Objective To develop a loop-mediated isothermal amplification method for rapid diag-nosing Vibrio cholerae O139 in inspection department and small-scale laboratory. Methods Four primers (2 inner primer and 2 outer primer) for the LAMP test were designed by targeting the wbfR gene of Vibrio chol-erae O139, and the reaction condition and reaction system of LAMP were optimized. Thirty Vibrio cholerae O139, 13 Vibrio cholerae reference strains, 10 O1 biotype Vibrio cholera* and 32 other enterobacterias were analyzed and the LAMP results were determined by visual inspection or electrophoretie analysis . Results All of the Vibrio cholerae O139's amplification products were observed as green by visual inspection and had a ladder-like pattern on the gel, but O1 biotype Vibrio cholera* and other enterobacteria's products were dis-played as orange by visual examination and had no ladder-like pattern on the gel. In addition, the reaction time of the LAMP method was only 1.5 h and the detection limit of this assay was 63 CFU/reaction. Con-clnsion LAMP assay targeting the wbfR gene of Vibrio cholera* O139 is rapid, specific, and sensitive for the detection of Vibrio cholerae O139. This method not only reduced the dependence of complicated equip-ment but also had a potential for wider use in inspection department, small-scale laboratory, emergency mo-tor vehicles and field survey.

Objective To evaluate effects of acitretin and interferon?α(INF?α)alone or in combination on the proliferative activity of and interleukin?15 expression in human cutaneous T?cell lymphoma Hut78 cells. Methods Cultured Hut78 cells were divided into several groups, including blank control group, negative control group, dimethyl sulphoxide (DMSO) group and experimental groups. Cells in experimental groups were additionally classified into several subgroups to be treated with acitretin(0.1-10μmol/L, acitretin groups)or INF?α(5 000-20 000 IU/ml, INF?αgroups) alone, or the combination of 1.0 μmol/L acitretin and IFN?α at concentrations of 5 000- 20 000 IU/ml (combination groups), for 24, 48 and 72 hours. Subsequently, cell counting kit 8(CCK8)assay was performed to assess the proliferative activity of Hut78 cells, and enzyme?linked immunosorbent assay(ELISA)to measure the expression of IL?15 in these cells. Results The proliferative activity of and IL?15 expression in Hut78 cells were both obviously suppressed in the acitretin groups and combination groups compared with the DMSO group, as well as in the INF?αgroups compared with the negative control group, and the inhibitory effects gradually increased with the increase in acitretin or INF?αconcentrations and treatment durations. As repeated measures analysis of variance revealed, there was a significant difference in both proliferation inhibition rates and IL?15 expression among different treatment durations and among different concentrations of acitretin or INF?α(all P<0.05), and there was an interaction effect between treatment durations and drug concentrations(all P<0.05). A significant difference was observed in both proliferation inhibition rates and IL?15 expression at 24, 48 and 72 hours when the 1.0?μmol/L acitretin+ 10 000/20 000?IU/ml IFN?αgroup was compared with the 1.0?μmol/L acitretin group and 10 000/20 000 IU/ml IFN?αgroup(all P<0.05). There was also a significant difference in IL?15 expression at 24, 48 and 72 hours between the 1.0?μmol/L acitretin+50 000?IU/ml IFN?αgroup and 5 000?IU/ml IFN?αgroup(all P<0.05). Conclusions Acitretin and IFN?αboth can inhibit the proliferation of and IL?15 expression in Hut78 cells, the inhibitory effects are enhanced with the increase in drug concentrations and treatment durations, and the combination of acitretin and IFN?α appears to have stronger inhibitory effects than acitretin or IFN?αalone.

Objective To explore whether hypertonic saline would partake in regulating Notch signaling in microglia in experimentally induced cerebral ischemic rats.Methods Male SD rats were randomly divided into sham group, cerebral ischemia group, normal saline group ( NS group ) , 10%hypertonic saline group (10%HS group) , the model of cerebral ischemia were established in all rats except the sham group by using middle cerebral artery occlusion ( MCAO) .After 2 hours of MCAO, the rats were through reperfusion for 24 h.In addition, rats in the normal saline group and 10% HS group were respectively treated with a continuous intravenous injection of normal saline (0.3 mL/h) and 10%HS (0.3 mL/h) by tail vein for 24 h.Immunofluorescence methods, RT-PCR and Western blot were used to detect the expression of Notch1 and intracellular Notch receptor domain ( NICD) .All data was analyzed by one-way analysis of variance ( ANOVA) , The intergroup comparisons were analyzed by the least-significant-difference (LSD) tests.Differences were considered statistically significant if P<0.05.Results Immunofluorescence showed that the expression of Notch1 and NICD were significantly increased in the microglia around peri-ischemia area in cerebral ischemia group and normal saline group compared to sham group;the expression of Notch1 and NICD in the microglia around peri-ischemia area were significantly reduced in 10% HS group compared to ischemia group and NS group.RT-PCR showed that the mRNA expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 1.000 ± 0.076; ischemia group: 2.203 ±0.283; NS group: 1.616 ±0.185; P <0.01 ); however, it was significantly reduced in 10% HS group compared to ischemia group and NS group ( ischemia group:2.203 ±0.283; NS group: 1.616 ±0.185; 10%HS group: 1.202 ±0.177; P <0.05 ) .Western blot showed that the protein expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.290 ±0.079; ischemia group: 0.750 ±0.029; NS group:0.765 ±0.182;P<0.01);but was significantly reduced in 10%HS group compared to ischemia group and NS group ( ischemia group:0.750 ±0.029; NS group:0.765 ±0.182;10%HS group:0.390 ±0.195;P<0.05 ) .The protein expression of NICD was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.401 ±0.196; ischemia group: 0.906 ±0.359; NS group:0.847 ±0.153;P<0.01);but was significantly reduced in 10%HS group compared to ischemia group and NS group ( ischemia group:0.906 ±0.359; NS group:0.847 ±0.153;10%HS group:0.561 ±0.165;P<0.05 ) .Conclusion Our results suggest that HS markedly suppresses Notch signaling in microglia around the ischemia tissue area in experimental induced cerebral ischemic rats.

Objective To explore the advantages and disadvantages of helicopter emergency medical services of South China in the long-distance transport for critical patients.Methods A total of 30 patients who received helicopter emergency medical services by Guangdong Generral Hospital from August 2004 to December 2014 were selected as the observation group,and the other 30 patients with similar conditions who received ground emergency medical services were selected as the control group.To analyses the difference between the two groups in the disease,transport distance,transportation time,costs and compliction by χ2-test,t-test and nonparametric test according types of data.Results There were significantly difference between two groups in transport distances (km) [578.0 (313.0,707.5)vs.214.5 (101.5,313.5),P <0.05],set-up time (min) [95.7 (56.7,133.4)vs.10.7 (6.8,15.7),P <0.05],transportation time (min) [112.3 (64.3,152.4) vs.146.8 (67.8,217.5),P <0.05],costs (yuan/h) [14378.5 (9887.0,16348.5)vs.557.0 (356.5, 787.5),P <0.05]and the distance/total time value [2.8 (1.3,4.8)vs.1.4 (0.8,2.8),P <0.05]. There was no significantly difference in the incidence of complications between two groups (χ2 =0.058,P >0.05).Conclusions Helicopter emergency medical services could shorten the transportation time of critical patients on long distance transportation,and improve the efficiency of first-aid.However,there were many disadvantages that need to be improved in the helicopter emergency medical service of China.

Hepatolithiasis is a common biliary tract disease in China and other Asian countries.It has insidious onset,recurrent disease,and may lead to biliary cirrhosis or even cancer in the late stage without inter-vention.At present,surgery is considered to be the best choice for treatment.In the 21st century of minimally inva-sive surgery,traditional procedures such as partial hepatectomy,choledochotomy,cholangioenterostomy and liver transplantation can be performed with the assistance of laparoscopy and da Vinci robots,and emerging endoscopic techniques such as endoscopic retrograde cholangiopancreatography(ERCP)and percutaneous transhepatic cho-ledochoscopic lithotripsy(PTCSL)have also been developed,bringing better treatment for patients with hepatoli-thiasis.As treatment options are becoming more varied,there are also some clinical problems that need to be addressed.In this article,we would like to briefly review the current surgical treatment modalities in order to provide a theoretical basis for optimizing the treatment modalities.

Objective:To explore the effect of emergency "zero channel" process on improving the efficiency of intravenous thrombolysis in stroke.Methods:Fifty-eight acute ischemic stroke patients admitted to our hospital from January 2020 to December 2020 were enrolled into experimental group; another 58 acute ischemic stroke patients admitted to our hospital from January 2019 to December 2019 and matched with age and gender were selected as control group. "Green channel" process was adopted for patients in the control group, and optimized "zero channel" process (moving the working passageway forward to the ambulance) was implemented for patients in the experimental group. Door to rescue room time (DRRT), door to consultation time (DCT), door to laboratory examination completion time (DLECT), door to CT report time (DCRT), and door to needle time (DNT) were used to evaluate the times of emergency treatment. The thrombolytic effect of the two groups was compared by evaluating the recanalization rate of occluded vessels and thrombolytic efficiency. Modified Rankin scale (mRS) was used to evaluate the prognoses 6 months after treatment in both groups, and mRS scores≤2 was defined as good prognosis.Results:The DCRT, DCT and DNT in the experimental group were significantly shorter than those in the control group ( P<0.05); the compliance rate of DNT≤60 min in the experimental group was significantly higher as compared with that in the control group ( P<0.05). The immediate recanalization rate of occluded vessels in the experimental group and control group was 60.3% and 27.6%, and the thrombolytic efficiency was94.83% and 82.76%; significant differences were noted between the two groups ( χ2=12.633, P<0.001; χ2=4.245, P=0.039). The good prognosis rate of the experimental group and control group was 36.2% and 15.5%, respectively, after 6 months of follow-up ( χ2=4.016, P=0.041). Conclusion:Emergency "zero channel" can further shorten DCT, DCRT, and DNT, and improve the efficiency of thrombolysis and prognoses of acute ischemic stroke patients.

Objective To evaluate the clinical application of Catalyst system in positioning patients during cervical cancer radiotherapy,and to analyze its correlation with the bladder volume and body mass index (BMI) of patients.Methods A total of 33 patients diagnosed with cervical cancer from July to December 2017 in our hospital were included in the study.All patients were auxiliary positioned by an optical surface imaging system (C-Pad Catalyst) before each treatment.The CBCT imaging was executed twice a week.The setup errors from Catalyst and CBCT in the anterior-posterior (AP),superior-inferior (SI) and leg-fight (LR) directions were recorded.Each patient's bladder volume and BMI were also recorded.Results The setup errors between Catalyst with masks and CBCT had the significant difference in SI (P＜0.05) and LR (P＜0.05).For Catalyst without masks,the setup errors with the bladder volume of 200-300 ml had the significant association in SI (R=-0.316,P＜0.05).For the bladder volume of＞300 ml,the setup errors for Catalyst with masks had the significant association in AP (R=-0.493,P＜0.05),and that without masks had the significant association in SI and LR (R=0.335,P＜0.05,R=-0.348,P＜0.05).For patients of＜25 kg/m2,setup errors for Catalyst with masks had the significant association with the BMI in LR (R=0.197,P＜0.05);for ≥ 25 kg/m2,that with masks had the significant association in AP and SI (R =0.818,P＜0.05;R=-0.498,P＜0.05),that without masks had the significant association in AP and LR (R=0.652,P＜0.05;R=-0.558,P＜0.05).Conclusion Unlike CBCT system,the patient positioning by Catalyst system was easily affected by the bladder volume and BMI of patients.

OBJECTIVETo study the severe acute respiratory syndrome (SARS)-associated coronavirus genotype and its characteristics.

METHODSA SARS-associated coronavirus isolate named ZJ01 was obtained from throat swab samples taken from a patient in Hangzhou, Zhejing province. The complete genome sequence of ZJ01 consisted of 29,715 bp (GenBank accession: AY297028, version: gi: 30910859). Seventeen SARS-associated coronavirus genome sequences in GenBank were compared to analyze the common sequence variations and the probability of co-occurrence of multiple polymorphisms or mutations. Phylogenetic analysis of those sequences was done.

RESULTSBy bioinformatics processing and analysis, the 5 loci nucleotides at ZJ01 genome were found being T, T, G, T and T, respectively. Compared with other SARS-associated coronavirus genomes in the GenBank database, an A/G mutation was detected besides the other 4 mutation loci (C:G:C:C/T:T:T:T) involved in this genetic signature. Therefore a new definition was put forward according to the 5 mutation loci. SARS-associated coronavirus strains would be grouped into two genotypes (C:G:A:C:C/T:T:G:T:T), and abbreviated as SARS coronavirus C genotype and T genotype. On the basis of this new definition, the ZJ01 isolate belongs to SARS-associated coronavirus T genotype, first discovered and reported in mainland China. Phylogenetic analysis of the spike protein gene fragments of these SARS-associated coronavirus strains showed that the GZ01 isolate was phylogenetically distinct from other isolates, and compared with groups F1 and F2 of the T genotype, the isolates of BJ01 and CUHK-W1 were more closely related to the GZ01 isolate. It was interesting to find that two (A/G and C/T) of the five mutation loci occurred in the spike protein gene, which caused changes of Asp to Gly and Thr to Ile in the protein, respectively.

CONCLUSIONAttention should be paid to whether these genotype and mutation patterns are related to the virus's biological activities,epidemic characteristics and host clinical symptoms.

Objective: To investigate herpesvirus infection in early stage of hematopoietic stem cell transplantation (HSCT) by multiplex polymerase chain reaction (PCR), and to explore the association between multiple herpesviruses infection and clinical characteristics in HSCT patients and its impact on post-transplant complications and prognosis. Methods: A total of 734 peripheral blood samples were collected from 90 patients undergoing HSCT in the Department of Hematology, the First Affiliated Hospital of Soochow University between February 2017 and August 2017. The peripheral blood specimens were obtained before and within 90 days after transplantation at different time points. Lab-Aid824 Nucleic Acid Extraction Mini Reagent was used to extract DNA and multiplex PCR assay was used to simultaneously detect 8 kinds of human herpesviruses from genomic DNA. The incidence of various herpesvirus infections, its correlation with clinical features and effects on post-transplant complications and prognosis were analyzed. Results: The median follow-up time was 192 (range: 35-308) days. Among the 90 patients before transplantation, the incidence of herpes virus infection was 35.6% (32/90), including 12.2% (11/90) with one herpes virus infection and 23.3% (21/90) with multiple viruses infection. The incidence of herpes virus infection after transplantation was 77.8% (70/90), including 20.0% (18/90) with one herpes virus infection and 57.8% (52/90) with multiple herpes virus infection. Among the 52 patients with multiple herpes viruses infection, 30 (57.7%) patients were infected by 2 kinds of viruses, 18 (34.6%) patients by 3 kinds of viruses and 4 (7.7%) patients by 4 kinds of viruses. There was a correlation between HHV-6 and HHV-7 herpesvirus infection (OR=13.880, Q=0.026). EBV infection was related to HHV-7 infection (OR=0.093, Q=0.044). The age of patients was correlated with the incidence of HHV-1 infection before transplantation. There were 24 patients in our study experienced clinical symptoms associated with viral infection. The main manifestations were hemorrhagic cystitis (HC), interstitial pneumonia, enteritis, viral encephalitis and fever of unknown origin. EBV infection was related to HLA incompatibility and the inconsistent of the ABO blood group and grade Ⅱ-Ⅳ aGVHD after transplantation. HLA incompatibility and the unrelated donor and grade Ⅱ-Ⅳ aGVHD were related to multiple viruses infection. Conclusion Multiple herpesviruses were common in patients undergoing HSCT, which were closely related to HLA mismatch, unrelated donor and grade Ⅱ-Ⅳ aGVHD.

Objective: To investigate the correlation between skin elasticity and setup error in optical surface image-guided radiotherapy. Methods: The skin elasticity (R7) data of the head, chest and abdomen were extracted and analyzed its correlation with age by systematic literature review. Fifty-four patients diagnosed with nasopharyngeal carcinoma, breast cancer and cervical cancer were recruited in this study. Firstly, the patients were positioned based on the room laser and markers. Subsequently, the patient position was verified by the Varian On-Board Imager, and then C-Rad Catalyst was adopted to obtain surface images in two states (mask or non-mask) as reference images. In the subsequent fraction treatment, after initial positioning, the local calibration was performed by Catalyst, and setup errors in three directions were recorded. Meanwhile, the patient setup was verified by CBCT twice a week. The Pearson correlation analysis was performed to analyze the correlation between setup error and age. Results: The skin elasticity was negatively correlated with aging (P<0.01). The correlation coefficient between random error and age in head-and-neck cancer were 0.645, 0.624 and 0.866 in the AP, SI and LR directions (all P<0.05) for male patients without mask, respectively. The system error was significantly correlated with age in the LR direction (P<0.05) for male patients, and in the AP direction (P<0.05) for female patients with head-and-neck cancer without mask. The setup error had a significant correlation with skin elasticity in male patients with head-and-neck cancer, and the sequence of absolute value of correlation coefficient was LR>SI>AP. Conclusion In optical surface-guided radiotherapy of head and neck cancer, skin elasticity may be a significant index for assessing the setup errors in male patients.