Objective To study the structures of the plasmids of Klebsiella pneumoniae KF3 at the genome metagenome level througth with whole plasmid DNA sequencing, to analyze the functional genes carried by plasmid and to identify the correlation of resistance and pathogenicity between the plasmids and the host strains. Methods The alkaline lysis method was used to extract plasmids. We constructed the small insert pUC18 library and the large insert Forsmid library, sequenced and used the Phred / Phrap / Consed package to assemble these sequences and gained a complete sequence. The open reading frame(ORFs) were predicted by the Glimmer software and annotated, analyzed the functions of these genes. Results We successfully constructed the pUC18 library and the Fosmid libraries for the plasmid DNA and obtained three circular double-stranded DNA plasmids: pKF3-70 (69 477 bp), pKF3-90 (91 327 bp) and pKF3-147 ( 147 416 bp). There were drug resistant genes, conjugative transfer genes and mobile DNA elements identified on three plasmids. Conclusion The three plasmids of KF3 could be transferred among different strains. It would lead to the dissemination of the resistant genes.

Objective To explore the best therapeutic methods for acute mesenteric venous thromboses(AMVT)with different degrees of intestinal ischemic lesions. Methods 6 cases of acute abdomen were preoperatively diagnosed as AMVT with imaging. After laparotomy, patients were classified as congestive lesion(n=3)and necrotic lesion(n=3)according to the degree of intestinal sichemia and were treated with mesenteric thrombectomy and bowel resection, respeetively. All cases received heparin and urokinase perioperatively. Results Of the 3 patients receiving mesenteric thrombectomy, 2 were cured and the other one with ischemic ascending colon was cured after right hemicolectomy due to the complication of colic dynamic ileus and perforation 10 days after thrombectomy. The other 3 patients recovered after bowel resection. Follow-up from 8 months to 6 years showed no recurrence. Conclusion Combined with anti-coagulation therapy, thrombectomy and bowel resection are rational and effective protocol for congestive lesion and necrotic lesion, respectively in AMVT patients.

Objective:To observe the effect of Ba Duan Jin(Eight-brocade Exercise)plus Tuina(Chinese therapeutic massage)in treating primary dysmenorrhea due to cold-induced blood stasis in female college students and on the score of fatigue scale-14(FS-14). Methods:Seventy-two female college students with primary dysmenorrhea due to cold-induced blood stasis were randomized into a Tuina group and a joint group,with 36 cases in each group.The Tuina group only received Tuina manipulations.In the joint group,besides the same Tuina manipulations,patients practiced Ba Duan Jin.For both groups,the once-daily intervention was conducted from 6 d before the menstrual period until menstrual day 1 for 3 menstrual cycles.Changes in the scores of COX menstrual symptom scale(CMSS),visual analog scale(VAS),and FS-14 after the intervention were observed.Clinical efficacy was also estimated. Results:During the process,1 case dropped out in the Tuina group,and 35 cases completed the intervention;2 cases dropped out in the joint group,and 34 cases completed the intervention.The total effective rate was 94.1%in the joint group,higher than 88.6%in the Tuina group(P<0.05).After treatment,the symptom duration and intensity scores in the scores of CMSS,VAS,and FS-14 declined in both groups(P<0.05 or P<0.01);the CMSS symptom duration score and FS-14 score were lower in the joint group than in the Tuina group(P<0.05). Conclusion:Tuina manipulations alone or combined with Ba Duan Jin practice can effectively treat primary dysmenorrhea due to cold-induced blood stasis in female college students;when combined with Ba Duan Jin practice,Tuina manipulations can more significantly improve pain duration and fatigue,suggesting the advantages of combining Tuina Gongfa and manipulations.

Objective To evaluate the efficacy and safety of XELOX regimen combined with hepatic artery chemoembolization in the treatment of gastric cancer with liver metastasis.Methods 50 cases of gastric cancer with liver metastasis were randomly divided into two groups,the experimental group (25 cases) received chemotherapy regimen of XELOX first:Xeloda tablets 1 000 mg/m2,orally,twice a day,days1-14;Oxaliplatin 130 mg/m2,intravenous drip,day 1.Hepatic artery chemoembolization was performed one a week later,and a cycle consists of 4 weeks.The control group (25 cases) received chemotherapy regimen of XELOX,3 weeks as a cycle.All patients were evaluated for efficacy and toxicity every 2 cycles.Results In the experimental group,the overall response rate was 56％,the tumor control rate was 80％,the increase rate of Karnofsky was 60％,and 10 patients got chance of tumor resection.In the control group,the overall response rate was 32％,the tumor control rate was 52％,the increase rate of Karnofsky was 48％,and 6 patients got chance of operation.The overall response rate,tumor control rate,surgical treatment rate and the increase rate of Karnofsky in the experimental group were significantly different from those in the control group (P ＜0.05).The median total survival time was 12.5 months in the experimental group and 10.5 months in the control group (P ＜0.05).There was no significant difference in toxicity and side effects between the two groups.Conclusion XELOX regimen combined with hepatic artery chemoembolization is effective and safe in the treatment of gastric cancer with liver metastasis.

Objective:To assess the usefulness of gray-scale ultrasonography and shear wave elastography in assessing the condition of skeletal muscle in people with chronic heart failure(CHF).Methods:In this prospective study, there were 50 CHF patients and another 50 healthy people undergoing physical checkups served as the control group.The rectus femoris and the gastrocnemius medialis of all participants were evaluated by shear wave elastography and gray-scale ultrasonography.Data were collected using quantitative parameters including muscle thickness, cross-sectional area, fascicle length, pinnation angle, gray value and Young's modulus.Results:Compared with the control group, the thickness(7.80±2.01)mm, cross-sectional area(3.27±0.91)cm 2 and gray value(70.07±6.06)of the rectus femoris and the thickness(12.23±2.42)mm, fascicle length(35.90±1.38)mm and pinnation angle(18.07±3.05)degrees of the gastrocnemius medialis in the CHF group were statistically different[(12.90±2.57)mm, (4.94±2.08)cm 2, 60.93±9.77]for the rectus femoris and(15.90±2.62)mm, (37.83±2.83)mm, (20.75±2.59)degrees for the gastrocnemius medialis of the control group, t=7.71, 3.78, 3.94, 4.99, 4.47, 3.06, P<0.01), but there was no significant difference in Young's modulus of the rectus femoris and the gastrocnemius medialis between the two groups( t=1.10, 0.51, P>0.05).In the CHF group, the thickness, cross-sectional area and gray value of the rectus femoris in different New York Heart Association(NYHA)heart failure grades were statistically different( F=56.36, 69.98, 55.96, P<0.01), but no difference was found in the gastrocnemius medialis with the same ultrasound parameters( P>0.05).When participants in the CHF group was divided into subgroups based on left ventricular ejection fraction(LVEF), there was no significant difference between the subgroups in values of the ultrasonic parameters for the rectus femoris and the gastrocnemius( P>0.05). Conclusions:Gray-scale ultrasound may quantitatively evaluate the skeletal muscle of CHF patients to guide early rehabilitation training to improve their prognosis.

Objective:To investigate differentially expressed genes in muscular tissue in Alzheimer's disease mice and normal mice, and to elucidate the role of differentially expressed genes in the pathogenesis of Alzheimer's disease.Methods:Muscle tissues of Alzheimer’s disease mice and normal mice were collected and the second-generation high-throughput RNA transcription sequencing(RNA-seq)technique was used to screen differentially expressed genes.Additionally, Gene Ontology(GO)functional annotation and Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis were conducted.Differences in the expression of candidate genes in muscle tissues between Alzheimer’s mice and normal mice were verified by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Results:A total of 4 222 genes in muscles had different expression between Alzheimer's disease mice and normal mice, with 2087 genes highly expressed and 2 135 lowly expressed in muscles of Alzheimer's disease mice.GO analysis showed that significantly up-regulated functional genes were mainly enriched in cellular, single biological, metabolic and catalytic processes and activities.KEGG pathway analysis showed that differentially expressed genes were significantly enriched in signal pathways related to metabolism and inflammation.Results from qRT-PCR showed that the mRNA expression of candidate genes was consistent with the trend of transcriptome sequencing results.Conclusions:Differentially expressed genes and related important regulatory signal pathways were screened at the transcriptome level in muscle tissues of Alzheimer's disease mice and normal mice.The differentially expressed genes are enriched in signal pathways related to metabolism and inflammation in muscle tissues of Alzheimer's disease mice and the findings provide an important basis for further exploration of the pathogenesis of Alzheimer's disease.

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 evaluate the effect of atmospheric fine particulate matter (PM2.5) on the expression of skin barrier-associated proteins and proinflammatory cytokines in human keratinocytes. Methods: Atmospheric PM2.5 samples were given by Professor Yufeng Zhou in Children′s Hospital of Fudan University. Human primary keratinocytes were isolated from circumcised foreskins of 5 males, and subjected to culture. These human primary keratinocytes were divided into several groups to be stimulated with PM2.5 at different concentrations of 0 (control group) , 10, 50, 100, 200 mg/L for 24 hours, and cell counting kit (CCK) -8 assay was performed to determine the survival rates of keratinocytes. Fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of filaggrin, keratin-14 and claudin-1 in these keratinocytes respectively, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin (IL) -1α, thymic stromal lymphopoietin (TSLP) and IL-33 in the culture supernatant of these keratinocytes. Statistical analysis was carried out with SPSS13.0 software by using one-way analysis of variance, least significant difference (LSD) -t test and Pearson correlation analysis. Results: After 24-hour treatment with different concentrations of PM2.5, there was no significant difference in the survival rate of keratinocytes between the 10-mg/L PM2.5 group and control group (P > 0.05) , while the survival rates of keratinocytes were significantly lower in the 50-, 100-, 200-mg/L PM2.5 groups than in the control group (all P < 0.05) . After the treatment with 50 mg/L PM2.5, the cell survival rate gradually decreased and then remained stable along with the increase of PM2.5 treatment duration, and the cell survival rate at 24 hours was 72.37% ± 3.12%. After 24-hour treatment with PM2.5, the mRNA expression of filaggrin and keratin-14 was significantly higher in both the 10- and 50-mg/L PM2.5 groups (filaggrin: 1.27 ± 0.15, 1.32 ± 0.09 respectively; keratin-14: 1.15 ± 0.13, 1.08 ± 0.16 respectively) than in the control group (all P < 0.05) , but lower in both the 100- and 200-mg/L PM2.5 groups (filaggrin: 0.84 ± 0.11, 0.42 ± 0.12 respectively; keratin-14: 0.67 ± 0.09, 0.74 ± 0.11 respectively) than in the control group (all P < 0.05) . The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly decreased mRNA expression of claudin-1 compared with the control group (all P < 0.05) . Western blot analysis showed that filaggrin expression was significantly higher in the 50- and 100-mg/L PM2.5 groups than in the control group (both P < 0.05) , while no significant difference was observed between the 10-, 200-mg/L PM2.5 groups and the control group (both P > 0.05) . The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly increased keratin-14 expression compared with the control group (all P < 0.05) . The claudin-1 expression did not differ between the 10-mg/L PM2.5 group and control group (P = 0.87) , but significantly higher in the 50-, 100- and 200-mg/L PM2.5 groups than in the control group (all P < 0.05) . The stimulation with PM2.5 at 10, 50, 100 and 200 mg/L could induce an increase in the supernatant levels of TSLP, IL-1α and IL-33 (all P < 0.01) . Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1α and IL-33 were positively correlated with the concentration of PM2.5 (r = 0.57, 0.67, 0.91 respectively, all P < 0.05) . Conclusion The exposure to PM2.5 can induce decreased survival rate of keratinocytes, aberrant expression of filaggrin, keratin-14 and claudin-1, and elevated secretion of the proinflammatory cytokines TSLP, IL-1α and IL-33, which may lead to impaired skin barrier function.