Objective To investigate the effects of different culture media and pretreatment methods on the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS) for the identification of Vibrio parahaemolyticus,and then provide the optimal conditions.Methods Forty strains of Vibrio parahaemolyticus were collected,and subcultured with the blood agar plate,thiosulfate citrate bile salts sucrose(TCBS) agar plate and double wash agar plate,respectively.The pretreatment methods before mass spectrometry included the smear method,extension method and extraction method.The effects of different culture media and pretreatment methods on the results of MALDI-TOF MS for the identification of Vibrio parahaemolyticus were analyzed by the logistics regression model and Chi-square test of SAS software.Results The detection rate of Vibrio parahaemolyticus was the highest in the combination of blood agar plate with the extraction method(genus level:100％;species level:67.5％),followed by the combination of double wash agar plate with the extraction method(genus level:70％;species level:37.5％).For the genus identification level of Vibrio parahaemolyticus,when the pretreatment method was the same,different culture media produced significantly different detection rates(P ＜ 0.01).However,when the culture medium was the same,there was no significant difference in detection rates for different pretreatment methods (P ＞ 0.05).The odd ratios (OR) of the smear method and extention method relative to the extraction method were 0.66 and 0.95,respectively,and there was no significant difference in detection rates for them(P ＞ 0.05),while the ORs of the TCBS agar plate and double wash agar plate relative to blood agar plate were 0.06 and 0.10,respectively,and there was significant difference in detection rates for them(P ＜0.05).The logistic regression equation was Y=2.95-0.41a-0.05b-2.83c-2.83d(a:smear method;b:extension method;c:TCBS agar plate;d:double wash agar plate).For the species identification level of Vibrio parahaemolyticus,when the extension method or the extraction method was used,different culture media produced significantly different detection rates(P ＜ 0.05).When the blood agar plate was used,different pretreatment methods also produced significantly different detection rates(P ＜ 0.01).The ORs of the smear method and extention method relative to the extraction method were 0.32 and 0.55,respectively,and there was significant difference in detection rates for them (P ＜ 0.05),while the ORs of the TCBS agar plate and double wash agar plate relative to blood agar plate were 0.18 and 0.49,respectively,and there was significant difference in detection rates for them(P ＜0.05).The logistic regression equation was Y =0.20-1.15a-0.61b-1.72c-1.72d(a:smear method;b:extension method;c:TCBS agar plate;d:double wash agar plate).Conclusion For improving the detection rate of Vibrio parahaemolyticus by MALDI-TOF MS,the selection of appropriate culture medium is more important than that of the pretreatment method.It is recommended that the extraction method may be as a conventional pretreatment method,and that the optimal medium is the blood agar plate,followed by the double wash agar plate and TCBS agar plate.

Objective To investigate the role of recombinant Vibrio vulnificus cytolysin (rVvhA) in inducing THP-1 cells damage and study the pathway of associated calcium influx .Methods Inverted mi-croscope, CCK-8 cell proliferation kit, Fluo3/AM staining and caspase activity detection were performed to analyze the damage of THP-1 cells induced by rVvhA and the pathway of calcium influx .Results rVvhA had cytotoxic effects on THP-1 cells in a dose-dependent manner .The concentrations of extracellular K +and LDH were respectively up-regulated after 1 h and 6 h of 12 μg/ml rVvhA intervention .Verapamil , Mibe-fradil and SKF-96365 could not prevent the influx of free Ca 2+induced by rVvhA .The activities of caspase-3 and caspase-9 were singanificantly enhanced by rVvhA in a time-dependant manner .Conclusion rVvhA can induce THP-1 cells damage through triggering extracellular calcium influx via porous channel on cell membrane.Moreover, rVvhA might induce THP-1 cell apoptosis through activating caspase-9/3-dependent pathway .

BACKGROUND:At present, scholars general y believe that the physical and chemical properties of titanium mesh are fine as easy plastic, easy cutting, good corrosion resistance and wear resistance, non-magnetic, less effect on CT or MRI results, good histocompatibility and resistance to stress. The titanium mesh is regarded as an ideal material for cranioplasty. ＜br> OBJECTIVE:To explore the clinical feasibility of primary reconstruction of comminuted and open skul fracture with titanium mesh. ＜br> METHODS:Sixty-two cases of comminuted and open skul fracture were analyzed retrospectively, including 49 males and 13 females, aged 7-69 years. Among these 62 patients, 30 patients underwent debridement and then after 6-12 months, they received titanium mesh reconstruction, serving as control group. Another 32 cases underwent debridement and titanium mesh reconstruction in the same time as observation group. Al the cases were fol owed-up for 1-2 years to observe wound healing, complications and hospitalization cost. ＜br> RESULTS AND CONCLUSION:During the fol ow-up, no wound infection and intracranial infection occurred in the two groups. The incidence of intracranial hematoma, epilepsy, and other complications was higher in the control group than the observation group (P＜0.05), and the hospitalization cost was also higher in the control group than the observation group (P＜0.05). These findings indicate that primary reconstruction with titanium mesh is feasible in the treatment of comminuted and open skul fracture and can significantly reduce the incidence of complications and hospitalization costs.

Objective:To explore the effect of self-learning ability and critical thinking ability in nursing students under mobile teaching mode based on portable classroom platform.Method:s Two classes of batch 2015 from nursing college were randomly selected and divided into control group ( n=58) and experimental group ( n=61). The control group adopted conventional teaching mode, while the experimental group used the same mode in addition to mobile teaching mode based on portable classroom platform. Self-learning ability questionnaire and critical thinking ability scale in Chinese were used to evaluate the teaching effect, and t test was performed to analyze the data using SPSS 19.0. Result:s After intervention, scores of different items and the total scores in the self-learning ability scale in the experimental group were higher than those in the control group, with statistical significance ( P<0.05); scores of different items and the total scores in the critical thinking ability questionnaire in the experimental group were higher than those in the control group, with statistical significance ( P<0.05). Conclusion:Mobile teaching mode based on portable classroom platform can improve the self-learning ability and critical thinking ability of nursing students.

Objective To explore the influence of WeChat platform rehabilitation guidance mode on refracture of osteoporotic fracture patients.Methods Totally 100 osteoporotic fracture patients admitted to the Department of Orthopedics of the hospital from 2018 to 2019 were selected and randomly divided into control group and experimental group with 50 patients in each group.The control group adopted the conventional discharge rehabilitation guidance mode,and the experimental group implemented WeChat platform rehabilitation guidance mode on this basis,including WeChat platform construction and detailed intervention mode.The changes of bone mineral density and the recurrence rate of osteoporotic fracture were compared between the two groups at admission and after 3 years of follow-up.Results There were 7 cases of lost follow-up in the experimental group,and there were 6 cases of lost follow-up in the control group.After 3 years,the bone mineral density of the experimental group was significantly higher than that of the control group,and the recurrence fracture rate was significantly lower than that of the control group(P<0.05).Conclusion The application of WeChat platform rehabilitation guidance mode can effectively improve bone density and reduce the incidence of refracture in osteoporotic fracture patients.

To investigate the neuroprotective effect and possible mechanism of masitinib on cerebral ischemia-reperfusion injury in rats, healthy adult male Sprague-Dawley rats were divided into sham group (n = 12), model group (n = 12), masitinib low dosage group (n = 12), masitinib middle dosage group (n = 12), and masitinib high dosage group (n = 12). All rats was subjected to middle cerebral artery occlusion (MCAO) for two hours and reperfusion except sham group, and received treatment twice per day for 7 days once reperfusion started.Neurological score, infarct volume, and brain water content were detected; some autophagic markers, apoptotic and inflammatory cytokines were evaluated by Western blot and PCR after 7 d of reperfusion. Treatment with masitinib significantly ameliorated neurologic deficit, infarct volume and brain water after I/R injury. Masitinib also decreased the ratio of LC3II/I and the expression of Beclin-1 and increased the expression of p62 in the brain tissues of rats with I/R injury.Furthermore, it could inhibit apoptosis-related proteins and NF-κB expression. Masitinib could relieve the cerebral ischemia-reperfusion injury in rats through inhibiting autophagy and apoptosis.

The aim of the present study was to investigate the effects and possible mechanism of tetramethylpyrazine(TMP)on morphine-induced microglia activation and tolerance. The antinociception and morphine tolerance were assessed in mice using hot-water tail flick test. IBA-1(ionized calcium binding adapter molecule 1), the marker of microglia, was detected by immumofluorescence method. The expression of p-p38 MAPK and total p38 MAPK(mitogen-activated protein kinase, MAPK)was analyzed by Western blot; real-time polymerase chain reaction(RT-PCR)was used to detect the expression level of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β). Results showed that TMP(15, 30, 60 mg/kg, ip)inhibited morphine-induced up-regulation of IBA-1, p-p38, TNF-α and IL-1β in a dose-dependent manner, yet with no effect on the expression of total p38 MAPK. In conclusion, TMP significantly inhibited the activation of microglia evoked by morphine via p38 MAPK signaling pathway, thus attenuating morphine antinociception tolerance.

Objective To summarize the clinical and the molecular genetic characteristics of type DYT11 dystonia by analyzing the clinical data and pathogenic gene mutation of type DYT11 dystonia of a myoclonus-dystonia syndrome (MDS) family.Methods A MDS family enrolled in the General Hospital of the People's Liberation Army Rocket Force in January 2018 was retrospectively analyzed.The clinical data of 11 affected family members were collected and genetic testing of four affected family members (including the proband) of the MDS family was conducted using a panel of dystonia-associated genes.Results The affected family members showed great differences in clinical characteristics and obvious clinical heterogeneity.Four affected family members had myoclonus and dystonia,two affected family members only had myoclonus and five affected family members only had dystonia.The results of genetic testing showed that the proband,his father and his grandfather had a mutation (c.835_839delACAAA) in SGCE gene,which is autosomal dominant and belongs to type DYT11 dystonia.Conclusions MDS shows clinical heterogeneity.Gene screening is of great importance for the diagnosis and treatment of dystonia with myoclonus.

Objective:To investigate the effects of TNF-α knockout on liver and spleen neutrophil responses to Vibrio vulnificus bloodstream infection in a mouse model. Methods:(1) TNF-α-knockout (TNF-α -/-) and wild-type (WT) C57BL/6J mice aged 6-8 weeks were randomly divided into four groups with six in each group: uninfected WT group, infected WT group, uninfected TNF-α -/- group and infected TNF-α -/- group. The mouse model of bloodstream infection was constructed by intraperitoneal injection of Vibrio vulnificus CGMCC1.1758 (2×10 8 CFU/200 μl), while the mice in the uninfected groups were injected intraperitoneally with equal amount of PBS. (2) Liver immune cells and splenocytes were isolated 4 h after infection and subjected to analyze the percentages and numbers of neutrophils, and the changes in cell viability, cellular reactive oxygen species (ROS) level and phagocytosis by flow cytometry. In addition, effects of Vibrio vulnificus bloodstream infection on mTOR signaling pathway in murine neutrophils were evaluated in vivo. Results:(1)Compared with the uninfected WT group, the percentages and numbers of neutrophils in liver and spleen tissues of the infected WT group increased significantly. The percentage and number of liver neutrophils were significantly higher in the infected TNF-α -/- group than in the infected WT group, but no significant difference in spleen neutrophils was detected between the two groups. (2) Compared with the infected WT group, the phagocytosis of liver neutrophils rather than that of spleen neutrophils was enhanced in the infected TNF-α -/- group. (3) The survival rates of neutrophils in both liver and spleen were decreased, while the cellular ROS level was significantly increased in the infected WT group compared with those of the uninfected WT group. Compared with the infected WT group, the infected TNF-α -/- group had increased survival rates of both liver and spleen neutrophils, but decreased level of ROS. (4) The levels of p-AKT (S473) in liver and spleen neutrophils of the infected WT group were lower than those of the uninfected WT group. Compared with the infected WT group, the infected TNF-α -/- group had lower level of p-AKT (S473) in liver neutrophils, but higher p-AKT (S473) level in spleen neutrophils. There were no significant differences in p-4E-BP1(T37/46) levels between the uninfected WT group and the infected WT group. The p-4E-BP1 (T37/46) level in liver neutrophils was lower in the infected TNF-α -/- group than in the infected WT group, but no significant difference in p-4E-BP1 (T37/46) levels in spleen neutrophils was observed between the two groups. Conclusions:TNF-α had different effects on the neutrophils in spleen and liver tissues of mice with Vibrio vulnificus bloodstream infection. It played a critical role in regulating the recruitment, phagocytic function and mTOR signaling of liver neutrophils after Vibrio vulnificus infection in vivo.

Objective To test the antimicrobial susceptibility of Staphylococcus aureus from children with impetigo,and to assess the differences in randomly amplified polymorphic DNA profiles between sensitive and resistant Staphylococcus aureus strains.Methods Secretion specimens were obtained from the impetiginous lesions of 178 children,and subjected to bacterial culture.The susceptibility of 162 Staphylococcus aureus isolates against 21 antibiotics was tested.Randomly amplified polymorphic DNA PCR(RAPD-PCR)was performed to characterize the genotype of Staphylococcus aureus.Results Totally,180 bacterial strains were isolated from 178 children with impetigo in Chengdu,including 162(90.00％)Staphylococcus aureus strains.Of the 162 Staphylococcus aureus strains,148 were methicillin sensitive Staphylococcus aureus(MSSA),14 methicillin resistant Staphylococcus aureus(MRSA).The most active antibiotic was minocycline,followed by teicoplanin,quinupristin,vancomycin and nitrofurantoin,while the resistance rate to penicillin was highest,followed by that to erythromycin,clindamycin,compound sulfamethoxazole and tetracycline.All the Staphylococcus aureus isolates were sensitive to fusidic acid,nitrofurantoin,vancomycin,minocycline and teicoplanin.According to RAPD-PCR,the 162 Staphylococcus aureus strains were divided into 8 genotypes,with the three most prevalent genotypes being Ⅲ(31.48％),Ⅱ(26.54％)and Ⅵ(25.93％),which accounted for 65.43％(106/162)in all the strains.The 148 MSSA strains fell into 8 genotypes,with genotype Ⅲ(50 strains,33.78％),Ⅵ(39 strains,26.35％)and Ⅱ(33 strains,22.30％)being the most prevalent genotypes;the 14 MRSA strains fell into 3 genotypes,i.e.,genotype Ⅱ(10 strains,71.43％),Ⅵ(3 strains,21.43％),and Ⅲ(1 strain,7.14％).Conclusions Staphylococcus aureus is the most prevalent pathogenic bacteria in children with impetigo in Chengdu area,which is highly sensitive to minocycline,teicoplanin and quinupristin,and falls into 8 genotypes according to RAPD-PCR with genotype Ⅲ being the most common genotype.