Objective:To analyze the antimicrobial resistance and genomic characteristics of Salmonella enterica serovar Derby strains isolated from human and food sources in Hangzhou. Methods:A total of 60 Salmonella enterica serovar Derby strains isolated in Hangzhou during the period from 2015 to 2020 were subjected to antimicrobial susceptibility testing, pulsed field gel electrophoresis (PFGE) typing and whole-genome sequencing. Multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST) and the identification of antimicrobial resistance genes were performed using the sequencing data. Phylogenetic tree based on the single nucleotide polymorphism (SNP) sites in the 60 genomes from Hangzhou and 379 genomes from public databases was constructed. Results:No significant difference was observed in the drug resistance rates between the clinical strains and food strains in Hangzhou. The multidrug resistance (MDR) rate was 76.7% (46/60). All of the 60 Salmonella Derby strains were positive for the antimicrobial resistance genes aac(6′)- Iaa and fosA7. The 60 strains were subtyped into 46 molecular types by PFGE and 53 molecular types by cgMLST(HC2). Except for one strain belonging to ST3220, the other Salmonella Derby strains were ST40. The phylogenetic analysis showed that some strains isolated in Hangzhou were close to the strains in Southeast Asia, suggesting the possibility of cross-border transmission of ST40 strains, with the main food sources being pork and fish; other strains were close to those circulating in Beijing, Guangzhou, Hubei, Chongqing and other provinces, suggesting the possibility of cross-province transmission of the strains, with the main food sources being pork, beef and chicken. Conclusions:The epidemic of Salmonella Derby in Hangzhou was mainly caused by the spread of ST40 strains and MDR was common. Clinical infections might be closely related to the consumption of pork, beef, chicken and fish. There was the possibility of cross-border transmission of Salmonella Derby between Hangzhou and Southeast Asia and cross-province transmission in China.

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine

Objective:To investigate the regulation of transcription factor CCCTC binding factor (CTCF) on the expression of B-cell lymphoma 2 ( Bcl-2) gene in pterygium and its molecular mechanism. Methods:Pterygium tissue samples from 22 primary pterygium patients who underwent pterygium excision combined with autologous limbal stem cell transplantation in The First Hospital of Changsha from June 2017 to February 2019 were collected during the operation as pterygium group.Normal conjunctival tissue from 20 patients with ocular trauma due to conjunctiva rupture, eyeball rupture or eyeball perforation in the same period were collected during the repair of ocular trauma as control group.Real-time PCR and Western blot were used to detect the expression levels of CTCF and Bcl-2 in the two groups.The DNA methylation level of the Bcl-2 promoter in the samples of the two groups was detected by bisulfite sequencing PCR (BSP). Pterygium fibroblasts were isolated and cultured.Fibroblasts were identified by immunohistochemistry using vimentin antibody.The cultured pterygium fibroblasts were divided into a CTCF interference group transfected with CTCF interference plasmid, and a control group transfected with control plasmid.The expression levels of CTCF and Bcl-2 in pterygium fibroblasts in CTCF interference and control groups were detected by real-time PCR and Western blot.The cell vitality was detected with cell counting kit-8 at 12, 24, and 48 hours after transfection.The DNA methylation level of the Bcl-2 promoter in the cells of the CTCF interference and control groups after transfection was determined by BSP.Differences of the indexes among groups were analyzed.Correlation between Bcl-2 mRNA and Bcl-2 gene promoter methylation level of CTCF protein in pterygium tissue was analyzed by Pearson linear correlation analysis.This study protocol was approved by the Ethics Committee of The First Hospital of Changsha (No.KL-2017021). Written informed consent was obtained from the patients from whom the specimens were collected.Results:The relative expression levels of CTCF mRNA and protein in pterygium group were 7.23±3.34 and 0.92±0.21, respectively, which were significantly higher than 1.10±0.44 and 0.28±0.07 in normal conjunctiva group ( t=-8.136, -13.025; both at P<0.01). The relative expression levels of Bcl-2 mRNA and protein in pterygium group were 10.27±4.64 and 0.95±0.27, which were higher than 1.10±0.41 and 0.32±0.14 in normal conjunctiva group, showing statistically significant differences ( t=-8.789, -10.782; both at P<0.01). The CTCF protein expression was significantly positively correlated with the Bcl-2 mRNA expression in pterygium group ( r=0.746, P<0.01). The DNA methylation level of the Bcl-2 promoter in pterygium group was 0.65±0.09, which was lower than 0.83±0.06 in normal conjunctiva group, with a statistically significant difference ( t=7.408, P<0.01). The DNA methylation level was significantly negatively correlated with the Bcl-2 mRNA expression in pterygium group ( r=-0.635, P<0.01). After the interference of CTCF expression in pterygium fibroblasts, the relative expression levels of CTCF and Bcl-2 mRNA in CTCF interference group were 0.37±0.03 and 0.53±0.06, which were significantly lower than 1.02±0.06 and 0.99±0.07 in control group ( t=20.035, 9.029; both at P<0.01). The relative expression levels of CTCF and Bcl-2 proteins in CTCF interference group were 0.23±0.06 and 0.56±0.07, which were lower than 0.52±0.05 and 0.92±0.12 in control group, showing statistically significant differences ( t=6.914, 4.719; both at P<0.01). The cell viability of pterygium fibroblasts in CTCF interference group was 0.10±0.01, 0.17±0.01, 0.38±0.04 at 12, 24, and 48 hours after interference, respectively, which were lower than 0.12±0.01, 0.29±0.01 and 0.85±0.06 in control group, and the differences were statistically significant ( t=3.718, 18.350, 15.621; all at P<0.01). The DNA methylation level of Bcl-2 promoter in CTCF interference group was 0.75±0.04, which was significantly higher than 0.61±0.03 in control group ( t=-4.472, P<0.05). Conclusions:CTCF is excessively expressed in pterygium, which may mediate the overexpression of Bcl-2 through down-regulating DNA methylation level.

Objective:To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB.Methods:Three full-length Mycobacterium ( M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M.tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results:Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance ( P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group ( P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group ( P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group ( P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group ( P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups ( P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant ( P>0.05). Conclusion:Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Objective:To investigate any anti-aging effect of repeated transcranial magnetic stimulation (rTMS) and explore the relationship between the effect and relief of clinical symptoms in patients with Parkinson′s disease (PD).Methods:A total of 108 PD patients were randomly divided into an rTMS group and a control group, each of 54, while another 54 healthy counterparts were selected to form a normal group. In addition to anti-PD drug therapy, the rTMS group was given daily rTMS treatment, 5 days a week for 4 weeks, while the control group received sham rTMS treatment, with no treatment of the normal group. Before the treatment and after 4 weeks of treatment as well as and 1 month after the ending of the treatment, the subjects′ clinical exercise symptoms were evaluated using the Unified Parkinson′s Disease Rating Scale (UPDRS), a timed exercise test and the 10m re-entry exercise test. Non-exercise symptoms were assessed using the Hamilton Depression Scale (HAMD), the Hamilton Anxiety Scale (HAMA) and the Mini-mental State Examination (MMSE). Fasting venous blood samples were analyzed to quantify the serum levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), interleukin-1β (IL-1β) and matrix metalloproteinase-3 (MMP-3).Results:Four weeks and 1 month after the treatment, the average UPDRS scores, exercise test times and 10m re-entry exercise test results of the rTMS group were significantly better than those before treatment and significantly better than those of the control group at the same time point. The rTMS group′s average HAMA, HAMD and MMSE scores, as well as its average P300 latency and amplitude were also significantly better than those of the control group at the same time point and significantly better than those before treatment. After 4 weeks, the average MMP-3 content in the rTMS group was significantly lower than the control group′s average, and after a month the average levels of TNF, IL-6, IL-1β and MMP-3 of the rTMS group were all significantly different from those before treatment and those of the control group. The TNF, IL-6, IL-1β and MMP-3 levels were all positively correlated with the average UPDRS total score.Conclusion:High-frequency rTMS therapy can change the phenotypes related to cell senescence, and thus has good therapeutic effect on motor and non-motor symptoms of PD.

Objective:To investigate the relationship of antinuclear antibody (ANA) status with clinical features and malignancy risk in adult patients with dermatomyositis.Methods:A retrospective analysis was performed to analyze clinical data from 101 inpatients with dermatomyositis in Department of Dermatology, the First Affiliated Hospital of Soochow University from April 2008 to April 2018. These patients were divided into ANA-positive group and ANA-negative group, and differences in myopathy and malignancy risks as well as other clinical features were analyzed between the 2 groups. A 2-year follow-up was undertaken among 92 patients. Chi-square test was used to analyze and compare clinical features between the 2 groups, and a multivariate regression model was used to analyze the relationship of ANA status with amyopathic dermatomyositis and malignancies.Results:Among the 101 patients with dermatomyositis, there were 42 males and 59 females, aged 55.13 ± 14.63 years; 14 patients had amyopathic dermatomyositis, 6 patients had hypomyopathic dermatomyositis, and 81 patients had myopathic dermatomyositis; 42 (41.58%) cases were positive for ANA, and 59 (58.41%) were negative for ANA. Compared with the ANA-negative group, the ANA-positive group showed significantly decreased incidence of cervical erythema (33.33% vs. 59.32%, P=0.010) and shawl sign (14.28% vs. 35.59%, P=0.017) . Twenty-eight (27.72%) patients with dermatomyositis were complicated by malignancies. Malignancies were found in 5 (11.9%) of ANA-positive patients, and in 23 (38.98%) of ANA-negative patients. Univariate analysis showed that ANA-negative patients with dermatomyositis had a higher risk of malignancies compared with ANA-positive patients with dermatomyositis, with an odds ratio of 7.52 (95% CI: 1.62-13.78, P=0.003) . In the multivariate regression model, the absence of ANA ( OR=4.34, 95% CI: 1.37-13.72, P=0.012) and cervical erythema ( OR=3.27, 95% CI: 1.20-8.91, P=0.020) were associated with high incidence of malignancies, while the absence of ANA was not significantly correlated with the occurrence of amyopathic dermatomyositis ( OR=0.99, 95% CI: 0.32-2.99, P=0.980) . Conclusions:ANA-negative adult dermatomyositis patients with cervical erythema had an increased risk of malignancies. Thus, close follow-up and regular tumor screening are necessary in these patients.

Objective To investigate the genomic characteristics and virulence factors of emetic-type Bacillus cereus strains isolated from food in Hangzhou for better understanding their pathogenic potential. Methods Real-time PCR was performed to detect the ces gene cluster ( cereulide) in 132 Bacillus cereus strains isolated from food from 2015 to 2017. Genomes of cereulide-positive strains were sequenced using Illumina MiSeq sequencing platform. Genome annotation, virulence factor detection, comparative and evolu-tionary analysis were performed after the sequences of genomes were assembled. Results Twelve strains (9. 09％) carried the ces gene. Their genome sizes ranged from 5. 35 to 5. 75 Mb and GC contents from 35. 25 to 35. 43 mol％. All of them harbored the full cereulide biosynthesis gene cluster, nonhemolytic ente-rotoxin ( NHE)-encoding gene cluster ( nheA, nheB and nheC) and hemolysinⅢ( hlyⅢ) . The average nu-cleotide identity ( ANI ) between the 12 isolates and the reference strain NC7401 ( Accession number:AP007209) was over 99. 35％. Phylogenetic analysis demonstrated these strains were clustered into the same branch with local clinical isolates and the emetic-type Bacillus cereus strains of NC7401 and AH187. Con-clusions The genomic sequences of the emetic-type Bacillus cereus strains isolated from food in Hangzhou area were highly similar to that of the reference strain NC7401. Results of the genomic analysis suggested that these isolates carried many virulence factors that were related to pathogenicity.

Objective To systematically evaluate the optimal dose of early enteral nutrition support in critically ill patients.Methods Systematic search database including PubMed,Web of science,Scopus,CINAHL,CBM,CNKI.RCTs about early enteral nutrition dose selections in critically ill patients were chosen according to include and exclude criteria by two researchers independently.Cochrane system evaluation manual bias risk assessment was used to evaluate quality of literature.RevMan5.3 Meta analysis software was used to analyze the data.Results A total of 1 571 literatures were retrieved and 8 RCT studies were included,2 713 subjects in total.Meta analysis results showed that there were statistically significant differences in mechanical ventilation time,incidence of diarrhea,and utilization rate of gastro dynamic drugs between trophic feeding and full feeding (P＜0.05).There were no statistically significant differences in mortality,length of stay,incidence of nosocomial infections,reflux,vomiting,constipation,etc.(P＞0.05).Conclusions Trophic feeding has familiar effects on mortality,length of hospital stay compared to full feeding,but it can help to shorten ICU mechanical ventilation time,improve the gastrointestinal tolerability.

Objective To explore the recyclability and safety of Celect retrievable filter placement in the prevention of pulmonary embolism in patients with deep venous thrombosis(DVT).Methods The data of 120 DVT patients with Celect retrievable filter were collected from the Second Hospital of Shanxi Medical University from August 2015 to March 2017 and analyzed retrospectively. The Celect filter was placed in the inferior vena cava(IVC)at the inferior margin of the renal vein for 1 to 2 cm by puncturing the contralateral femoral vein or right internal jugular vein.The filter retrieve risk was assessed within 8 weeks after being implanted. The filters would be recovered through the right jugular vein when meeting the recovery standard, and the retrieve methods included conventional method, removing the guide wire into a loop trap method and guiding wire into a loop combined with balloon assisted method.The perforation of the vena cava was observed and the tilt angle of the filter was measured.The success rate of Celect filter retrieve was evaluated by the Kaplan-Meier method. Results Celect filters were successfully implanted in 120 patients with DVT.The IVC filters were implanted through femoral vein in 111 patients and 2 cases via right internal jugular vein.No complications,asymptomatic pulmonary embolism and related death was found in all patients.Twenty four patients did not reach the standard of filter retrieve,and were follow-uped.Ninety six cases were treated with Celect retrievable filter,among which,93 cases were successfully recovered with the filter indwelling time ranging from 7 to 144 days and the median being 50 days.The failure of the filter retrieve occurred in 3 cases because of the serious tilt of the filter or the encapsulation of filter by inferior vena cava thrombus.Perforation of vena cava with no clinical symptoms occurred in 21 cases.Filter tilt was found in 35 cases,among which,15 cases had inclined angle>15 degrees or the recovery hook closed to the IVC wall.Thirteen cases with filter tip or recovery hook attached to the wall were successfully removed by using the guide wire into a loop or trap guide wire into a loop combined with balloon assisted method instead of routine removal method.The retrieve rate was 100% when the retention time of Celect filter in the body was within 106 days. Conclusion Celect retrievable filter can be implanted in DVT patients with long retrieve time window and high retrieve rate,but the filter inclination rate and vena cava perforation rate are high.

Objective: To analyze genomic features of pathogens based on next generation sequencing technique in a food-borne disease event. Methods: A total of 11 blood samples, stomach contents before gastric lavage from the death and patients' foods were collected. S. aureus, B. cereus and toxic substances were detected. B. cereus detected in foods were counted. The conserved region of 16 S rDNA gene and ces gene(cereulide) of B. cereus isolates were detected by real-time PCR. Next Generation Sequencing (NGS) technology was applied to acquire genome sequences of isolates. Different plasmids distribution and comparative genomics analysis with reference sequences in public databases were analyzed. Results: Only B. cereus tested positive in all samples. The counts of B. cereus in Egg fried rice, one food samples, were 1.9×10⁷ CFU/g, and the counts of B. cereus in dried and fried fish and brine pork head meat samples were 3.0×10³ CFU/g both. Ten isolates were carrying hlyⅢ, nheA, nheB, inlA and inhA genes, and nine isolates carried the plcR gene and nine isolates carried the nheC gene. The PCR result of 16 S rDNA gene and ces gene of all isolates were positive. All carried the complete ces genes cluster sequence which were identical to the sequence of plasmid pCER270 (NC_01 0924.1) from strain AH187 in United Kingdom and pNCcld (NC_016792.1) from NC7401 in Japan. The alignment of plasmids turned out the sequence of the isolate differed from the pXO1 and pXO2 plasmids of B. anthracis, but carried the pNCcld plasmid containing the ces genes cluster. The phylogenetic tree based on genomic sequences of ten isolates showed high similarity (distances in phylogenetic tree from 2.0×10^-6-9.0×10^-6) to each other and to the B. cereus strains AH187 and NC7401 (MLST ST26 type, distances in phylogenetic tree from 3.8×10^-5-4.5×10^-5). Conclusion The foodborne disease event was caused by vomiting type Bacillus cereus without plasmid pXO1 and pXO2 contaminated egg fried rice. The vomiting-type food poisoning caused by B. Cereus globally is probably associated with ST26, ST164 and other strains harboring ces gene.