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 investigate the risk factors for identifying local tumor progression(LTP)in patients with hepatocellular carcinoma(HCC)after radiofrequency ablation(RFA)and to establish a predictive model.Methods The clinical data of 122 HCC patients treated by RFA were analyzed retrospectively,and then the patients were divided into positive and negative LTP groups according to that whether LTP occurred within 12 months after RFA.The risk factors of LTP were determined using univariate and multivari-ate analysis,and the predictive model was constructed based on these factors and the internal validation was conducted.Results The results of this study showed that multiple number,diameter＞2 cm,rough margin,and adjacent to large blood vessels of the tumor could be independent predictors of LTP,which were further incorporated into constructing the predictive model.Internal validation results showed that the area under the curve(AUC)of receiver operating characteristic(ROC)curve was 0.815[95％confidence interval(CI)0.735-0.895],indicating the model with high differentiation ability.The calibration curve was drawn and the Hosmer-Lemeshow goodness-fit test showed that the model had good stability(P＞0.05).The decision curve suggested that the model had good clinical application value.Conclusion The independent risk factors of LTP of HCC after RFA are multiple number,diameter＞2 cm,rough margin,and adjacent to large blood vessels of the tumor.When the predictive model is integrated with the above factors,it can poten-tially predict the risk of local tumor and may offer useful guidance for individual treatment and follow-up.

Objective To explore the application of Chinese Drug-Related Problems Classification System in the pharmaceutical care of inpatients in neurology department.Methods A total of 222 inpatients admitted to the Department of Neurology of Affiliated Jinhua Hospital,Zhejiang University School of Medicine from March to June 2022 were selected as the study subjects,and the work records of clinical pharmacists were retrospectively analyzed.The drug-related problems(DRP)were sorted and analyzed according to the Chinese Drug-Related Problems Classification System.Binomial Logistic regression model was used to analyse the influencing factors of DRP.Results Among the 222 patients,76(34.23%)had DRP and a total of 104 DRP were found.The main problem type of DRP was treatment safety(39.42%).The involving objects of DRP were all doctors.In the severity assessment,the most involved level was"DRP reached the patient,but the patient was not harmed"(81.73%).The main reason for DRP was"choice of drugs"(67.31%),followed by"usage and dosage"(26.92%).A total of 62(59.62%)DRP intervention schemes were accepted,and the overall acceptance rate of intervention schemes was 87.32%.In the end,61(58.65%)DRPs were resolved.The drug class with the largest number of DRP cases was antiplatelet drugs.Binomial Logistic regression analysis showed that the number of drug varieties[OR=1.097,95%CI(1.030,1.167)]was the influencing factor for the occurrence of DRP in neurology inpatients(P<0.05).Conclusion DRP is common in inpatients of neurology department.Chinese Drug-Related Problems Classification System is helpful for clinical pharmacists to review and sort pharmaceutical care data,summarize common problems and solving experience,and improve the effect of pharmaceutical care.

Objective To evaluate biomechanical properties of the nickel-titanium (NiTi) memory alloy stent and its in vitro biomechanical properties for lumbar interbody fusion. Methods The mechanical properties of the NiTi memory alloy stent were tested on mechanical testing machine. Moreover, lumbar interbody fusion was simulated on fresh lumbar specimens, and biomechanical properties of the NiTi memory alloy stent with matching bone graft for used for lumbar interbody fusion were analyzed and compared with the traditional box-shape cage. Results The maximum compressive strength of the NiTi memory alloy stent was ( 12 964 ± 962) N. The maximum deformation within the effective range of memory characteristics was (4. 68±0. 03) mm. The recovery rate of the NiTi memory alloy stent was up to 99. 86% . Compared with the intact lumbar model, the stability of the operative segment after the simulated lumbar interbody fusion using NiTi memory alloy stent alone was increased in the direction of anterior flexion, posterior extension, lateral flexion and rotation, which was equivalent to the box shape cage group (P>0. 05). After the combined use of autogenous bone granule and absorbable bone cement the ROM of the operative segment was further reduced (P<0. 05), which was equivalent to the box-shape cage+ unilateral posterior fixation group (P>0. 05). The pull-out strength of the NiTi memory alloy stent with matching bone graft group was significantly stronger than that of the box-shape cage group (P<0. 05). Conclusions The NiTi memory alloy stent in this study was designed with a matched bone granule-absorbable bone cement graft,which provided a new idea for the further optimization and development of lumbar interbody fusion. With excellent support and deformation properties, this NiTi memory alloy stent is biomechanical equivalent to the traditional box shape cage for lumbar interbody fusion, and can greatly improve the stability of surgical segment and the pull-out strength of implants after the combined use of autogenous bone granule and absorbable bone cement.

BackgroundQuality of life， as a major criterion for judging the clinical outcome of ovarian cancer patients， can be affected by adverse psychological symptoms of patients. Meanwhile， fear of disease progression， as a frequent psychological symptom among cancer survivors， is significantly influenced by metacognition， while there is a paucity of research into the specific correlation among the three in patients with ovarian cancer. ObjectiveTo explore the correlation among fear of disease progression， metacognition and quality of life in patients with ovarian cancer， and to test the role of fear of disease progression in the relationship between metacognition and quality of life， so as to provide references for improving the quality of life in patients with ovarian cancer. MethodsA total of 135 patients with ovarian cancer hospitalized in Cangzhou People's Hospital of Hebei Province from January 2019 to December 2022 were selected. All subjects were requested to complete the Functional Assessment of Cancer Therapy-Ovarian Cancer （FACT-O）， Fear of Progression Questionnaire-Short Form （FoP-Q-SF） and Metacognition Questionnaire （MCQ） to assess their quality of life， fear of disease progression and metacognitive level. Pearson correlation analysis was adopted to examine the correlation among the above scales. Process v3.5 macro program was utilized to determine the mediating effect of fear of disease progression on the relationship between metacognition and quality of life， and nonparametric Bootstrap with bias-correction was used to test the mediating effect. ResultsA total of 122 patients （90.37%） with ovarian cancer completed the effective questionnaire survey. Patients scored （90.52±17.13） on FACT-O， （68.52±16.31） on MCQ， and （37.72±8.91） on FoP-Q-SF. Pearson correlation analysis denoted that FoP-Q-SF score was negatively correlated with FACT-O score （r=-0.412， P<0.05） and positively correlated with MCQ score （r=0.241， P<0.05）， and MCQ score was negatively correlated with FACT-O score （r=-0.453， P<0.05）. Analysis demonstrated that the total effect of metacognition on quality of life was -0.298 （95% CI： -0.402~-0.186）. The direct effect of metacognition on quality of life was -0.219 （95% CI： -0.504~-0.277）， accounting for 73.49% of the total effect， and the indirect effect of metacognition on quality of life via fear of disease progression was -0.079 （95% CI： -0.162~-0.037）， accounting for 26.51% of the total effect. ConclusionQuality of life is reduced in patients with ovarian cancer， and fear of disease progression plays a partial mediating role in the relationship between metacognition and quality of life.

Objective : To analyze the expression and immune infiltration levels of the BMP7 gene ( BMP7) in e- sophageal squamous cell carcinoma(ESCC) ． Methods : Initially，in 274 cases of ESCC and 242 cases of normal tissues，the level of BMP7 was verified by immunohistochemistry ，and the relationship between the expression difference and the survival cycle and clinical pathological characteristics of patients with ESCC was explored，and BMP7 overexpression plasmid transfection of ESCC cells was established，and the effect of BMP7 on the biological behavior of ESCC cells was examined by CCK-8，Clone，and Transwell. Results : BMP7 expression in normal e- sophageal tissues was higher than that of ESCC(P＜0. 001) ，the expression level of BMP7 was correlated with the degree of differentiation of patients(P = 0. 006) and TNM staging(P ＜0. 001) ，and the survival of patients with high expression of BMP7 exceeded that of patients with low BMP7 (P = 0. 041) ，and the experiments of CCK-8 and Clone showed that the proliferation effect of cells in the overexpressed BMP7 group was lower than that of the control group．Transwell experiments confirmed that the cell invasion migration capacity of the overexpressed BMP7 group was less than that of the control one．The immune infiltration results showed that BMP7 was positively correlated with macrophages(P = 0. 008) and negatively correlated with γ-δT cells(P = 0. 028) ． Conclusion BMP7 is low in ESCC and associated with poor prognosis and immune infiltration levels in patients.

OBJECTIVE To analyze the influential factors of cardioto xicity in patients with positive breast cancer of human epidermal growth factor receptor 2（HER-2）treated by trastuzumab combined with chemotherapy. METHODS From April 2017 to January 2021，200 HER-2 positive breast cancer patients receiving pirarubicin + cyclophosphamide combined with sequential paclitaxel+trastuzumab were collected from our hospital. According to the presence or absence of cardiotoxicity ，the patients were divided into cardiotoxicity group and non-cardiotoxicity group. The clinical data and echocardiographic results of the patients were collected，and the influential factors of cardiotoxicity were analyzed. RESULTS Among 200 patients，43 patients suffered from cardiotoxicity with the incidence of 21.5％. The proportion of patients with cardiotoxicity during pirarubicin+cyclophosphamide therapy accounted for 5.5％（11/200），and the proportion of patients with cardiotoxicity during sequential paclitaxel+trastuzumab therapy accounted for 20.5％（41/200）；the latter was significantly higher than the former （P＜0.01）. At the same time ，the decrease of left ventricular ejection fraction during sequential therapy of paclitaxel and trastuzumab was significantly higher than that during pirarubicin+cyclophosphamide therapy [ 14％（12％，17％）vs. 7％（3％，10％），P＜0.001]. Compared with patients without cardiotoxicity ，the proportion of patients with cardiotoxicity with a history of hyperlipidemia was significantly higher （P＜ 0.01），while the proportion of patients receiving dexrazoxane was significantly lower （P＜0.01）. Results of binary Logistic regression analysis showed that the history of hyperlipidemia [OR ＝3.672，95％ CI（1.499，8.992），P＝0.004] and the use of dextrazoxane [OR ＝0.154，95％ CI（0.072，0.330）， P＜0.001] were associated with the occurrence of cardiotoxicity. CONCLUSIONS Hyperlipidemia is an independent risk factor for cardiotoxicity induced by pirarubicin + cyclophosphamide combined with sequential paclitaxel+trastuzumab in HER 2 positive breast cancer patients ，while the use of dextrazoxane is a protective factor.

Humans ; Infant, Newborn ; Intensive Care Units, Neonatal ; Logistic Models ; Phenotype ; Risk Factors

Objective:To explore the genetic etiologies of newborn deaths.Methods:A total of 98 newborns who were recruited to the Neonatal Genome Project of the Children′s Hospital of Fudan University and died in the hospital from January 2018 to August 2020 were enrolled in this study. The genetic information and the interventions based on the genetic findings were retrospectively analyzed. T-test, Mann-Whitney U test, Chi square test and Fisher′s exact probability test were used to compare the demographic features and clinical characteristics between the patients with or without a genetic finding. Results:Among 98 newborns (55 males and 43 females), there were 63 preterm and 35 term infants, with a gestational age of (33±5) weeks, a birth weight of (2 107±975) g and the age at death of 12 (2,34) days. Sixteen (16%)patients were identified with genetic variants, including 11 with single nucleotide variants, 4 with copy number variants and 1 with both single nucleotide variant and copy number variant. The detected single nucleotide variants were spanning 12 genes, among which 3 were multiple disorders-related, 2 metabolic disorder-related, 2 hematological disorder-related, 2 respiratory disorder-related, 2 cardiovascular disorder-related and 1 skeletal disorder-related. The patients with a positive genetic finding had significant differences in the birth weight ((2 605±940) vs. (2 009±957) g, t=2.283, P=0.025), the gestational age ((36±5) vs. (33±5) weeks, t=2.131, P=0.036), the age at death ((37 (5, 69) vs. 11 (2, 29) days, Ζ=-2.245, P=0.025) and the history of asphyxia at birth (1/16 vs. 46% (38/82), P=0.002)when compared to those without a genetic finding. In addition, the genetic etiology rates of patients who were born term or with a birth weight ≥ 2 000 g were significantly higher than those who were born preterm (29% (10/35) vs. 10% (6/63), P=0.022) or with a birth weight<2 000 g (25% (13/51) vs. 7% (3/46), χ 2=5.016, P=0.025), respectively. Six cases were medically actionable based on the genetic findings and the treatments included special diet, applying specific medicine, hematopoietic stem cell transplantation and lung transplantation. Conclusions:Genetic etiologies are not rare in newborn deaths and mainly associated with metabolic disorder, multi-system disorders, hematological disorder, respiratory disorder, cardiovascular disorder and skeletal disorder. Some findings are medically actionable, based on which the specific treatments could be scheduled timely. A genetic etiology should be investigated in newborn deaths especially in those who are term birth or with a birth weight ≥2 000 g or without a history of asphyxia at birth.

Objective:To analyze the efficacy and safety of enzyme therapy in late-onset Pompe disease (LOPD) patients, so as to provide reference for the treatment and prognosis of LOPD.Methods:The effect of α-glucosidase (GAA) on a patient diagnosed with LOPD in the Affiliated Hospital of Jining Medical University was observed and analyzed. Besides, literature related to enzyme therapy in LOPD patients were searched in PubMed, Web of Science, Medline databases. Twenty-one studies containing clinical data from 910 LOPD patients related to enzyme therapy were finally included for analysis.Results:The patient developed muscle weakness since he was 16 years old. The GAA activity in peripheral blood was 0. Electromyography suggested myogenic lesions in both lower extremities. Compound heterozygous mutations of GAA gene were found by next- generation sequencing. Muscle biopsy revealed characteristic vacuolar changes. After eight years of diagnosis, the patient was given enzyme therapy for 18.5 months, 20 times in total. The symptoms of muscle weakness were slightly improved in the early stages of treatment without obvious adverse reactions. Most of the 910 LOPD patients were stabilized or had improved muscular and (or) respiratory function following treatment with GAA.Conclusion:GAA treatment is effective and well tolerated. In patients with advanced severe LOPD, enzyme replacement therapy remains effective even years after onset.