Objective To investigate the molecular mechanism of the inhibitory effects of rhizoma coptidis on multi-drug resistant uropathogenic Escherichia coli.Methods High-throughput RNA sequencing ( RNA-seq ) was performed to investigate the transcriptome in a multi-drug resistant uropathogenic Escherichia coli strain (NB8) treated with water decoction of rhizoma coptidis .Agar dilution test was used to determine the minimal inhibitory concentration ( MIC) of water decoction of rhizoma coptidis against the NB 8 strain.A growth curve was drawn to evaluate the effects of water decoction of rhizoma coptidis on the growth of NB8 strain.Total RNAs were extracted from the NB 8 strain after treated with the water decoction of rhizo-ma coptidis for 30 minutes and then synthetized to cDNA by reverse transcription after screening out the rRNAs.The HiSeq 2000 sequencing system was used for transcriptome sequencing .The TopHat software was used to map and analyze the RNA-Seq reads, and then Cufflinks was run to assemble transcripts and es-timate their abundances .The differential expression , GO enrichment and KEGG metabolic pathway were fur-ther analyzed .The NB8 strain dealt with normal saline was used as negative control .Results The MIC of water decoction of rhizoma coptidis to NB 8 strain was 12.5 mg/ml.There were 3665 genes expressed in NB8 strain treated with water decoction of rhizoma coptidis and 3430 genes expressed in NB8 strain treated with normal saline .The number of differentially expressed genes was 1428 including 921 up-regulated genes and 507 down-regulated genes .Those differentially expressed genes mainly enriched in the modules of binding and catalysis.The genes concerning cell adhesion , apoptosis and multicellular process were up-regulated, while those concerning the regulation of enzyme activities were down-regulated.Results of the KEGG meta-bolic pathway enrichment analysis showed that the genes concerning synthetic pathway of LPS were signifi -cantly up-regulated as well as those encoding the repair polymerase Ⅲthat was involved in DNA replication . However , the genes concerning fatty acid metabolism , histidine metabolism , thiamine metabolism , folate metabolism and iron carrier in ribosome synthesis showed overall down-regulation.Conclusion The tran-scriptome in uropathogenic Escherichia coli strain treated with rhizoma coptidis was profiled .The main mo-lecular mechanism of the inhibitory effects of rhizoma coptidis on uropathogenic Escherichia coli was to de-stroy the cell wall of Escherichia coli, affect the replication of DNA and regulate the transcription and transla-tion of proteins .This study illustrated that the inhibitory effects of rhizoma coptidis on uropathogenic Esche-richia coli were achieved in multiple levels .

Horizontal gene transfer (HGT), also Lateral gene transfer (LGT), is any process in which an organism transfers genetic material to another species that is not its offspring. With the increase of available genomic data, it has become more convenient to study the way to detect the genes, which are products of horizontal transfers among a given genome. There are few data about known horizontal gene transfers in three bacterium genomes under consideration, so the experiments, which simulated gene transfer by artificially inserting phage genes, were carried out. Combining the feature analysis methods of gene sequences with support vector machine (SVM), a novel method was developed for identifying horizontal gene transfers (HGT) in 3 fully sequenced bacterium genomes (Escherichia coli K12, Borrelia burgdorferi, Bacillus cereus ZK). According to our previous work, codon use frequency (FCU) was selected as the sequence feature, in respect that it is inherently the fusion of both codon usage bias and amino acid composition signals. In addition, another computational method was proposed considering strand asymmetry and predicting horizontal gene transfers of leading strand and lagging strand of genomes under consideration, respectively. To avoid the occasionality of simulating gene transfer through artificially inserting phage genes, 100 times of the transfer-and-recover experiment were repeated and arithmetic average of measurement for each genome being considered were reported to evaluate algorithm's performance. Ten-fold cross-validation was used for both parameter and accuracy estimation. The best results were obtained for C-Support Vector Classification (C-SVC) type by using the radial basis function kernel with ?=100, while for one-class SVM type the best performance was obtained using the polynomial kernel of three degree. The performance of the approach was compared with that of Tsirigos' method ,which is one of the best predictive approachs to date in detecting of horizontal transfer genes. Firstly, for the original method that did not consider the strand asymmetry, the C-SVC type has a high relative improvement(RI) of 31.47% on hit ratio for Escherichia coli K12, while the one-class SVM type has RI of 11.61% for Borrelia burgdorferi. Moreover, as theoretically expected, the method considering the strand asymmetry resulted in higher RI than the original method. In order to examine the approach's performance in detecting factual gene transfer events, the approach was applied in genome of Enterococcus faecalis V583. It is not only succeed in recovering all the seven factual horizontally transferred genes, also found that the whole segment from 7 kb upstream of gene EF2293 to 38 kb downstream of gene EF2299 was probably transferred into E. faecalis V583 genome simultaneously with the above seven genes.

Objective To investigate the distribution of β-lactamase genes in a pan-drug resistant Klebsiella pneumoniae isolate JM45.Methods Klebsiella pneumoniae JM45 was isolated from the blood sample of a patient admitted in the intensive care unit,the Second Affiliated Hospital,Zhejiang University School of Medicine on April 7,2010.The susceptibilities to 26 antibiotics were tested using E-test method.Cica-β-Test was performed to detect β-lactams,and modified Hodge test was performed to detect carbapenemase.Resistant genotypes were detected using PCR,DNA sequencing and BLAST algorithm.Whole genome sequencing (complete graph) was performed by high throughput Roche 454 sequencing approach to analyze the distribution of β-lactamase genes.Results Except polymyxin B and tigecycline,JM45 was resistant to other 24 kinds of antibiotics including cephalosporins and carbapenems.Several β-lactamases were positive in Cica-β-Test,and modified Hodge test was positive.Based on PCR typing,TEM-1,SHV-11,CTX-M-24 and VEB-3 were positive,but carbapenemase genes and metallo-β-lactamase genes were negative.A complete genome (chromosome) sequence (GenBank accession number:CP006656) and 2 plasmids sequences (GenBank accession number:CP006657,CP006658) were obtained by wholegenome sequencing.CTX-M-24 (Locus tag:N559_5233),TEM-1 (Locus tag:N559_5242) and VEB-3 (Locus tag:N559_5248) were positive in plasmid 1.CTX-M-24 located in insertion sequence (IS903-CTXM-24-ISEcp1),while TEM-1 and VEB-3 located in transposons (tnpA-TEM-1-rmtB and VEB-3-tnpA).SHV-11 (Locus tag:N559_2715) was positive in genome (chromosome),and 4 putative β-lactamase genes or β-lactamase domains were obtained:(1) metallo-β-lactamase domain protein (Locus tag:N559_0119,780 bp) ; (2) putative β-lactamase (Locus tag:N559_1633,1308 bp) ; (3) β-lactamase domain protein (Locus tag:N559_2279,813 bp); (4) β-lactamase domain protein (Locus tag:N559_3769,1101 bp).No insertion sequence or transposase gene was observed near SHV-11.Conclusion The resistance to antibiotics including cephalosporins and carbapenems is correlated with TEM-1,SHV-11,CTX-M-24,VEB-3 and 4 kinds of putative β-lactamase genes or β-lactamase domains.

To study the biological characteristics and mutations of influenza A(H1N1)pdm09 virus isolated from one case of pneumonia of unknown etiology (PUE),which would provide references for clinical treatment and disease control,the throat swab specimen from the PUE case was isolated in the Madin-Darby Canine Kidney (MDCK) cells,and then the antigenicity,pathogenicity and drug resistance of influenza A (H1N1) pdm09 virus were analyzed after sequencing.As a result,one influenza virus strain was isolated from the specimen and named as A/FujianGulou/SWL64/2016(H1N1).The similarities of nucleotide sequences and amino acids sequences compared with the vaccine strain A/California/07/2009 (H1N1) were 96.9％-98.9％ and 96.7％-99.5％,respectively.Eighteen amino acids had mutated in the HA and 4 mutations,K163Q,S185T,S203T and D222N,were involved in 3 different epitopes,which indicated that the antigenic drift had occurred in the influenza virus.The D222N mutation associated with receptor binding site made the virus infect lower respiratory tract more easily.The virus was still amantadine-resistance and oseltamivir-sensitive.In conclusion,the influenza A (H1N1) pdm09 virus in this study have occurred antigenic drift and has the molecular characterization of causing severe pneumonia,so further surveillance should be performed to prevent and control the influenza epidemic.

Objective:To compare the biomechanical characteristics of screw only spatial weaving fixation and calcaneal plate fixation in calcaneal fractures.Methods:Sanders type III calcaneus fracture model was established by using calcaneus model specimens: the physiological model group were the normal calcaneal models; the steel plate group were conventional steel plate fixation fracture models; the metal screw group were fracture models with only metal screw weaving fixation; seven and nine absorbable screw spatial weaving groups (seven absorbable screw group, nine absorbable screw group) were used to weave and fix fracture models with seven and nine absorbable screws. Cyclic test and mechanical compression test were carried out, and load-displacement curves were recorded. The material properties of metal screw spatial weaving and calcaneal anatomical plate system were replicated, finite element fracture models were established, and the calcaneal internal fixation models of plate screw group and spatial weaving screw group were completed by reverse processing. The changes of biomechanical characteristics of calcaneal bone in human (70 kg) standing on one foot were simulated, and the distribution of structural strength was analyzed by Von Mises equivalent stress cloud diagram and displacement cloud diagram.Results:In the cyclic test of 20-200 N load, the physiological model group, the plate group, the metal screw group, the absorbable 7 screw group, the absorbable 9 screw group were 0.87±0.22, 0.82±0.08, 0.70±0.12, 1.04±0.13 and 0.83±1.76 mm, the difference in model gap was statistically significant ( F=3.16, P=0.037). Among them, the absorbable 7 screws group was larger than the metal screws group ( t=4.28, P=0.003), and the other pin-two comparisons were not statistically significant ( P>0.05). The deformation of the five groups was 0.37±0.06, 0.38±0.07, 0.38±0.06, 0.52±0.07 and 0.42±0.07 mm, and the difference was statistically significant ( F=4.39, P=0.010). The deformation of absorbable 7 screws group was greater than that of physiological model group, the plate group and metal screw group ( t=3.69, P=0.006; t=3.25, P=0.012; t=3.51, P=0.008). In static test, compression displacement was 7.14±0.79, 7.30±0.66, 6.95±0.28, 8.19±0.61 and 7.16±0.55 mm, the difference was statistically significant ( F=3.28, P=0.032). The displacement of the absorbable 7 screws group was greater than that of the metal screws group ( t=4.13, P=0.003). The stiffness changes were 570.60±122.62, 512.86±80.77, 497.40±66.50, 456.21±58.19 and 560.39±94.40 N/mm, respectively, with no statistical significance ( F=1.44, P=0.258). The results of finite element analysis showed that under 3 500 N axial pressure load, the maximum compression displacement and stiffness of the plate and screw set were 6.47 mm, 540.96 N/mm, and the Von Mises equivalent stress peaks were 450.31 and 353.15 MPa, respectively. The maximum compression displacement and stiffness of the braided screw group were 5.25 mm, 666.67 N/mm, and the peak Von Mises equivalent stress of the screw was 396.20 MPa. Conclusion:Compared with lateral plate fixation, spatial weaving fixation can provide sufficient biomechanical stability for calcaneal healing and is superior to plate fixation in terms of structural stability, which may help to improve the effectiveness of calcaneal fracture fixation.

Objective:To evaluate the clinical significance of the coincidence test with 3D mirror image of unaffected side in the treatment of contralateral calcaneal fracture.Methods:A retrospective analysis was performed of the 35 patients who had been treated for single calcaneal fracture by coincidence test with 3D mirror image of unaffected side from January 2016 to June 2018 at Department of Trauma Orthopedics, The Sixth Hospital of Ningbo. They were 29 males and 6 females with an average age of 44 years (from 18 to 71 years). By Sanders classification, 19 cases were type Ⅱ and 16 cases type Ⅲ. CT scan and reconstruction of both calcaneuses was performed. The image of unaffected calcaneus was mirrored by Mimics software to guide virtual treatment of contralateral calcaneal fracture. Preoperatively the virtual bilateral calcaneuses and the reduced calcaneus were 3D printed to guide the actual operation. The coincidence test with 3D mirror image of unaffected side was used to evaluate fracture reduction after operation. Postoperative parameters such as B?hler Angle, Gissane Angle, calcaneal width, and coincidence of the affected calcaneal bone were recorded at 12 months after surgery. The clinical outcomes were assessed by American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score and visual analogue scale (VAS) score.Results:In this cohort, operation time averaged 43.9 min (from 30 to 55 min), time from admission to operation 3.6 d (from 0 to 14 d), and hospital stay 6.3 d (from 3 to 11 d). The patients were followed up for an average of 18.8 months (from 13 to 24 months). All patients achieved bone union after an average of 17 weeks (from 12 to 26 weeks). Re-collapse of subtalar articular surface, talocalcaneal joint fusion, loss of calcaneal height or length, and calcaneal width increase were observed in none of the patients. The VAS scores averaged 2 (from 0 to 4), and the AOFAS scores 93.3 (from 76 to 98). The total coincidence was 95.2%±3.5%. Spearman test indicated a positive correlation between postoperative total coincidence and AOFAS score ( ρ=0.588, P<0.05). Conclusion:The coincidence test with 3D mirror image of unaffected side can be used to treat contralateral calcaneal fracture, with advantages of reduced surgical time, increased surgical accuracy and fine clinical outcomes.

Objective:To study a safe anatomical range of the lateral calcaneus for safe surgical approaches.Methods:In 15 cadaveric specimens of the calcaneus, the coordinate axes X, Y, Z were established on the apex of the fibula (the lowest point) as the origin on the lateral side of the calcaneus. In the main part of the lateral calcaneus in the third quadrant of coordinates, the spatial quantification was conducted of the 4 important anatomical structures on the lateral calcaneus (calcaneofibular ligament insertion, common peroneal tendon sheath, lateral calcaneal artery and nerve). The trend trajectory of each structure was summarized. Based on the summary of above measurements, the safety ranges were coincided to find a safe anatomical range for a surgical approach that might lead to the least probability of damaging the important lateral calcaneal structures.Results:The distances from the 4 important anatomical structures to the X, Y, and Z axes of the main part of the lateral calcaneus in the third quadrant are respectively: (21.40±3.38) mm, (18.47±3.91) mm, and (25.06±3.45) mm for the lateral calcaneal artery; (16.53±4.77) mm, (16.27±3.68) mm, and (23.13±4.00) mm for the lateral calcaneal nerve; (9.73±1.73) mm, (11.47±2.13) mm, and (10.87±1.59) mm for the common peroneal tendon sheath; (22.33±2.84) mm for the calcaneofibular ligament insertion. The above 4 important structures mainly converge at the origin O and the anterior 1/3 of the tangent to the outer edge of the calcaneus. In this range, a safe surgical approach can be designed that is located at >2.5 cm below the lowest point of the fibula and 2.1 cm behind the rear side of the arc range.Conclusion:A safe surgical approach can be designed in the safe convergence range of important anatomical structures of the lateral calcaneus to reduce iatrogenic injury to the important structures on the lateral side of the calcaneus.

Objective To investigate the characteristics and influencing factors of infection in elderly patients with oral and maxillofacial surgery,and to explore the effect of infection on inflammatory response-related proteins,in order to guide the clinical treatment.Methods A total of 653 cases of patients aged more than 60 who were treated with oral and maxillofacial surgery during January 2004 to December 2015 in Shenzhen People's hospital was involved and divided into infected group (n =48) and uninfected group (n =605) according to whether postoperative nosocomial infection was occurred.The characteristics and influencing factors of infection were observed.A total of 171 cases of healthy volunteers whose age and sex were matched with patients were collected as control group.The expressions of inflammatory responserelated proteins in 3 groups were compared.Results The main site of postoperative nosocomial infection was lower respiratory tract,with 15 cases of patients,accounting for 31.3％.A total of 48 strains of pathogens was isolated,the main pathogenic bacteria were Streptococcus pyogenes,with 8 strains,accounting for 16.7％.Streptococcus pyogenes had high drug resistance to erythromycin and clindamycin,and had high sensitivity to penicillin G and meropenem.Aged more than 70,operation time more than 3 h,the primary diseases as malignant tumor were the independent risk factors for postoperative nosocomial infection (P ＜0.05).Blood interleukin (IL)-1,IL-2,IL-6,IL-10,tumor necrosis factor α (TNF-α),transforming growth factor β (TGF-β) and interferon-γ (IFN-γ) in infected group were more than uninfected group and control group.The differences were statistically significant (P ＜ 0.05).Conclusions The rate of infection in elderly patients with oral and maxillofacial surgery is still high in our hospital.The main site of infection is lower respiratory tract and the main pathogens were Streptococcus pyogenes.Aged more than 70,operation time more than 3 h,the primary diseases as malignant tumor were the independent risk factors for postoperative nosocomial infection.Infection can lead to peripheral inflammatory factor levels increase significantly.

Purpose To design PCR combined probes u-sing TaqMan technology to detect the expression of major driver genes in a variety of soft tissue sarcomas at one time,and to dis-cuss whether the combined probes can better assist clinicopatho-logical diagnosis based on histological features and FISH results.Methods Our research group designed 32 pairs of fusion gene probes related to soft tissue sarcoma based on TaqMan tech-nique,involving 10 types of sarcoma.The histopathological specimens of 70 patients with common fusion gene soft tissue sarcoma in our hospital were examined by fusion gene combina-tion,and the histopathological specimens of 30 patients with oth-er soft tissue sarcoma without fusion gene were set as controls.Individual common sarcoma types were analyzed with FISH probe detection.At the same time,the detection performance of the combined probe was evaluated by various methods.Results The soft tissue sarcoma-related fusion gene probe designed by our research group was used to detect the confirmed soft tissue sarcomas,and the results showed that the highest sensitivity was 100％.Among the three types of tumors,protuberant dermatofi-brosarcoma,synovial sarcoma and mucinous liposarcoma were verified by FISH,and the coincidence rate of the two methods was high,with no statistical significance(P＞0.05).The re-sults of interlot and intralot reproducibility of protuberous derma-tofibrosarcoma,mucinous liposarcoma and synovial sarcoma were consistent.Three different concentration limits were used to de-tect the positive plasmid of all the fused gene RNA,and 25 cop-ies/μL was the lowest concentration limit.Conclusion Com-bined with the pathological diagnosis results,TaqMan technology can be used to design PCR combined probes for soft tissue sarco-ma,which have high sensitivity and high specificity and good methodological performance,and meet the needs of primary medical institutions for one-time and rapid auxiliary pathological diagnosis of common soft tissue sarcoma.It provides a rapid and reliable method for the detection of multiple fusion genes in clin-ical soft tissue sarcoma.

Objective: To explore the clinical manifestations, histopathological features, diagnosis, treatment and prognosis of Rosai-Dorfman′s disease (RDD) in the maxillofacial region and to review the relevant literature in order to improve the understanding, diagnosis and treatment of oral and maxillofacial RDD. Methods : The clinical manifestations, histopathological features, diagnosis, treatment, and prognosis of a patient with RDD in the maxillofacial region admitted to Shenzhen People′s Hospital were analyzed, and the literature was reviewed for analysis. Results: The clinical manifestations were palpable masses of 3.5 cm × 2.0 cm × 1.0 cm in the right cheek and 3.0 cm × 2.0 cm × 1.0 cm in the right submaxillary area, with clear boundaries, good mobility, medium and hard textures, respectively, no tenderness, smooth surfaces, and no obvious nodules. On contrast-enhanced and plain CT scans of the maxilla and neck, a diffuse soft tissue shadow was seen in the right maxillofacial region with an unclear boundary and uniform density, and the contrast-enhanced scan also showed moderate and uniform enhancement. The primary diagnosis was right maxillofacial lesions. The tumor was resected surgically. The pathological report was right buccal and right submaxillary extranodal RDD. Under light microscopy, nodular lesions in the fibrous fat tissue were found, which were composed of light and deep staining areas. The light staining areas consisted of patchy, polygonal cells with large volumes and rich cytoplasm, in which lymphocytes and neutrophils could be seen stretching into the movement; the deep staining areas were composed of lymphocytes and plasma cells. IHC: S-100 (+), CD68 (+), CD163 (+), CD1a (-), CD21FDC (+), langerin (-), IgG (+), IgG4 (+). No recurrence was found 11 months after the operation. RDD is a rare, benign and self-limited tissue and cell disease and consists of multiple lesions in the maxillofacial region. Its imaging features are similar to those of lymphoma. Its pathological features are large volumes, rich cytoplasm and phagocytosis of lymphocytes and plasma cells. Generally, RDD only needs to be observed, and individuals with symptoms or the involvement of important organs need to be treated; the first choice for the extranodal type is drug treatment, with radiotherapy administered if the central nervous system is involved. Surgery is recommended if involvement of important organs and compression of the trachea are observed; chemotherapy should be used for diffuse RDD. Most patients with RDD experienced relapse or remission of the disease; a few patients died because of the involvement of important organs or complications. Conclusion The clinical manifestations of maxillofacial RDD vary and lack specific imaging features, and pathological immunohistochemistry is the gold standard for diagnosis. The etiology is not completely clear, the treatment methods are varied, and the prognosis is related to the involved range of the disease.