Objective To establish RNase L gene knockout HEK 293 cell lines using CRISPR/Cas9 system.Methods Small guide RNA ( sgRNA) sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA(gRNA) vectors were obtained.The donor DNA sequences of the homologous arm were designed for RNase L knockout .In the presence of the right homologous arm , the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair , the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained .The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout .Cells were cultured with hygromycin B , while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome .Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed.Five RNase L gene knockout HEK293 cell lines were generated,contributing to the study of the biological function and molecular mechanism of RNase L .

Objective To investigate the effects of combination of tandospirone,mosapride and Bella ray on the levels of oxygen free radicals and inflammatory cytokines in patients with reflux esophagitis.Methods From March 2016 to March 2017,ninety cases with reflux esophagitis treated in Nanchong Central Hospital were involved in this study.The patients were divided into the observation group and the control group according to different treatment methods,45 cases in each group.Patients in the observation group were treated with tandospirone and mosapride combined with ray Bella with treatment,patients in the control group were treated with Mosapride combined with Bella ray,the therapeutic effect of two groups were observed and compared,oxygen free radical and inflammatory cytokines before and after treatment of the two groups were compared.Results After treatment,MDA and AOPP of the patients in the observation group were(16.51±2.6)U/L and(36.5 ±4.5)μmol/L,significantly lower than those in the control group(MDA:(20.8±2.9)U/L,AOPP:(59.9 ±4.8)mol/L).Glutathione peroxidase(GSH-PX)and superoxide dismutase(SOD)in the observation group were significantly higher than those in the control group((249.4 ± 32.3)mg/L vs.(228.6 ± 17.2)mg/L, (27.3± 3.4)mg/L vs.(18.8 ± 2.7)mg/L),the differences were statistically significant(t=33.5,32.3, 31.4.32.7,P<0.05).After treatment,the levels of TNF-a,IL-6,IL-8 were significantly lower than those in the control group(17.7±2.8)μg/L vs.(26.3±1.5)μg/L,(4.9±0.3)ng/L vs.(6.5±1.8)ng/L,(5.4±0.7) μg/L vs.(6.6±0.8)μg/L),the differences were statistically significant(t=36.3,31.5,32.4,P<0.05).In the observation group,22 cases were significantly effective,19 cases were effective and 4 cases were ineffective, the effective rate was 91.1%,which was significantly higher than that of the control group(86.7%),(χ2=29.5,P=0.02).Conclusion Tandospirone combined with Bella ray can reduce the levels of oxygen free radicals and inflammatory cytokines in patients with reflux esophagitis.

Objective:To analyze the correlation between frailty and health literacy in elderly patients with chronic cardiac insufficiency.Methods:The convenience sampling method was used to select 290 elderly patients with chronic cardiac insufficiency who were hospitalized in the Department of Geriatrics and Department of Cardiovascular Medicine of a tertiary first-class hospital in Wuhu City from Mar 2022 to Jun 2022.The patients were investigated with the general information questionnaire,FRAIL scale,Health Literacy Management Scale,etc.Spearman analysis was used to analyze the correlation between frailty and health literacy.Binary logistic regression were used to analyze the risk factors of frailty in elderly patients with chronic cardiac insufficiency.Results:The incidence of frailty in elderly patients with chronic cardiac insufficiency was 22.8% .Spearman analysis showed that the total score of health literacy was negatively correlated with frailty(r=-0.291,P= 0.000).Results of binary logistic regression analysis showed that health literacy score(OR=0.419,95% CI:0.266-0.908),long-term insomnia(OR=6.466,95% CI:2.099-19.914),nutritional risk(OR=11.202,95% CI:3.983-31.508),depression risk(OR=10.014,95% CI:1.963-51.075),chronic disease types≥5(OR=12.784,95% CI:3.811-42.878),exercise self-efficacy(OR=0.512,95% CI:0.304-0.956),and chronic disease information acquisition ability(OR=0.512,95% CI:0.304-0.956)were independent predictors of frailty in elderly patients with chronic cardiac insufficiency(P＜0.05).Conclusions:The incidence of frailty in elderly patients with chronic cardiac insufficiency is high,and clinical staff should pay more attention to the elderly with frailty,especially patients with long-term insomnia,risk of nutrition and depression,coexistence of chronic diseases,low level of health literacy and exercise self-efficacy.Targeted measures should be actively taken to improve the quality of life of patients and reduce the readmission rate.

Objective To investigate the prognostic significance of the lymphovascular invasion (LVI) in patients with upper tract urothelial carcinoma (UTUC) after radical nephmureterectomy (RNU).Methods A retrospective review was performed on 812 patients who underwent radical nephroureterectomy for UTUC in our hospital from January 1998 to March 2016.Among all the patients,534 were male and 278 female,with median age 67 years old (ranged 25 to 89 years).Three hundred and three patients had hypertension and 119 patients had diabetes.Hydronephrosis was present in 393 patients.445 patients had tumor in left side and the remaining 367 in right side.The tumor was located in the renal pelvis in 422 patients and was ureteric in 319 patients and multifocal in 71 patients.Low pathological grade and high pathological grade was diagnosed in 239 and 573,respectively.The x2 test was used to detect the association between lymphovascular invasion (LVI) and several clinicopathological features.Kaplan-Meier method with the log-rank test was used to assess overall survival (OS) and cancer-specific survival (CSS).Multivariate analysis was conducted using Cox proportional-hazards regression model.Results There were 396 cases with pathological stage Tis +Ta +T1,135 cases T2,257 cases T3 and 24 cases T4.Of all patients,52 had lymph node metastasis.The median follow-up time was 41 months (ranged 2 to 206 months).Of all 812 patients included,110 patients (13.5％) had LVI,while 702 patients (86.5％) were LV1 negative.The 5-year OS and CSS was 44.8％ and 48.9％ for LVI positive group while 70.1％ and 76.0％ for LVI negative group (P ＜ 0.001).Furthermore,there were statistically significant differences between LVI positive group and LVI negative group in hydronephrosis,tumor grade,tumor stage,muscle invasion and lymph node metastasis (P＜0.05).Cox regression showed LVI,advanced age (≥65 year),higher tumor grade,advanced tumor stage (≥ pT2),lymph node metastasis and multifocal tumor were significant prognostic factors in patients with UTUC after RUN.Conclusion UTUC may have a poor prognosis and LVI could be an independent predictor of both OS and CSS.

Objective To investigate the preventive and inhibitory effects of tumor cell lysate(TCL) combined with IL-2 on melanoma and the potential immune mechanism. Methods The B16F10 melanoma TCL cell were prepared using an ultrasonic disruptor. Twenty-four C57BL/6 mice were randomly divided into four groups which were immunized with PBS, IL-2, TCL and TCL+IL-2 for three weeks, and contra lateral tumors were implanted in the fourth week. We observed onset time of tumor and tumor size, collected peripheral blood continuously and monitored the expression of CD4⁺T and CD8⁺T cell subsets dynamically by flow cytometry. Spleen and tumor tissues of mice were also tested for CD4⁺T and CD8⁺T cell subsets by flow cytometry and immunohistochemistry, respectively. Results The preventive immunization of the TCL+IL-2 group significantly delayed the onset time of tumor (P=0.034); moreover, the tumor volume (P=0.023) and tumor weight (P=0.0015) were also significantly smaller than those in the control group. The expression of CD8⁺T cell subsets in the TCL+IL-2 group and the TCL-only group were significantly higher than that in the control group (P=0.0016, P=0.012). However, the CD4⁺T cell subsets of the TCL+IL-2 group decreased after tumor implantation, compared with the control group (P=0.0089). The expression of CD4⁺T and CD8⁺T cell subsets in spleen and tumor tissues were as same as those in peripheral blood. Conclusion The tumor vaccine of TCL combined with IL-2 could prevent the occurrence of melanoma in mouse and effectively inhibit tumor growth by activating CD8⁺T cells.

Objective:To investigate the prognostic significance of tumor architecture in patients with upper tract urothelial carcinoma (UTUC) after radical nephroureterectomy.Methods:A retrospective study was performed on 958 patients who underwent nephroureterectomy in Second Affiliated Hospital of Zhejiang university (156) and Renji Hospital (802) between January 1998 and June 2019. There were 630 males and 328 females with median age 67 years old, ranging 30-89 years old. Among them, 499 patients suffered with preoperative hydronephrosis, 370 patients suffered with hypertension, 120 patients suffered with diabetes, 252 patients had history of smoking and 119 patients had history of non-muscle invasive bladder cancer (NMIBC) or with NMIBC. 489 patients had tumor in renal pelvic, 394 patients had tumor in ureter and 75 patients had tumor in both sites. Laparoscopic surgery was performed in 543 patients while open surgery was performed in 415 patients. The χ 2 test was used to detect the association between tumor architecture and several clinicopathological features. Kaplan-Meier method with the log-rank test was used to assess survival analysis. Multivariate analyses were conducted using Cox proportional-hazards regression model. Results:516 cases (53.9%) showed papillary architecture(Group A) and 442 cases (46.1%) showed sessile architecture(Group B). 543 patients had a tumor ≤3 cm and 415 had a tumor >3 cm. Low pathological grade and high grade was diagnosed in 275 and 683 patients, respectively. The distribution of pathological stage was pT a-1 in 441 cases, pT 2 in 180 cases, pT 3 in 308 cases and pT 4 in 29 cases. Lymphadenectomy was performed in 227 patients and 62 patients were pathologically confirmed lymph node metastasis. 48 patients were found squamous or glandular differentiation. Lymphovascular invasion (LVI) was observed in 150 patients. 134 patients were multifocality. Positive surgical margin was found in 43 patients. Median follow-up was 39 (ranging, 2-206) months. During follow-up, a total of 304 patients died and 236 died of UTUC. 5-year OS and CSS were 76.6% and 81.8%, respectively, in patients with papillary architecture (group A), which were significantly higher than 54.4% and 60.5% in patients with sessile architecture (group B, all P<0.001). Patients in group B had more female patients (38.9% vs.30.3%, P=0.005), ureteral location (47.1% vs. 36.1, P=0.002), hydronephrosis (55.9% vs.48.8%, P=0.030) and postoperative adjuvant chemotherapy (27.1% vs. 14.7%, P<0.001), higher pathological grade (89.6% vs.55.6%, P<0.001) and stage (79.4% vs.32.4%, P<0.001), lymph node metastasis rate (12.0% vs.1.7%, P<0.001), squamous or glandular differentiation (9.5% vs.1.2%, P<0.001) and LVI (24.4% vs.8.1%, P<0.001) than patients in group A. Cox multivariate regression analysis showed that sessile architecture ( P=0.022, 0.028), age ≥65 years ( P<0.001, <0.001), history of diabetes ( P=0.008, 0.043), history of NMIBC or with NMIBC ( P<0.001, <0.001), higher grade ( P=0.002, <0.001), advanced tumor stage ( P=0.003, 0.005), lymph node metastasis ( P=0.003, 0.044), squamous or glandular differentiation ( P=0.008, 0.027) and positive surgical margin ( P=0.003, 0.010) were independent risk factors for OS and CSS. However, tumor >3 cm ( P=0.013, 0.131) and positive LVI ( P=0.045, 0.174) were independent risk factors for CSS rather than OS. Conclusions:UTUC is high malignancy. Tumor architecture was one of an independent risk factor for OS and CSS in UTUC patients and sessile tumors were more malignant, more aggressive and have worse prognosis.

Objective To investigate the effect of subclinical epileptiform discharges (SED) on the cognition of adult patients with epilepsy,exploring the mechanism of SED that leads to cognitive impairment in adult patients with epilepsy to raise physicians' attention about SED.Methods Patients were collected in the Department of Neurology,China-Japan Union Hospital of Jilin University from March 2016 to March 2017.Sixty adult patients with epilepsy without clinical episodes in the last three months were selected as SED group and 40 healthy volunteers as control group.Medical history of the SED group was recorded in detail.All patients were examined by Self-rating Anxiety Scale,Self-rating Depression Scale,Pittsburgh Sleep Quality Index scale in order to exclude organic brain disorders,metabolic diseases,anxiety,depression,sleep disorders and drug-induced cognitive dysfunction.Subjects in the two groups received Montreal Cognitive Assessment (MoCA),electroencephalogram and blood oxygen level dependent functional magnetic resonance imaging examination.Finally,the results were compared between the two groups.Results ①SED had different effects on cognitive function in adult patients with epilepsy,and the MoCA score (26(22,27)) showed statistically significant difference compared with the control group (29 (28,29),Z =-6.26,P =0.00).②Different discharges indexes showed different effects on cognitive function aspects.Cognitive impairment was significant when the discharges indexes were ＞ 10％ (discharges indexes 1％-10％:MoCA score 26(26,28),discharges indexes 10％-50％:MoCA score 22(19.5,25),Z =-4.74,P =0.00).③The cognitive function of epilepsy patients was positively correlated with the duration of education (r =0.41,P =0.00) and the time interval to recent seizure (r =0.31,P =0.02),and negatively correlated with SED (r =-0.57,P=0.17).There was no correlation between cognitive function and duration of disease and onset age.The SED was the main influencing factor of cognitive function in epilepsy.④Compared with healthy people,epilepsy patients with SED showed differences in resting brain function network connection,with strong connective regions at the right inferior temporal gyrus,right hippocampus,bilateral thalamus,with weak connective regions at the double medial upper frontal gyrus,lateral dorsal frontal gyurs.Conclusions SED had an effect on the cognitive function of adult patients with epilepsy.The mechanism of cognitive impairment in adult epilepsy with SED may be related to abnormal brain function in cognitive-related areas.

Objective:To assess the therapeutic efficacy and safety of the gemcitabine combined with nedaplatin (GN) chemotherapy for metastatic human epidermal growth factor receptor-2 (HER-2) negative breast cancer patients.Methods:Forty-five patients with HER-2 negative recurrent metastatic breast cancer who had received prior adjuvant or neoadjuvant therapy with anthracycline and/or taxanes were enrolled. All the patients received GN regime from January 2014 to February 2019. The therapeutic efficacy was evaluated according to response evaluation criteria in solid tumors (RECIST) 1.1. The adverse response was evaluated and monitored according to common terminology criteria for adverse events (CTCAE). The progression-free survival (PFS) and overall survival (OS) and prognostic factors were also analyzed.Results:All of the 45 patients received 4 course GN, 1 of them achieved complete response, 21 achieved partial response. The objective response rate was 48.9 (95% CI: 33.7%-64.1%). Grade 3-4 hematological toxicities include leukopenia occurred in 10 (22.2%) of patients, neutropenia in 13 (28.9%) patients, and thrombocytopenia in 8 (17.6%) patients. The grade 3-4 hematological toxicities mainly manifested as nausea and vomiting, and the incidence was 4.4% (2/45). Among the 45 patients, 34 died, the median PFS was 5.1 (95% CI: 3.9-6.1) months and the median OS was 17.6 (95% CI: 13.1-20.9) months. Conclusion:The combination of gemcitabine and nedaplatin is an effective and tolerable treatment for metastatic breast cancer patients previously treated with anthracyclines and/or taxanes.

Objective:To summarize the clinical features of a family of neurofibromatosis type I (NF1) and its NF1 gene mutation characteristics. Methods:The clinical data of a family of NF1 admitted to our hospital in May 2020 were collected. The proband was sequenced with NF1/ NF2 panel using second-generation sequencing. Sanger sequencing verification analysis was performed on the family members. The clinical characteristics of the proband and other family members were summarized and their gene mutations were analyzed. Results:The proband (V 2), a 1-year and 2-month old girl, had multiple Café au lait spots on the skin at birth, normal mental and motor development, and focal epileptic seizures in infancy. The father (IV 3), grandmother (III 2), and other 2 family members (II 2, I 2) in the family all had Café au lait spots or neurofibromatous changes without seizures. The mother's phenotype was normal. The proband had a heterozygous mutation in the NF1 gene, and the mutation site C.83-84delAG (P.N29Hfs*8) was a frameshift heterozygous mutation. After verification analysis by Sanger sequencing, the pathogenic genes of the father and grandmother were consistent with the proband, which was in line with the characteristics of heterozygous mutation in NF1 gene, dominant inheritance. Conclusion:NF1 is caused by NF1 gene mutation; the early clinical manifestations mostly include café-au-lait spots, and some have seizures; patients with multiple café-aulait spots with seizures should be diagnosed by genetic analysis as soon as possible.

Objective:To assess the therapeutic efficacy and safety of the gemcitabine combined with nedaplatin (GN) chemotherapy for metastatic human epidermal growth factor receptor-2 (HER-2) negative breast cancer patients.Methods:Forty-five patients with HER-2 negative recurrent metastatic breast cancer who had received prior adjuvant or neoadjuvant therapy with anthracycline and/or taxanes were enrolled. All the patients received GN regime from January 2014 to February 2019. The therapeutic efficacy was evaluated according to response evaluation criteria in solid tumors (RECIST) 1.1. The adverse response was evaluated and monitored according to common terminology criteria for adverse events (CTCAE). The progression-free survival (PFS) and overall survival (OS) and prognostic factors were also analyzed.Results:All of the 45 patients received 4 course GN, 1 of them achieved complete response, 21 achieved partial response. The objective response rate was 48.9 (95% CI: 33.7%-64.1%). Grade 3-4 hematological toxicities include leukopenia occurred in 10 (22.2%) of patients, neutropenia in 13 (28.9%) patients, and thrombocytopenia in 8 (17.6%) patients. The grade 3-4 hematological toxicities mainly manifested as nausea and vomiting, and the incidence was 4.4% (2/45). Among the 45 patients, 34 died, the median PFS was 5.1 (95% CI: 3.9-6.1) months and the median OS was 17.6 (95% CI: 13.1-20.9) months. Conclusion:The combination of gemcitabine and nedaplatin is an effective and tolerable treatment for metastatic breast cancer patients previously treated with anthracyclines and/or taxanes.