mouse serum(P<0.01)and promoted the secretion of specific IFN-γ in splenocytes(P<0.01),indicating that TBM Ⅲ-NE could enhance specific cellular immune responses.Conclusion A stable and highly effective TBMⅢ-NE that can induce humoral and cellular immune responses is successfully prepared.

Objective:To explore the therapeutic effect of compound Kuijie Ankang Decoction on ulcerative colitis (UC) model mice by non targeted metabonomics; To explore its mechanism.Compound Kuijie Ankang.Methods:The mice were randomly divided into blank control group, model group, Kuijie Ankang Decoction group and sulfasalazine group, with 12 mice in each group. Except the blank control group, the other groups were given 1.5% DSS solution for free drinking to prepare UC model. After successful modeling, Kuijie Ankang Decoction group was intragastrically administered with compound Kuijie Ankang Decoction of 9.68 g/kg, sulfasalazine group was intragastrically administered with sulfasalazine capsule suspension of 320 mg/kg, model group and blank control group were intragastrically administered with equal volume of purified water, once a day, for 7 consecutive days. The body mass and disease activity index (DAI) score of mice were measured. ELISA was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) in the colon tissue of mice; the protein expressions of Claudin-1 and Zo-1 in colon tissue were detected by immunofluorescence method. HE staining was used to observe the pathological changes in the colon, and UHPLC-OE-MS technology was used to analyze the endogenous metabolite structure of mouse colon tissue, differential metabolites and related metabolic pathways were screened.Results:Compared with the model group, the colon length in Kuijie Ankang Decoction group and sulfasalazine group increased ( P<0.01), the DAI score decreased ( P<0.01), the levels of TNF-α, IL-1β and IL-6 in colon tissue decreased ( P<0.01), the level of IL-10 increased ( P<0.01), and the average optical density of Claudin-1 and Zo-1 protein increased ( P<0.01 or P<0.05). Metabolomics analysis identified 26 potential differential metabolites, including nicotinamide adenine dinucleotide, guanine, gamma aminobutyric acid, and thiamine, affecting 26 key metabolic pathways, including lysine biosynthesis, thiamine metabolism, cysteine and methionine metabolism. Conclusion:Kuaijie Ankang Decoction may improve metabolites such as Gamma aminobutyric acid and thiamine through metabolic pathways such as lysine biosynthesis to alleviate inflammatory reactions, thereby exerting therapeutic effects on ulcerative colitis in mice.

Ulcerative colitis(UC)is a common disease of the digestive system.Phosphatidylinositol-3-kinase(PI3K)/synuclein/threonine kinase(AKT)is closely related to cell survival,apoptosis,inflammation and other biological processes,and the expression levels of PI3K and AKT significantly increase during the course of UC,with accelerated apoptosis,improved inflammation,and damaged intestinal mucosal barrier function.In recent years,a large number of basic and clinical trials have been conducted on PI3K/AKT signaling pathway in TCM,and the results indicate that PI3K/AKT signaling pathway is expected to be an important potential target for UC treatment.This article analyzed the mechanism of the regulation of PI3K/AKT signaling pathway in TCM from monomer,extract,compound and acupuncture,and suggested that the regulation of this signaling pathway is of great significance for the prevention and treatment of UC,and provide reference for drug development.

Objective:To explore the influencing factors of clinical pregnancy outcomes in patients with hydrosalpinx undergoing in vitro fertilization-embryo transfer(IVF-ET) after interventional embolization and whether residual hydrops has an adverse impact on pregnancy outcomes.Methods:Clinical data from 65 patients who underwent interventional embolization and IVF-ET for hydrosalpinx at the Third Affiliated Hospital of Zhengzhou University from March 2021 to October 2022 were collected retrospectively. The hydrops index was quantified by the ratio of the widest diameter to the pelvic transverse diameter of the intraoperative hydrops, and the patient′s age, body mass index(BMI), follicle-stimulating hormone(FSH), lutenizing hormine(LH), estradiol(E2), automated matetials hangling(AMH), endometrial thickness at the time of transplantation, the number of transplanted embryos, embryo type, and location of the hydrosalpinx were recorded. In addition, the clinical pregnancy outcomes of the first transplantation after embolization were followed. Two independent samples t-test, rank sum test and chi-square test were used to analyze the difference of the above indexes among different clinical pregnancy outcomes by SPSS 25.0. The receiver operating characteristic (ROC) curves and Youden index were used to calculate the cut-off value of the water accumulation index. Results:Among 65 patients, the clinical pregnancy rate was 63.1%(41/65), among the 45 patients who underwent embryo transfer before embolization without success, the clinical pregnancy rate after embolization was 62.2%(28/45). Based on data analysis, it showed that IVF-ET clinical pregnancy outcomes were not associated with age, BMI, FSH, LH, E2, AMH, endometrial thickness at the time of transplantation, the number of transplanted embryos, embryo type, as well as location of hydrosalpinx( P>0.05), but associated with hydrosalpinx index( P<0.001). ROC curve analysis showed that the hydrops index could be used as a predictor of pregnancy outcome, and the area under the curve was 0.825, and the optimal cut-off value of the hydrops index was 12.925% based on the Youden index analysis result. Conclusions:Interventional embolization of hydrosalpinx may improve clinical pregnancy rates. When the hydrosalpinx is large enough, it could adversely affected IVF-ET clinical pregnancy, and further aspiration of hydrosalpinx should be performed prior to transplantation.

Objective:To investigate the correlation between the clinical features and endoscopic ultrasound-guided portal pressure gradient (EUS-PPG) in patients with cirrhosis.Methods:A total of 148 patients with cirrhosis and portal hypertension who underwent EUS-PPG measurement at the Third Xiangya Hospital of Central South University from March 15, 2022 to June 20, 2023 were selected. The clinical data of patients collected before EUS-PPG measurement were analyzed. Variations in the EUS-PPG across different clinical data subgroups were analyzed. Multivariate linear regression analysis was used to explore the independent factors influencing EUS-PPG.Results:The EUS-PPG was significantly elevated in patients exhibiting red signs (16.62±5.33 mmHg VS 13.44±5.34 mmHg, t=3.616, P<0.001), gastroesophageal varices (15.78±5.30 mmHg VS 9.70±4.77 mmHg, t=4.247, P<0.001), hepatic encephalopathy (20.83±7.52 mmHg VS 14.92±5.35 mmHg, t=2.606, P=0.010), thrombocytopenia (15.66±5.39 mmHg VS 13.29±5.83 mmHg, t=2.136, P=0.034), hypoproteinemia (16.13±5.86 mmHg VS 14.12±5.03 mmHg, t=2.230, P=0.027), and an increased international normalized ratio (16.25±6.00 mmHg VS 14.40±5.11 mmHg, t=2.022, P=0.045). Conversely, the EUS-PPG was significantly reduced in patients with a history of splenectomy and devascularization (13.17±5.88 mmHg VS 15.73±5.34 mmHg, t=-2.379, P=0.019). The EUS-PPG in patients with varying degrees of ascites (no VS slight VS moderate or severe: 13.40±5.48 mmHg VS 15.90±5.49 mmHg VS 16.69±5.17 mmHg, F=5.188, P=0.007) and different Child-Pugh classifications (A VS B VS C: 14.07±5.05 mmHg VS 15.69±5.74 mmHg VS 17.64±5.99 mmHg, F=3.066, P=0.049) increased gradually. Multivariable linear regression analysis showed that red signs ( β=2.44, t=2.732, P=0.007), gastroesophageal varices ( β=4.45, t=2.990, P=0.003), ascites ( β=1.75, t=2.368, P=0.019), and hepatic encephalopathy ( β=5.82, t=2.644, P=0.009) were independent factors for the elevated EUS-PPG. Conclusion:There is a significant correlation between EUS-PPG and the clinical features related to the severity of cirrhotic portal hypertension, which indicates the feasibility of EUS-PPG in evaluating cirrhotic portal hypertension.

Objective:Discipline construction is the key and effective method for the high-quality development of hospitals. This study summarized the practical and valuable experience of discipline construction with the support of the ″Sanming Project of Medicine″ in Shenzhen, to provide a worthy reference for similar projects and hospital specialty development.Methods:Taking the introduction of the hematology-oncology team of the Canadian Children′s Hospital as an example, we analyzed the background, the process, and the effectiveness of discipline construction of hematology and oncology of Shenzhen Children′s Hospital under the support of the ″Sanming Project of Medicine″, and explored the common rules for rapid specialty development.Results:Through the implementation of two levels supporting policies from cities and hospitals and quantifiable and verifiable annual progressive program, the adoption of the international advanced medical management mode and concepts, and the transformation and application of cutting-edge clinical technologies, we successfully built the largest pediatric hematologic oncology diagnosis and treatment center in South China and the largest pediatric hematopoietic stem cell transplant center in China. By executing a multi-level talent developing program, the number of specialists with MD and PhD degrees increased by 6.5 times, and 23 doctors and nurses were trained abroad, leading to the development of provincial key clinical specialties and municipal key scientific research platforms.Conclusions:This project concluded the beneficial experience of introducing high-level overseas medical teams to boost discipline construction, including internationally advanced medical concepts and management modes, tiered talent cultivation schemes, the construction of clinical and basic research platforms, etc., providing references for the rapid improvement of discipline construction in terms of health care for other hospitals in Shenzhen and similar cities.