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:The minimal ablative margin (MAM) after radiofrequency ablation (RFA) was evaluated based on magnetic resonance imaging (MRI) image registration to analyze its effect on the prognosis of patients with hepatocellular carcinoma (HCC).Methods:Clinical data of 120 patients with HCC undergoing RFA in the General Hospital of Ningxia Medical University from January 2017 to April 2022 were retrospectively analyzed, including 88 males and 32 females, aged (58.4±8.5) years. The enhanced MRI images of patients before and after treatment were imported into a 3D Slicer software to show the ablative margin, and patients were divided into two groups according to whether MAM exceeded the peritumor safety boundary of 5 mm: MAM<5 mm group ( n=75) and MAM≥5 mm group ( n=45). Clinical data were recorded such as gender, age, tumor length and location. Patients were followed up by outpatient review to record whether local tumour progression occurred. Kaplan-Meier method was used for survival analysis, and log-rank test was used for survival comparison. Cox regression analysis was performed to analyze the risk factors of local tumour progression after RFA in patients with HCC. Results:There were significant differen-ces in tumor volume, whether the tumor is located around the vessels, and the mode of RFA guidance between the two groups (all P<0.05). The cumulative local tumour progression-free survival rates at 6, 12 and 24 months after RFA were 100%, 100% and 98% in MAM ≥5 mm group, superior to those in MAM<5 mm group (92%, 84% and 69%, respectively, χ2=47.22, P<0.001). Multivariate Cox regression analysis showed that MAM<5 mm ( OR=9.992, 95% CI: 4.358-22.913), tumor diameter ≥2 cm ( OR=1.758, 95% CI: 1.025-3.015) and perivascular tumor ( OR=2.344, 95% CI: 1.379-3.985) were risk factors for local tumour progression after RFA in patients with HCC (all P<0.05). Conclusion:The MAM evaluated based on MRI image registration is an influential factor on prognosis of patients with HCC. Patients with MAM<5 mm suffer an increased risk of postoperative local tumour progression.

Objective:To construct a prediction model for local tumor progression (LTP) in patients with hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) based on the radiomics features of enhanced MRI.Methods:Clinical data of 120 patients with HCC undergoing RFA in the General Hospital of Ningxia Medical University from June 2017 to June 2022 were retrospectively analyzed, including 90 males and 30 females, aged (58.2±8.2) years. The patients were divided into training set ( n=84) and validation set ( n=36) in a ratio of 7∶3. According to whether LTP occurred within 2 years after RFA, the patients in training set were divided into LTP positive group ( n=32) and LTP negative group ( n=52). Logistic regression analysis was performed to analyze the risk factors for LTP after RFA in patients with HCC in training set. In the advanced arterial phase of preoperative enhanced MRI, the region of interest of tumor and peritumoral 5 mm area were mapped, and the radiomics features were extracted. The maximum correlation-minimum redundancy algorithm, the minimum absolute value shrinkage and selection operator algorithm were used to screen the radiomics features closely related to LTP, and the radiomics score was established. A nomogram model was constructed by combining the radiomics score with clinical tumor characteristics. The predictive performance and clinical practical value of different models were compared by the area under the receiver operating characteristic curve, calibration curve, clinical decision curve analysis (DCA) and clinical impact curve (CIC). Results:Tumor located around the blood vessels ( OR=4.574, 95% CI: 1.454-14.393, P=0.009) and ablation margin <5 mm ( OR=5.724, 95% CI: 1.996-16.420, P=0.001) were independent risk factors for LTP in patients with HCC after RFA. Five higher-order radiomics features were extracted and screened, including three tumoral features (glrlm_ShortRunHighGrayLevelEmphasis, ngtdm_Complexity and glcm_Imc1) and two peritumoral features (firstorder_Mean and glszm_SmallAreaHighGrayLevelEmphasis). Delong test showed that the area under curve of the combined model was higher than that of the radiomics model ( Z=2.90, P=0.004) and the clinical tumor characteristic model ( Z=2.56, P=0.010). Calibration curves, DCA and CIC curves all show that the combined model had a better clinical net benefit. Conclusion:Combining the radiomics features extracted from enhanced MRI images with clinical tumor characteristics can effectively predict the risk of LTP in patients with HCC after RFA.

OBJECTIVE To explore the distribution and drug resistance of pathogens isolated from the diabetes mel-litus patients complicated with skin soft tissue infections(SSTI)so as to provide guidance for reasonable clinical use of antibiotics.METHODS A total of 1177 diabetes mellitus patients with SSTI who were hospitalized in endo-crinology department of Zhongda Hospital Affiliated to Southeast University from Jan.2016 to Dec.2023 were re-cruited as the research subjects.The wound secretion specimens were cultured for pathogens,and the drug suscep-tibility testing was performed.The pathogens isolated in the 8 years and the results of drug susceptibility testing for the main pathogens were divided into the first 4-year group(from 2016 to 2019)and the last 4-year group(from 2020 to 2023).RESULTS Among the 1177 patients with SSTI,the patients with diabetic foot infections ac-counted for 76.10％.Among the 1383 strains of isolated pathogens,there were 815(58.93％)strains of gram-positive bacteria,529(38.25％)strains of gram-negative bacteria,and 39(2.82％)strains of fungi.The constitu-ent ratio of Staphylococcus aureus strains of the last 4-year group was lower than that of the first 4-year group,while the constituent ratio of Pseudomonas aeruginosa strains of the last 4-year group was higher than that of the first 4-year group(P＜0.05).The drug resistance rate of the S.aureus strains to oxacillin and the drug resistance rate of Streptococcus agalactiae strains to erythromycin were increased,and the drug resistance rate of Escherichia coli strains to ampicillin-sulbactam was increased(P＜0.05).The P.aeruginosa strains that were resistant to piperacillin-tazobac-tam,ceftazidime,cefepime,tobramycin,gentamycin,amikacin,ciprofloxacin and levofloxacin were detected in the last 4-year group.Among 7 strains of Candida glabrata,1 strain was resistant to fluconazole,and 2 strains were resistant to itraconazole.CONCLUSIONS There are a great variety of pathogens isolated from the diabetes mellitus patients complicated with SSTI,the gram-positive bacteria are dominant.The drug resistance rates of the major species of pathogens are remarkably higher in the last 4-year group than in the first 4-year group.

Objective:To analyze the clinical features of postnatal cytomegalovirus (pCMV) infection in very preterm infants or very low birth weight infants.Methods:This was a case-control study. A total of 50 very preterm or very low birth weight infants who were hospitalized and diagnosed with pCMV infection in the Neonatal Intensive Care Unit of Children′s Hospital, Zhejiang University School of Medicine from January 2019 to June 2024, were enrolled as the pCMV group. Meanwhile, through propensity score matching, each infant in the pCMV group was paired with a very preterm or very low birth weight infant without cytomegalovirus infection during the same period, constituting the control group, also consisting of 50 cases. Subsequently, the pCMV group was divided into a treated subgroup and an untreated subgroup according to antiviral treatment. Clinical data of all enrolled infants, including clinical features, laboratory test results, and clinical outcomes were collected. Differences in relevant parameters were analyzed using with χ2 test or continuity-corrected χ2 test or Fisher′s exact test, independent-samples t test, Mann-Whitney U test as appropriate. Logistic regression was employed to analyze the risk factors, and Spearman correlation analysis was applied for non-normal distribution data or ordinal data. Results:There were no significant differences between the pCMV group and the control group in terms of gestational age, birth weight, proportion of male infants, Apgar score at the 1 st minute and 5 th minute and days of breastfeeding during the first 3 weeks of life (all P>0.05). Compared with the control group, the duration of hospital stay and invasive mechanical ventilation were both longer in the pCMV group (both P<0.05). The risks of bronchopulmonary dysplasia, retinopathy of prematurity, and hearing impairment were all higher in the pCMV group when compared with the control group(all P<0.05). The body weight and body length of the infants in the pCMV group were both lower than those of in the control group at the corrected gestational age of 36 weeks (both P<0.05). pCMV infections were associated with the increased incidence of both necrotizing enterocolitis ( OR=11.50, 95% CI 1.94-68.30, P=0.007) and severe intraventricular hemorrhage ( OR=6.82, 95% CI 1.19-38.97, P=0.031) in very preterm infants or very low birth weight infants. In the treated group, the platelet count was significantly improved after 6-8 weeks of antiviral treatment compared with that before treatment ((245±19)×10 9/L vs. (119±14)×10 9/L, t=5.37, P<0.001). Conclusions:Very preterm infants or very low birth weight infants with postnatal cytomegalovirus infection have longer hospital stay and duration of invasive mechanical ventilation, and are highly susceptible to bronchopulmonary dysplasia, retinopathy of prematurity, hearing impairment, and growth restriction. Antiviral treatment can effectively ameliorate thrombocytopenia in these infants.

OBJECTIVE To analyze the pathogenic characteristics and clinical features of patients with invasive pul-monary aspergillosis(IPA)and summarize the risk factors for poor prognosis.METHODS The clinical data of 89 patients diagnosed with IPA based on microbial identification of Aspergillus from sputum or bronchoalveolar lav-age fluid cultures and diagnostic criteria,who were admitted to Zhongda Hospital Southeast University from Aug.2022 to Jul.2024 and were queried by electronic case system,were analyzed retrospectively.The distribu-tion of Aspergillus pathogens and clinical features of patients with IPA were analyzed,and the risk factors for poor prognosis were summarized.RESULTS A total of 107 Aspergillus strains were isolated from 89 cases,including 55 strains of Aspergillus fumigatus(51.40％),30 strains of Aspergillus flavus(28.04％)and 10 strains of As-pergillus niger(9.34％).There were 63 m ale patients(70.79％),and 34 cases had poor prognosis.Baseline pe-ripheral blood lymphocyte count(＜0.645×109)(OR=6.653,95％CI:2.013-21.990,P=0.002)and ICU admission(OR=8.303,95％CI:1.283-53.741,P=0.026)were identified as risk factors for poor prognosis in patients with IPA.Receiver operating characteristic curve analysis showed that the cut-off point for baseline pe-ripheral blood lymphocyte count in predicting prognosis in patients with IPA was 0.645 ×109/L,with a sensitivity of 85.22％and a specificity of 69.67％.CONCLUSIONS Baseline peripheral blood lymphocyte count and ICU ad-mission are risk factors for poor prognosis in patients with IPA.The alert value for baseline peripheral blood lym-phocyte count is 0.645 × 109/L,which has good predictive value for the occurrence of poor prognosis.

Objective To establish a technical system for allogeneic kidney transplantation surgery in pigs using the Banna miniature pig inbred strain,and to evaluate it through routine blood tests,liver and kidney function tests,thus providing reference data for the preparation of allogeneic kidney transplantation models.Methods A total of 4 cases of allogeneic kidney transplantation surgeries were performed,including 1 case of single kidney transplantation in a healthy pig,2 cases of kidney transplantation after unilateral nephrectomy,and 1 case of kidney transplantation after bilateral nephrectomy.Before kidney transplantation,cross-matching and complement-dependent cytotoxicity(CDC)tests were used for matching between donor and recipient pigs.After kidney transplantation,peripheral blood samples were regularly collected from pigs for routine blood tests,liver function tests,and kidney function tests,and color Doppler ultrasound technology was used to detect blood supply to the transplanted kidneys.After reaching the experimental endpoint,both kidneys of pig DR1 and the left kidney of pig DR3 were collected and hematoxylin-eosin(HE)staining was performed to evaluate pathological changes in the transplanted kidneys.Results Recipient pigs DR1 and DR3 died at 17 days and 30 days after surgery respectively,while recipient pigs R and DR2 remained in good condition during the 30-day observation period.The results of liver and kidney function test showed that in pig DR1,alanine aminotransferase(ALT)levels increased on postoperative day 1(＞1 000 U/L),peaked on postoperative day 7(1 300 U/L),and aspartate aminotransferase(AST)levels peaked on postoperative day 1(＞3 000 U/L).On postoperative day 17,ALT and AST levels remained high(ALT,500 U/L;AST,700 U/L).In pigs R,DR2,and DR3,ALT and AST levels returned to normal around day 17.Serum creatinine(Crea)levels in pig R remained stable without postoperative increase.Crea levels in pigs DR1 and DR2 showed transient elevation on postoperative day 1,then gradually returned to normal(＜100 μmol/L).Crea levels in pig DR3 remained below 500 μmol/L from postoperative days 2-10,but increased between days 11-28,reaching up to 1 500 μmol/L,indicating gradual loss of kidney function.Ultrasound results showed that the preoperative resistive index(RI)of recipient pig R was 0.91.On postoperative day 24,renal cortex and medulla showed abundant blood flow signals with RI value of 0.88,which was close to the pre-transplantation RI value.For pig DR2,the RI value on postoperative day 17 was 0.89,with poor renal cortex blood flow and relatively good renal medulla blood flow.In pig DR1 on postoperative day 17,no blood flow signals were detected in the transplanted kidney.HE staining results showed that the non-transplanted healthy right kidney of pig DR1 had normal structure,while the transplanted left kidney showed blurred glomerular structure and nuclear dissolution,indicating that the left kidney had lost function before removal.In the transplanted left kidney of pig DR3,large numbers of red blood cells and lymphocyte infiltration were observed in glomeruli and renal tubules,indicating possible coagulation dysfunction and rejection reactions after kidney transplantation.Conclusion Banna miniature pig inbred strain is used as experimental animals to perform four cases of allogeneic pig-to-pig kidney transplantation.The physiological parameters of the recipient pig and the function of the transplanted kidney are monitored after surgery using routine blood tests,liver and kidney function tests,color Doppler ultrasound,and pathological examinations.The allogeneic pig-to-pig kidney transplantation technical system established in the study can provide a foundation for clinicians to conduct kidney transplantation surgeries.

Objective To establish a technical system for allogeneic kidney transplantation surgery in pigs using the Banna miniature pig inbred strain,and to evaluate it through routine blood tests,liver and kidney function tests,thus providing reference data for the preparation of allogeneic kidney transplantation models.Methods A total of 4 cases of allogeneic kidney transplantation surgeries were performed,including 1 case of single kidney transplantation in a healthy pig,2 cases of kidney transplantation after unilateral nephrectomy,and 1 case of kidney transplantation after bilateral nephrectomy.Before kidney transplantation,cross-matching and complement-dependent cytotoxicity(CDC)tests were used for matching between donor and recipient pigs.After kidney transplantation,peripheral blood samples were regularly collected from pigs for routine blood tests,liver function tests,and kidney function tests,and color Doppler ultrasound technology was used to detect blood supply to the transplanted kidneys.After reaching the experimental endpoint,both kidneys of pig DR1 and the left kidney of pig DR3 were collected and hematoxylin-eosin(HE)staining was performed to evaluate pathological changes in the transplanted kidneys.Results Recipient pigs DR1 and DR3 died at 17 days and 30 days after surgery respectively,while recipient pigs R and DR2 remained in good condition during the 30-day observation period.The results of liver and kidney function test showed that in pig DR1,alanine aminotransferase(ALT)levels increased on postoperative day 1(＞1 000 U/L),peaked on postoperative day 7(1 300 U/L),and aspartate aminotransferase(AST)levels peaked on postoperative day 1(＞3 000 U/L).On postoperative day 17,ALT and AST levels remained high(ALT,500 U/L;AST,700 U/L).In pigs R,DR2,and DR3,ALT and AST levels returned to normal around day 17.Serum creatinine(Crea)levels in pig R remained stable without postoperative increase.Crea levels in pigs DR1 and DR2 showed transient elevation on postoperative day 1,then gradually returned to normal(＜100 μmol/L).Crea levels in pig DR3 remained below 500 μmol/L from postoperative days 2-10,but increased between days 11-28,reaching up to 1 500 μmol/L,indicating gradual loss of kidney function.Ultrasound results showed that the preoperative resistive index(RI)of recipient pig R was 0.91.On postoperative day 24,renal cortex and medulla showed abundant blood flow signals with RI value of 0.88,which was close to the pre-transplantation RI value.For pig DR2,the RI value on postoperative day 17 was 0.89,with poor renal cortex blood flow and relatively good renal medulla blood flow.In pig DR1 on postoperative day 17,no blood flow signals were detected in the transplanted kidney.HE staining results showed that the non-transplanted healthy right kidney of pig DR1 had normal structure,while the transplanted left kidney showed blurred glomerular structure and nuclear dissolution,indicating that the left kidney had lost function before removal.In the transplanted left kidney of pig DR3,large numbers of red blood cells and lymphocyte infiltration were observed in glomeruli and renal tubules,indicating possible coagulation dysfunction and rejection reactions after kidney transplantation.Conclusion Banna miniature pig inbred strain is used as experimental animals to perform four cases of allogeneic pig-to-pig kidney transplantation.The physiological parameters of the recipient pig and the function of the transplanted kidney are monitored after surgery using routine blood tests,liver and kidney function tests,color Doppler ultrasound,and pathological examinations.The allogeneic pig-to-pig kidney transplantation technical system established in the study can provide a foundation for clinicians to conduct kidney transplantation surgeries.

OBJECTIVE To analyze the pathogenic characteristics and clinical features of patients with invasive pul-monary aspergillosis(IPA)and summarize the risk factors for poor prognosis.METHODS The clinical data of 89 patients diagnosed with IPA based on microbial identification of Aspergillus from sputum or bronchoalveolar lav-age fluid cultures and diagnostic criteria,who were admitted to Zhongda Hospital Southeast University from Aug.2022 to Jul.2024 and were queried by electronic case system,were analyzed retrospectively.The distribu-tion of Aspergillus pathogens and clinical features of patients with IPA were analyzed,and the risk factors for poor prognosis were summarized.RESULTS A total of 107 Aspergillus strains were isolated from 89 cases,including 55 strains of Aspergillus fumigatus(51.40％),30 strains of Aspergillus flavus(28.04％)and 10 strains of As-pergillus niger(9.34％).There were 63 m ale patients(70.79％),and 34 cases had poor prognosis.Baseline pe-ripheral blood lymphocyte count(＜0.645×109)(OR=6.653,95％CI:2.013-21.990,P=0.002)and ICU admission(OR=8.303,95％CI:1.283-53.741,P=0.026)were identified as risk factors for poor prognosis in patients with IPA.Receiver operating characteristic curve analysis showed that the cut-off point for baseline pe-ripheral blood lymphocyte count in predicting prognosis in patients with IPA was 0.645 ×109/L,with a sensitivity of 85.22％and a specificity of 69.67％.CONCLUSIONS Baseline peripheral blood lymphocyte count and ICU ad-mission are risk factors for poor prognosis in patients with IPA.The alert value for baseline peripheral blood lym-phocyte count is 0.645 × 109/L,which has good predictive value for the occurrence of poor prognosis.

OBJECTIVE To analyze the clinical characteristics of the 12 patients tested with Candida auris in a three-A hospital so as to provide guidance for prevention and control of nosocomial C.auris infection.METHODS The specimens sources and departments where the C.auris strains were isolated as well as the drug resistance were retrospectively collected from Zhongda Hospital Affiliated to Southeast University between Sep.2023 and Jun.2024.The clinical characteristics of the patients with C.auris infection were summarized.The epidemiological survey was conducted for the possibility of its transmission in hospitals,and the corresponding modified preven-tion measures were put forward.RESULTS Among the 12 patients who were tested with C.auris,10 were male,and 2 were female;the age ranged between 21 and 92 years old.Most of the patients were complicated with multi-ple underlying diseases and had the history of repeated hospitalization as well as the history of invasive operation procedures such as tracheotomy,urinary catheterization,gastric tube indwelling and venoarterial catheterization;in the meantime,other multidrug-resistant organisms(MDROs)and fungi were tested in those patients,and the patients used multiple types of antibiotics.Totally 16 strains of C.auris were isolated from the 12 patients with C.auris tested,62.50％(10/16)of which were isolated from urine specimens,18.75％(3/16)were isolated from sputum specimens,and 6.25％(1/16)were isolated from deep venous catheter,tissue biopsy and wound swab specimens.All of the 16 strains of C.auris were resistant to fluconazole and sensitive to caspofungin;4 strains were resistant to amphotericin B.Totally 2 patients had Candida auris infection,one was pulmonary infection,and the other was urinary tract infection,and the infection rate was 16.67％;both patients were cured.There were 10 cases of colonization,and the colonization rate was 83.33％.The result of epidemiological survey indicated that the C.auris that was tested in the No.8 patient may originate from the No.4 patient who carelessly brought from outside the hospital due to poor implementation of standardized prevention measures.CONCLUSIONS The patients tested with C.auris usually have complicated medical backgrounds.Although the colonized C.auris strains are dominant among the strains in the hospital,the related departments need to intensify the implementation of pre-vention measures so as to prevent the transmission of C.auris and pay special attention to the early identification and management of imported pathogens.