Objective To establish a method for acquisition, perfusion, preservation and transportation of the genetically modified pig kidneys. Methods An eight genetically modified pig was utilized as experimental subject. Prior to kidneys procurement, the health status of the pig was assessed through hematology examination, and the vascular structure of the kidneys was examined using imaging techniques. Following kidneys acquisition, the pig kidneys were perfused and subsequently packaged into the cryogenic storage container labeled "For Organ Transportation Only" for interprovincial transport after communicating the transportation process with transportation department. To evaluate pathological damage to the pig kidneys, a serious of methods were employed such as hematoxylin-eosin (HE) staining, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorescence staining and enzyme-linked immune absorbent assay (ELISA). Results The preoperative examination of the eight genetically modified pig showed that the serum creatinine was 73.2 μmol/L, blood urea nitrogen was 2.8 mmol/L and hemoglobin was 116 g/L, all within the normal range, indicating normal renal function. CT angiography revealed no lesions in the pig kidneys, and no dilation, stenosis or premature branching of the blood vessels. The total time of obtaining the left and right kidneys from the eight genetically modified pig was (125 ± 10) min, with a blood loss of (20 ± 2) mL. The warm ischemia times were 3 min and 7 min, respectively. The perfusion and trimming times of the left and right kidneys were 36 min and 41 min, respectively. After perfusion, both kidneys were white and moist. The cold preservation and transportation time was 8 h. HE staining showed that some glomeruli were shrunk, and the lumens of the surrounding renal tubules were slightly depressed and swollen with partial inner membrane shedding and microvacuoles formed when the kidneys were preserved for 8 h. The level of cysteinyl aspartate-specific proteinase-3 messenger RNA in the kidneys tissue gradually increased with the extension of cold preservation time after 2 h (P<0.05). TUNEL fluorescence staining showed that only a small number of cells underwent apoptosis after 8 h of cold preservation, which was not significantly different from that at 0 h (P>0.05). ELISA results showed that the contents of lactate dehydrogenase (LDH) and creatinine in the preservation solution remained relatively stable, but the content of kidney injury molecule 1 (KIM-1) gradually increased with the extension of preservation time, suggesting that the pig kidneys had mild injury. Conclusions By establishing methods for acquisition, perfusion, preservation and transportation of the kidneys from genetically modified donor pig, it is possible to effectively and reliably use genetically modified pig kidneys for xenotransplantation.

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.

BACKGROUND:Bibliometrics and visual analyses based on thematic literature are particularly important for understanding the foundation and frontiers of postmenopausal osteoporosis research. OBJECTIVE:To perform bibliometric,citation,and visualization analyses of highly cited SCI papers in postmenopausal osteoporosis research over the last 20 years. METHODS:The top 100 highly cited papers on postmenopausal osteoporosis published between 2003 and 2022 included in SCI-EXPANDED catalog of the Web of Science database were obtained for bibliometric measure and visual analysis using CiteSpace software. RESULTS AND CONCLUSION:The top 100 highly cited papers have a total of 67 377 citations in the Web of Science Core Collection,with an annual average of 49.17 citations per paper.Postmenopausal osteoporosis research primarily involves medical,engineering,biological,and multidisciplinary fields.The subcategories are dominated by endocrinology and metabolism,and medicine:internal medicine.Stable and close cooperative network relationships have been formed globally.United States,University of California System,Cummings,and Steven R are the country,research institution,and author,respectively,with the most highly-cited publications.The frontiers of postmenopausal osteoporosis research mainly include calcium and vitamin D supplementation and fracture risk,clinical studies of bisphosphonates in the treatment of postmenopausal osteoporosis,atypical femur fracture,clinical studies of new drugs and sequential treatment of postmenopausal osteoporosis,predictors of fracture risk,mid-and long-term follow-up of osteoporotic vertebral compression fractures,genetic polymorphisms and hereditary factors,formulation and updating of clinical practice guidelines for postmenopausal osteoporosis.Large cohort studies,high-quality randomized controlled trials,systematic reviews,meta-analyses,and clinical practice guidelines are the great engines that drive the development of clinical research in postmenopausal osteoporosis.We should make efforts in the above areas to improve China's international influence in the field of osteoporosis.

Objective To evaluate the efficacy of endovascular treatment for ruptured descending thoracic aortic aneurysm(rDTAA),and to summarize the treatment experience.Methods The clinical data of 8 patients with rDTAA,who were admitted to the Department of Vascular Interventional Surgery,Zhangjiajie Municipal People's Hospital of China to receive treatment between January 2016 and June 2023,were retrospectively analyzed.The patients included 4 males and 4 females,with a median age of 70.5 years(61-78 years).Preoperative computed tomography angiography(CTA)was performed in all the 8 patients,and the diagnosis of rDTAA complicated by hematoma in the thoracic cavity and/or mediastinum was confirmed.Emergency thoracic endovascular aortic repair(TEVAR)was carried out in all the 8 patients.Results Complete isolation of the thoracic descending aortic aneurysm was achieved in 6 patients,and 2 of them received reconstruction of left subclavian artery by using chimney stent technique.The remaining two patients had a small amount of type Ⅰa endoleak,which disappeared after implantation of a cuff stent at the proximal site.During hospitalization,one patient died of septic shock caused by thoracic aortic-esophageal fistula,and 3 patients developed respiratory failure after operation,which was recovered after thoracic drainage,anti-infection medication and ventilator-assisted breathing therapy.Two patients developed postoperative ischemic stroke,one of them presented as grade Ⅲ motor muscle weakness on the affected side accompanied by speech impairment,and the another patient presented as grade Ⅳ motor muscle weakness on the affected side accompanied by speech impairment.During the follow-up period,one patient died of sudden chest pain 7 months after surgery,and another patient who had not received chimney stent implantation developed type Ⅰa endoleak 18 months after operation,which disappeared after implantation of a cuff stent at the proximal site.The remaining 5 patients showed good clinical condition during the follow-up period.Conclusion For the treatment of rDTAA,TEVAR is clinically safe and effective.

Humans ; Clavicle ; Fractures, Bone/surgery*

Objective To summarize our experience with surgical management of renal neoplasm involving inferoir veno cava.Methods We review the data of 115 patients,including 74 male patients and 41 female patients,with renal neoplasm involving venous system between March 1993 and December 2015.The mean age was 52 years old,ranging 22 to 77 years old.The tumor was found in right side in 77 cases and left side in 38 cases.There were 15 patients (13.0％) with renal vein thrombus,38 (33.1％) with infrahepatic thrombus,29 (25.2％) with low retrohepatic thrombus,20 (17.4％) with high retrohepatic thrombus,and 13 (11.3％) with supradiaphragmatic thrombus.The mean age was 52 years old,ranging 22 to 77 years old.The tumor was found in right side in 77 cases and left side in 38 cases.All patients accepted the radical nephrectomy.Primary outcomes were overall survival (OS),and cancer special survival (CSS) in patients with renal cell carcinoma (RCC) estimated by Kaplan-Meier method.Secondary outcome included operative and oncological features,past-operative complications and hospital mortality.Cox proportional hazard model was used to univariate and multivariate analysis for risk factor impacting on OS of RCC patients.Results Complete resections of renal neoplasm with tumor thrombus were achieved in 113 patients (98.3％),2 patients died intraoperatively due to the dropping of thrombus.Postoperative complication rate was 16.8％.Hospital mortality was 2.6％.Mean follow-up interval was 48 months.OS rates at 5-,and 10-year in RCC patients were 66.5％,and 61.8％,respectively.Metastasis was an independent risk factor affecting on OS (P =0.000).However,the level of thrombus was not an risk factor affecting the prognosis.Conclusions Radical resection of renal tumor and caval thrombus is an effective treatment for prolonging survival in patients with RCC extending into venous system.Retrohepatic caval tumor thrombus below or above the main hepatic vein should be dichotomized and managed respectively with diverse techniques.Metastatic rather than the level of thrombus was a risk factor impacting on RCC patient survival.

Objective To investigate prevalence and epidemiologic features of Saffold virus (SAFV) in hospitalized children with acute respiratory infection or digestive tract infection Tianjin area. Methods Nasopharyngeal aspirates from children with acute respiratory infection and fecal samples from children with digestive tract infection in Tianjin Children ’s Hospital were collected from January 2013 to December 2013. Viral nucleic acid was extracted, and SAFV infection was determined by using real-time quantitative PCR. Positive PCR products were sequenced. The sequencing results were aligned with known gene sequences of SAFV sequences in GenBank. The positive viral infection rate of nasopharyngeal aspirates and fecal samples, viral positive constituent ratio and positive detection rate in different age groups, seasonal distribution of SAFV infection were calculated. Other common respiratory tract or digestive tract viruses were also detected. Results Fourty-three (11.9%) nasopharyngeal aspirates from children with acute respiratory infection tested positive for SAFV. There was no significant difference between male and female infected children (aged between 6 d and 12 years old). The 79%(34/43) of the patients with SAFV infection aged under 1 year old. The infection most occurred in summer and winter. The 63 (16.4%) fecal samples from children with digestive tract infection tested positive for SAFV. There was significant difference between male and female infected children (aged between 5 h and 11 years old). SAFV infection was found to be year round. There was no significant difference in different age groups of nasopharyngeal aspirates and fecal samples. The mixed infection rate with SAFV and other respiratory tract or digestive tract viruses were 7.0%(3/43)and 12.7%(8/63), respectively. Conclusion Infection of SAFV had occurred in children with acute respiratory infection or digestive tract infection in Tianjin. SAFV has high detection rate in these children and is more common in children
aged under 1 year old. The data suggest that some of acute respiratory infection or digestive tract infections in pediatric patients are related to SAFV. The Clinical doctors should pay attention to them .

Adenoma, Liver Cell ; Gallbladder Diseases ; Histiocytosis, Sinus ; Humans ; Liver Neoplasms

Humans ; Male ; Middle Aged ; Tuberculoma ; Tuberculosis, Hepatic

Carbonyl reductases catalyze carbonyl compounds to chiral alcohols that are important building blocks in fine chemical industry. To study carbonyl reductase from Pichia pastoris GS115 (ppcr), we discovered a new gene (ppcr) encoding an NADPH-dependent carbonyl reductase by genomic data mining. It was amplified by PCR from the genomic DNA, and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity. The optimum temperature was 37 degrees C and the optimum pH of PPCR was 6.0. PPCR was stable below 45 degrees C. The Km and k(cat) value of the enzyme for ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s, respectively. The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, a-ketoesters, beta-ketoesters and aryl ketones to give the corresponding alcohols with >97% ee with only a few exceptions, showing its application potential in the synthesis of chiral alcohols.
Alcohol Oxidoreductases ; biosynthesis ; chemistry ; genetics ; Amino Acid Sequence ; Biotechnology ; methods ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Pichia ; enzymology ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Stereoisomerism ; Substrate Specificity ; Temperature