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 To investigate the association between D-dimer levels within 2 hours of admission and in-hospital major adverse events (MAEs) in patients with acute type A aortic dissection (ATAAD) who underwent total arch replacement combined with frozen elephant trunk (FET) implantation. Methods This retrospective study included patients with ATAAD who underwent total arch replacement with FET implantation at Fuwai Yunnan Cardiovascular Hospital from September 2017 to December 2022. Patients were divided into two groups based on the occurrence of in-hospital MAEs: a MAEs group and a non-MAEs group (control). Perioperative data were compared between the two groups. Univariate and multivariate logistic regression analyses were performed to identify risk factors for in-hospital MAEs, which included in-hospital death, gastrointestinal bleeding, paraplegia, acute kidney injury, low cardiac output syndrome, stroke, respiratory failure, multiple organ dysfunction syndrome, and severe infection. The predictive value of D-dimer was evaluated using the area under the receiver operating characteristic curve (AUC). Results A total of 218 patients were included (157 males, 61 females), with a mean age of (51.54±9.79) years. There were 152 patients in the non-MAEs group and 66 in the MAEs group. The overall incidence of in-hospital MAEs was 30.3%, and the in-hospital mortality rate was 2.8% (6/218). Compared to the non-MAEs group, the MAEs group had significantly higher levels of D-dimer and lactate, as well as longer cardiopulmonary bypass time, aortic cross-clamp time, and ICU length of stay (all P<0.05). Multivariate logistic regression analysis identified D-dimer as an independent risk factor for in-hospital MAEs [OR=1.077, 95%CI (1.020, 1.137), P=0.013]. The AUC for the D-dimer level within 2 hours of admission to predict in-hospital MAEs was 0.83 [95%CI (0.736, 0.870), P<0.001]. The optimal cutoff value was 2.2 μg/mL, with a sensitivity of 84.8% and a specificity of 73.0%. Conclusion The serum D-dimer level is an independent risk factor for in-hospital MAEs in patients with ATAAD following total arch replacement with FET implantation. D-dimer levels on admission can help clinicians optimize risk stratification and perioperative management, potentially reducing the incidence of early adverse events.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent inflammation and joint damage, accompanied by the accumulation of plasma cells, which contributes to its pathogenesis. Understanding the genetic alterations occurring during plasma cell differentiation in RA can deepen our comprehension of its pathogenesis and guide the development of targeted therapeutic interventions. Here, our study elucidates the intricate molecular mechanisms underlying plasma cell differentiation by demonstrating that PRDX1 interacts with DOK3 and modulates its degradation by the autophagy-lysosome pathway. This interaction results in the inhibition of plasma cell differentiation, thereby alleviating the progression of collagen-induced arthritis. Additionally, our investigation identifies Salvianolic acid B (SAB) as a potent small molecular glue-like compound that enhances the interaction between PRDX1 and DOK3, consequently impeding the progression of collagen-induced arthritis by inhibiting plasma cell differentiation. Collectively, these findings underscore the therapeutic potential of developing chemical stabilizers for the PRDX1-DOK3 complex in suppressing plasma cell differentiation for RA treatment and establish a theoretical basis for targeting PRDX1-protein interactions as specific therapeutic targets in various diseases.

Osteoporosis is a prevalent skeletal condition characterized by reduced bone mass and strength, leading to increased fragility. Buqi-Tongluo (BQTL) decoction, a traditional Chinese medicine (TCM) prescription, has yet to be fully evaluated for its potential in treating bone diseases such as osteoporosis. To investigate the mechanism by which BQTL decoction inhibits osteoclast differentiation in vitro and validate these findings through in vivo experiments. We employed MTS assays to assess the potential proliferative or toxic effects of BQTL on bone marrow macrophages (BMMs) at various concentrations. TRAcP experiments were conducted to examine BQTL's impact on osteoclast differentiation. RT-PCR and Western blot analyses were utilized to evaluate the relative expression levels of osteoclast-specific genes and proteins under BQTL stimulation. Finally, in vivo experiments were performed using an osteoporosis model to further validate the in vitro findings. This study revealed that BQTL suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and osteoclast resorption activity in vitro in a dose-dependent manner without observable cytotoxicity. The inhibitory effects of BQTL on osteoclast formation and function were attributed to the downregulation of NFATc1 and c-fos activity, primarily through attenuation of the MAPK, NF-κB, and Calcineurin signaling pathways. BQTL's inhibitory capacity was further examined in vivo using an ovariectomized (OVX) rat model, demonstrating a strong protective effect against bone loss. BQTL may serve as an effective therapeutic TCM for the treatment of postmenopausal osteoporosis and the alleviation of bone loss induced by estrogen deficiency and related conditions.
Animals ; NFATC Transcription Factors/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Ovariectomy ; Osteoclasts/metabolism* ; Female ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; NF-kappa B/genetics* ; Osteoporosis/genetics* ; Signal Transduction/drug effects* ; Bone Resorption/genetics* ; Cell Differentiation/drug effects* ; Humans ; RANK Ligand/metabolism* ; Mitogen-Activated Protein Kinases/genetics* ; Transcription Factors

ObjectiveTo explore the current situation and influencing factors of humanistic care experience among patients visiting traditional Chinese medicine （TCM） outpatient clinics in China， and to provide a basis for optimizing TCM-characterized services in both TCM and Western medicine hospitals. MethodsA multi-center cross-sectional study was conducted using convenience sampling to select 35 hospitals across 13 provinces in China （including 3 TCM hospitals and 32 TCM outpatient clinics in general hospitals）. A total of 3，430 patients were surveyed using the general information questionnaire and the Outpatient Humanistic Care Experience Questionnaire， with data collected via Questionnaire Star. Univariate analysis and multiple linear regression were employed to examine the impacts of patient characteristics， visit characteristics， hospital type （TCM hospital/general hospital）， and geographic region （eastern/central/western） on humanistic care experience. ResultsThe total score of humanistic care experience was 194 （188， 233）. Univariate analysis showed that gender， educational level， current residence， per capita monthly household income， location attribute of medical institutions， number of previous visits to this hospital， payment method of medical expenses， previous hospitalization history in this hospital， frequency of outpatient visits within the past 12 months， self-rated disease severity， familiarity with the outpatient procedures， implementation of the follow-up service provided by the hospital， satisfaction with follow-up services， the grade of the hospital visited， geographical region of the hospital visited， and the department visited had an impact on the humanistic care experience during outpatient visits （P<0.05）. Multivariate analysis demonstrated that educational level （β=0.609， P=0.011）， self-rated disease severity （β=-0.646， P=0.047）， familiarity with outpatient procedures （β=4.784， P<0.001）， satisfaction with follow-up services （β=6.365， P<0.001）， and the grade of the hospital visited （β=-5.487， P<0.001） affected the humanistic care experience in outpatient medical treatment， explaining 24.4% of the total variation. ConclusionHumanistic care experience in TCM outpatient clinics is influenced by multiple factors. It is recommended to optimize the medical treatment process， strengthen doctor-patient communication training， and establish a precise follow-up mechanism， with a focus on improving care perceptions among patients with lower education levels and those attending primary-level hospitals， to refine the TCM-characterized service system.

Objective To evaluate the effectiveness of a 1470 nm semiconductor laser therapeutic instrument(referred to as a curestar therapeutic instrument)for prostatectomy in Beagle dogs.Methods Twenty-eight adult male Beagle dogs were randomly divided into three groups:sham(n=3),experimental(n=15),and control(n=10).The experimental group was further divided into three subgroups:120 W/50 W,150 W/50 W,and 160 W/50 W for vaporization cutting/coagulation hemostasis.The control group was divided into two subgroups:120 W/50 W and 150 W/50 W with five in each subgroup.Experimental and control groups underwent canine prostatectomy through the entrance of the bladder neck under electrocision.The operational suitability and effectiveness of the product during surgery were assessed.After the operation,the general condition of the dogs was observed,and blood biochemical and hematological indicators were measured before,immediately after,and at 3,7,and 28 days after the operation.At 1 h and 4 weeks after surgery,B-ultrasound and electric resection were performed under anesthesia to observe the conditions of the urethra and prostate,and prostatic tissue was subjected to HE staining for pathological observations.The thickness of the coagulation layer at 1 h after the operation and repair of the urothelial epithelium at 4 weeks were analyzed.Results During the operation,experimental and control groups had good operability and showed good vaporization cutting and coagulation hemostasis performance.After the operation,no significant effects were observed on the general condition,and blood biochemical and hematological indicators of the dogs.Ultrasound showed that the urethral expansion was visible immediately after the operation,and the echo of the urethral epithelium was slightly enhanced.At 4 weeks,the prostate tissue had a slightly low echo with uniformly distributed small point-like echoes inside,and the capsule had a linearly high echo,consistent with the sham group.The weight of the vaporized prostate tissue in experimental and control groups was 0.91～1.33 g with a resection rate of 17.11％～20.27％.As the power of vaporization cutting increased,the laser emission time gradually decreased,while the vaporization cutting speed and efficiency both increased.However,no significant difference was found between experimental and control groups(P＞0.05).Under the electrocision microscope,a burn-like change was observed in the surgical wounds of the prostate urethra in experimental and control groups at 1 h after surgery,and the boundary between the wound and normal urothelium was visible.At 4 weeks,the urothelium of the prostate had been repaired and flattened,and the boundary with the surrounding normal urothelium was blurred.Similarly,pathological observations showed that experimental and control groups had significant damage to the prostate urethral orifice at 1 h after surgery with a small amount of carbonization and coagulative necrosis on the surface of the wound,a small amount of inflammatory cell infiltration,and a coagulation layer thickness of approximately 0.4 mm.At 4 weeks,the prostate urethral morphology of the sham group was normal,whereas experimental and control groups showed new epithelial growth covering the wound with a uniform thickness and no coagulative necrosis tissue attached to the wound.A mild inflammatory reaction was still present in the surrounding area,fibroblast proliferation was obvious,and stromal and epithelial cell proliferation was visible in the surrounding prostate,some of which showed squamous metaplasia.The prostate capsule was intact and the morphology of the surrounding nerves and blood vessels was normal.Conclusions The curestar therapy instrument is effective for prostatectomy in Beagle dogs with good vaporization cutting and coagulation hemostasis performance.No significant difference was found in postoperative physiological indicators compared with the sham group.

Objective:To construct a comprehensive material management system for public hospitals to meet the needs of material homogeneity and refined management under the hospital's multi-campus development model.Methods:By systematically sorting out the process and each link of the whole life cycle of hospital material management,using Java programming language,browser and server(B/S)architecture,setting up the integrated management platform module of medical equipment,the process module of each link of the material management system and the authority management module,the comprehensive material management system of public hospitals was constructed.The information call,mutual connection and restriction mechanism between the component modules were determined,and the dynamic real-time usage data and status of hospital materials were uniformly supervised.The homogeneity and fine management effects of hospital materials in Beijing Children's Hospital Capital Medical University before and after the application of the system were compared.Results:The integrated material management system of public hospitals realized full lifecycle coverage of hospital materials,the collaboration between various business modules and the smooth operation of the system,and realized the coordination of basic information,business and resources of hospital materials,as well as the homogeneity,refinement and integrated management of cross-campus materials.After the application of the system,seamless real-time data docking between different hospital campuses was realized,the average time of receiving materials in clinical departments was shortened by 2 hours compared with that before the application of the system,the review efficiency of material warehousing data,consumption data and inventory data was increased by 30%,and the backtracking error rate of the whole process of materials was reduced to 0.2%.Conclusion:The comprehensive material management system of public hospitals can improve the efficiency of material management in the operation of multiple hospital campuses,reduce management costs,and realize the homogeneity,refinement and scientificization of hospital material management.

ObjectiveSpatial working memory (SWM) is an important function in cognitive behavior, and working memory impairment can seriously affect the patient’s life and cause great stress to the patient. Intermittent theta burst stimulation (iTBS) has been shown to regulate working memory function by entrainment of neural oscillations in different frequencies of the brain, but its regulation of working memory-related neural oscillations and their synchronization is not clear. The purpose of this study was to study the effect of iTBS on neural oscillation and synchronization in local and transbrain regions of rats, and to explore the mechanism of iTBS in regulating working memory. MethodsTwenty-four rats were randomly divided into four groups according to their age and whether they received iTBS stimulation (AS: adult stimulation group, AC: adult control group, ES: elderly stimulation group, EC: elderly control group). Using the methods of time-frequency distribution, phase synchronization and phase-amplitude coupling analysis, the changes of local field potential signal neural oscillations in the prefrontal and hippocampal brain regions of theta and gamma bands in the process of spatial working memory behavioral tasks in each group of rats were compared and analyzed, and the relationship between the changes of neural oscillations in the two brain regions and the changes in spatial working memory ability of rats was judged based on the Pearson correlation coefficient. ResultsWith the increase of age, the time taken by the elderly rats to learn the spatial working memory task rules increased significantly (P=0.005 6), and the time taken by iTBS stimulation to learn the SWM task rules in adult rats (P=0.001 1) and elderly rats(P=0.009 0) was shortened. At the same time, compared with adult rats, the time-frequency energy of theta and gamma band neural oscillations in the prefrontal and hippocampal brain regions of elderly rats (theta: P<0.000 1; gamma: P<0.000 1) and phase-amplitude coupling across brain regions (PFC-HPC: P=0.000 2; HPC-PFC:P=0.027 7) decreased to a certain extent, and iTBS stimulation could increase the time-frequency energy of neural oscillations of adult rats (theta: P<0.000 1; gamma: P<0.000 1) and elderly rats (theta: P=0.014 4; gamma:P=0.000 6) and the phase-amplitude coupling effect across brain regions in elderly rats (PFC-HPC: P=0.018 0; HPC-PFC: P=0.022 1). In addition, the time-frequency energy and phase-amplitude coupling of signals in each frequency band of the two brain regions were positively correlated with the behavioral accuracy of rats, while the phase synchronization of theta band and gamma band neural oscillations in the two brain regions during working memory was not correlated with the behavioral accuracy. ConclusioniTBS can enhance SWM ability and cognitive function in elderly rats, and this improvement is associated with increased coupling of time-frequency energy and cross-brain phase amplitude of neural oscillations across theta and gamma bands during SWM tasks. Similarly, in adult rats, iTBS enhances SWM ability and cognitive function by increasing the time-frequency energy of theta and gamma band neural oscillations in both brain regions during SWM tasks. Furthermore, in addition to the main findings, this study provides evidence supporting the state-dependent effects of iTBS stimulation to some extent.