Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Objective To develop GTKO (α-1,3-galactosyltransferase gene-knockout, GTKO)/hCD55 (human CD55) gene-edited xenotransplant donor pigs and verify their function. Methods In this study, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated nuclease 9), PiggyBac transposon technology and somatic cell nuclear transfer technology were used to construct GTKO/hCD55 gene-edited Diannan miniature pigs. The phenotype and function of GTKO/hCD55 pigs were analyzed by Sanger sequencing, real-time fluorescence quantitative PCR, flow cytometry, immunofluorescence, bisulfite sequencing, antigen-antibody binding assays, and complement-dependent cytotoxicity assays. Results After transfection of PX458 and PiggyBac gene editing vectors into wild-type fetal pig fibroblasts, 48 single-cell colonies were obtained through puromycin drug screening. Two single-cell colonies were selected for somatic cell nuclear transfer, resulting in two fetal pigs at 33 days of gestation. The GGTA1(α-1,3-galactosyltransferase) genotypes of fetal pig F01 were -17 bp and wild type (WT), while the GGTA1 genotypes of fetal pig F02 were -26 bp/+2 bp and -3 bp. The hCD55 mRNA expression levels of both fetal pigs were significantly higher than those of WT pigs (P＜0.01). The fetal pig F02 was selected as the donor cell source for recloning, 11 surviving piglets were obtained, all identified as GTKO/hCD55 gene-edited pigs. These pigs showed absence of α-Gal antigen expression, but weak or no expression of hCD55 was observed. Methylation analysis of the hCD55 gene's CpG island showed hypermethylation in kidney tissue lacking hCD55 expression, whereas it was not methylated or partially methylated in kidney tissue expressing hCD55. Moreover, codon optimization of the CpG island of the hCD55 gene to reduce CG content could achieve stable expression of the hCD55 gene. In addition, antigen-antibody binding experiment showed that the amount of human IgM binding to GTKO/hCD55 gene-edited pig fibroblasts was significantly lower than that of WT pigs (P＜0.01). Complement-dependent cytotoxicity experiment showed that the survival rate of fibroblasts in GTKO/hCD55 pigs was significantly higher than that in WT pigs (P＜0.01). Conclusion This study demonstrates the successful generation of GTKO/hCD55 gene-edited xenotransplant donor pigs. Methylation-induced gene silencing of the hCD55 gene can be effectively avoided by reducing the CG content of the CpG island through codon optimization. This study provides a reference for the development of xenotransplant donor pigs and guides subsequent research on xenotransplantation.

Objective This study aims to assess the current status of pharmaceutical management in county medical communities in Hubei province,and provide recommendations for the homogenization,standardization,and regulation of pharmaceutical management in these communities.It also intends to offer decision-making support for health administrative departments,and provide reference experiences for management in other regions.Methods The current status of pharmaceutical management in county medical communities in Hubei province was conducted through a questionnaire survey and field research.Existing problems were analyzed,key management areas were identified,and reasonable recommendations were proposed.Results Pharmaceutical management in county medical communities has significant shortcomings in organizational structure,system construction,personnel allocation,key link control,and the leading unit's outreach capabilities.These deficiencies are not aligned with the high-quality development of pharmacy in the new era.Conclusions It is recommended that county medical communities should establish a comprehensive pharmaceutical management quality control system.This can be achieved by improving organizational management,strengthening talent development,enhancing core systems,setting monitoring indicators,and increasing outreach capabilities.Additionally,evaluation standards for the quality control system of pharmaceutical management should be established to enhance management capabilities through scientific assessment and positive feedback.

Traumatic supraorbital fissure syndrome (TSOFS) is a symptom complex caused by nerve entrapment in the supraorbital fissure after skull base trauma. If the compressed cranial nerve in the supraorbital fissure is not decompressed surgically, ptosis, diplopia and eye movement disorder may exist for a long time and seriously affect the patients′ quality of life. Since its overall incidence is not high, it is not familiarized with the majority of neurosurgeons and some TSOFS may be complicated with skull base vascular injury. If the supraorbital fissure surgery is performed without treatment of vascular injury, it may cause massive hemorrhage, and disability and even life-threatening in severe cases. At present, there is no consensus or guideline on the diagnosis and treatment of TSOFS that can be referred to both domestically and internationally. To improve the understanding of TSOFS among clinical physicians and establish standardized diagnosis and treatment plans, the Skull Base Trauma Group of the Neurorepair Professional Committee of the Chinese Medical Doctor Association, Neurotrauma Group of the Neurosurgery Branch of the Chinese Medical Association, Neurotrauma Group of the Traumatology Branch of the Chinese Medical Association, and Editorial Committee of Chinese Journal of Trauma organized relevant experts to formulate Chinese expert consensus on the diagnosis and treatment of traumatic supraorbital fissure syndrome ( version 2024) based on evidence of evidence-based medicine and clinical experience of diagnosis and treatment. This consensus puts forward 12 recommendations on the diagnosis, classification, treatment, efficacy evaluation and follow-up of TSOFS, aiming to provide references for neurosurgeons from hospitals of all levels to standardize the diagnosis and treatment of TSOFS.

Objective To explore the relationship and internal path between activities of daily living(ADL),sleep quality and mental health of community elderly people in Shanghai.Methods A questionnaire survey was conducted among community residents aged 60 years and older seeing doctors in community health care center of five streets in Shanghai during Sept to Dec,2021 using convenience sampling.Activities of Daily Living(ADL),Pittsburgh Sleep Quality Index(PSQI)and 10-item Kessler Psychological Distress Scale(K10)were adopted in the survey.Single factor analysis,correlation analysis and multiple linear regression were used to analyze the data.The effect relationship between the variables was tested using Bootstrap's mediated effects test.Results A total of 1 864 participants were included in the study.The average score was 15.53±4.47 for ADL,5.60±3.71 for PSQI and 15.50±6.28 for K10.The rate of ADL impairment,poor sleep quality,poor and very poor mental health of the elderly were 23.6%,27.3%,11.9%and 4.9%,respectively.ADL and sleep quality were all positively correlated with mental health(r=0.321,P＜0.001;r=0.466,P＜0.001);ADL was positively correlated with sleep quality(r=0.294,P＜0.001).Multiple linear results of factors influencing mental health showed that ADL(β= 0.457,95%CI:0.341-0.573),sleep quality(β =0.667,95%CI:0.598-0.737)and mental health were positively correlated(P＜0.001).Sleep quality partially mediated the relationship between ADL and mental health(95%CI:0.078-0.124)with an effect size of 33.0%.Conclusion Sleep quality is a mediator between ADL and mental health among community elderly people.Improving ADL and sleep quality may improve mental health in the population.

Currently,human health due to corona virus disease 2019(COVID-19)pandemic has been seriously threatened.The coronavirus severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19.However,the efficacy is compromised by the SARS-CoV-2 evolvement and mutation.Here we report the SARS-CoV-2 S protein receptor-binding domain(RBD)inhibitor licorice-saponin A3(A3)could widely inhibit RBD of SARS-CoV-2 variants,including Beta,Delta,and Omicron BA.1,XBB and BQ1.1.Furthermore,A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells,with EC50 of 1.016 pM.The mechanism was related to binding with Y453 of RBD deter-mined by hydrogen-deuterium exchange mass spectrometry(HDX-MS)analysis combined with quan-tum mechanics/molecular mechanics(QM/MM)simulations.Interestingly,phosphoproteomics analysis and multi fluorescent immunohistochemistry(mIHC)respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 mitogen-activated protein kinase(MAPK)path-ways and rebalancing the corresponding immune dysregulation.This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.

Objective:To investigate the correlation between triglyceride-glucose (TyG) index on admission and unfavorable outcomes of patients with moderate-to-severe traumatic brain injury (msTBI) at 6 months postinjury.Methods:A retrospective cohort study was conducted to analyze the clinical data of 277 patients with msTBI admitted to Affiliated Jiangyin Hospital of Nantong University from January 2019 to December 2022, including 208 males and 69 females, aged 18-88 years [(57.0±15.1)years]. Glasgow Coma Scale (GCS) scores on admission were 3-8 points in 168 patients and 9-12 points in 109. According to the Glasgow Outcome Scale-Extended (GOSE) assessment at 6 months after injury, there were 121 patients with unfavorable outcomes (GOSE≤4 points) and 156 with favorable outcomes (GOSE≥5 points). The following indicators of the patients were recorded, including gender, age, history of diabetes, cause of injury, admission GCS, GCS motor score (GCSM), pupillary light reflex, worst Marshall CT classification within the first 24 hours after admission, admission TyG index, Mean Amplitude of Glycemic Excursions (MAGE) within 24 hours after admission, GCSM decline≥2 points within 72 hours after admission, craniotomy or not after admission, and prognosis, etc. TyG index served as the exposure variable focused in this study, which was calculated with fasting triglycerides and fasting blood glucose within 24 hours after admission. The 6-month prognosis of the patients was designated as the outcome variable of the study. After the patients were divided into different groups according to the three quantiles of the TyG index and unfavorable or favorable outcomes, the univariate analysis was conducted on watch variables, and variables with statistically significant differences were included in directed acyclic graphs (DAGs) for further identification of confounding variables. Factors which were found with no statistical significance in the univariate analysis but might affect insulin resistance after injury according to the authors′ previous researches were also included in the DAGs analysis. Three Logistic regression models were designed (Model 1 without correction, Model 2 with core variables of International Mission for Prognosis and Analysis of Clinical Trials in Traumatic Brain Injury (IMPACT) corrected, and Model 3 with confounding variables screened by DAGs corrected) to analyze whether the TyG index was an independent risk factor for the prognosis of msTBI patients. The optimal Logistic regression model was selected and then restricted cubic spline (RCS) was employed to investigate the relationship between the TyG index and the unfavorable outcomes.Results:The univariate analysis suggested that there were significant differences in gender, history of diabetes, MAGE, GCSM decline, and prognosis among the three quantiles of the TyG index ( P<0.05 or 0.01). Significant differences in age, history of diabetes, GCSM, pupillary light reflex, Marshall CT classification, TyG index, MAGE and GCSM decline were observed between unfavorable and favorable outcome groups ( P<0.05 or 0.01). The results of Logistic regression analysis that identified the confounding variables that influenced the correlation between the TyG index and unfavorable prognosis with DAGs suggested that a high TyG index level was significantly correlated with unfavorable outcomes in msTBI patients. Moreover, Model 3 that was corrected with confounding variables screened by DAGs had an optimal goodness-of-fit and adaptability. Model 3-based further RCS analysis indicated that the risk of unfavorable outcomes following msTBI may increase approximately linearly with the increase in TyG index within a certain range (TyG index<9.79). Conclusions:A high TyG index level on admission is the identified as an independent risk factor for unfavorable outcomes of patients with msTBI at 6 months postinjury. As the TyG index level increases, the risk of unfavorable outcomes also rises and may show a linear increasing trend within a certain range (TyG index<9.79).

Objective:To construct a teaching ability evaluation system for diversified general medicine course teachers under the guidance of "one practice and three learning".Methods:A research group was established to search for relevant documents and literature. A framework of evaluation index system was established through drafting and repeated discussion and modification by members of the group, as well as further discuss and modification by experts in general medicine. The framework was used to develop an expert consultation questionnaire on the teaching ability evaluation system for diversified general medicine course teachers under the guidance of "one practice and three learning". Through two rounds of expert consultation, a teaching ability evaluation system for diversified general medicine teachers under the guidance of "one practice and three learning" was constructed. A questionnaire was developed according to the system. The rationality and scientificity of the evaluation index system were verified by questionnaire survey.Results:The teaching ability evaluation system for diversified general medicine teachers under the guidance of "one practice and three learning" included 3 first-level indicators, 15 second-level indicators, and 41 third-level indicators. The general medicine curriculum evaluation questionnaire developed on the system showed that the Cronbach's α coefficient of the overall evaluation system was 0.981. The Cronbach's α coefficients of first-level indicators, including general medicine teaching plan, diversified theory and practice teaching, and comprehensive ability cultivation under the guidance of "one practice and three learning" ideology, were 0.920, 0.919, and 0.923, respectively. The content validity index (S-CVI) of the system was 0.981, and the content validity index (I-CVI) of indicators were 0.826-1.000. The correlation coefficients of first-level indicators and the system were 0.837-0.942 (all P<0.05). The correlation coefficients of second-level indicators and their corresponding first-level indicators were 0.586-0.971 (all P<0.05). The correlation coefficients of third-level indicators and their corresponding first-level indicators were 0.412-0.904 (all P<0.05). Conclusions:Under the guidance of "one practice and three learning", the teaching ability evaluation indicators for diversified general medicine course teachers have high specificity, rationale structure, high feasibility, high reliability, and high practicability. This evaluation system can provide theoretical reference for the training of undergraduate students in general medicine.

Objective To construct a three-dimensional statistical shape model of the pelvis and analyze the individual variation and gender differences of the three-dimensional shape of the pelvis.Methods We collected CT data from 201 Chinese individuals and used deep learning to automatically reconstruct three-dimensional models of the pelvis.Through three-dimensional model registration,dense correspondence mesh mapping,and the use of statistical shape modelling(SSM)and principal component(PC)analysis method,we extracted models of variations(MoV)of pelvic shape changes and statistically compared the shape MoV between males and females.Results We analysed the top 10 principal components of shape variations,which accounted for 86.1％of the total variability.Among them,PC01,PC02,and PC04 showed significant differences between genders(P＜0.001),accounting for a total variability of 60.1％.PC08 and PC 10 demonstrated pelvic asymmetry,accounting for a total variability of 3.8％.Conclusion We constructed a three-dimensional statistical shape model of the pelvis in Chinese individuals,revealing the morphological variation and sex differences of Chinese pelvis.

In order to guarantee the quality of traditional Chinese medicines (TCMs), the crystallization transformation of complex extracts of TCMs and the influence of solid form on their physicochemical properties were studied.The extract of total flavonoids from Pueraria lobata was taken as a model.Crystallization transformation happened when lofting under different conditions, and the intrinsic dissolution tests were carried out.It was found that humidity was the key factor to induce crystallization of total flavonoids from Pueraria lobata.The greater the wettability was, the more the crystallization was.The dissolution rate of total flavonoids from Pueraria lobata with the most crystallization amount significantly decreased by 96.51% compared to the sample without crystallization.After further simulating the preparation process of total flavonoids from Pueraria lobata, it was found that the wet granulation process with introduced water would also lead to crystallization and reduced dissolution rate.As for all crystallization samples, there was an inversely proportional relationship between the dissolution rates and the amount of crystallization.The risk of crystallization existed both in the storage and preparation process of TCM extracts.Crystallization would significantly affect the dissolution rate, and thus the quality of TCM products.In this study, the crystallization transformation of amorphous complex TCM extracts was discovered, and the effect of the crystallization transformation on its dissolution behavior was systematically studied, which provides a new research idea for assuring the quality of TCM products and promoting the improvement of TCM preparation level.