Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.
Antigens, Viral ; genetics ; immunology ; metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Mutagenesis, Site-Directed ; methods ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Envelope Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo analyze the factors affecting the occurrence and severity of crush syndrome (CS) after crush injury (CI) in pediatric trauma victims in the Wenchuan earthquake.

METHODSMedical records of 98 patients who were transferred to our hospital were retrospectively reviewed. The risk factors, such as age, gender, time being besieged, type of injury, wound infection, hemodialysis, etc., which were assessed with T-test/chi(2)/Fisher's exact tests and logistic regression analysis for the occurrence of crush syndrome after crush injury. Possible risk factors influencing CS severity were analyzed.

RESULTSThere were 15 patients with CS, and all these cases were from 59 patients with extremities crush injury. The incidence of CS reached 15.3% in pediatric trauma victims after earthquake and 25.4% in extremities crush injury. Six risk factors were assessed with logistic regression analysis for three outcomes relating to crush syndrome, they are age, time being szeged and closed CI, whose log-odds ratio (log-OR) respectively was 1.049, 1.221, and 0.068 (P < 0.05 for all). And no correlation was found between CS and gender, upper or lower limbs injury or wound infection. There was no significant difference in wounds infection rate between patients with open injury and those who underwent CS fasciotomy (P = 0.754), but there was significant difference between those patients who underwent CS fasciotomy and those who underwent other operative incisions (P < 0.05). Wound infection had a significant association with severity of CS (P = 0.041) as compared with other factors such as age, gender, and time being szeged.

CONCLUSIONThe occurrence of crush syndrome is mainly because of extremities crush injury and also has significant relations with age, time being szeged and closed crush injury in children. Infection of incisional wound after CS fasciotomy is a risk factor for aggravation of CS.

Adolescent ; Child ; Child, Preschool ; China ; Crush Syndrome ; Disasters ; Earthquakes ; Female ; Humans ; Injury Severity Score ; Male ; Risk Factors

OBJECTIVETo investigate the distribution and the drug resistance pattern of pathogenic bacteria isolated from pediatric cases suffering from wounds infection following the Wenchuan earthquake.

METHODSOf the ninety-eight injured children, 50 had wound infection diagnosed by clinical examination. Specimens for culture were collected from the fifty injured children and the results of bacterial identification and antibiotic resistance were retrospectively reviewed.

RESULTSIn the fifty injured children with wound infection, microbial growth was detected in 31 (62.0%) and 21 children suffered from mixed infections (the infection rate was 67.7%). Ninety-nine pathogens were isolated, gram positive bacteria accounted for 16.16% (16 strains), Gram negative bacteria accounted for 81.82% (81 strains), and fungus 2.02% (2 strains). Staphylococcus aureus (5 strains, 5.05%), Enterococcus faecalis (3 strains, 3.03%) and Enterococcus faecium (2 strains, 2.02%) were the primary Gram-positive bacteria identified and Gram-negative infections typically included Acinetobacter baumanii (27 strains, 27.27%), Enterobacter cloacae (18 strains, 18.18%) and Pseudomonas aeruginosa (13 strains, 13.13%). Acinetobacter baumanii was the most common organism isolated from wounds. Duration of being szeged and complications had a significant association with wound infection with Acinetobacter baumanii. Drug sensitivity tests displayed that the isolated bacteria were highly resistant to common antibiotics. One strain of Acinetobacter baumanii-calcoaceticus complex and six strains of Acinetobacter baumanii were resistant to all common antibiotics including imipenem/cilastatin. Vancomycin-resistant Gram-positive bacteria were not identified.

CONCLUSIONFollowing the Wenchuan earthquake disaster, wound infection profiles of pediatric patients were significantly different, Acinetobacter baumanii was the main common organism isolated from wounds in contrast to the previous low isolation rate. The isolated bacteria were highly and multiple drug resistant and it was difficult to treat. Knowing the distribution and the drug resistance pattern of pathogen is of paramount importance in guiding the clinical treatment.

Adolescent ; Child ; Child, Preschool ; Disasters ; Drug Resistance, Bacterial ; Earthquakes ; Female ; Gram-Negative Bacteria ; drug effects ; isolation & purification ; Gram-Positive Bacteria ; drug effects ; isolation & purification ; Humans ; Male ; Microbial Sensitivity Tests ; Wound Infection ; microbiology ; Wounds and Injuries ; microbiology

Objective To study the epidemic tendency of emerging influenza A (H1N1) in mainland China, and to explore the different patterns of spread on the disease under the following contexts: (1) To stop the temperature screening program at the border areas of the country; (2)To stop measures of prevention and control on those identified cases and their close contacts; (3) To strengthen programs for the foreign immigrants on 'home quarantine'. Methods Under relevant parameters and information on the transmission link from different reference data, the patterns of influenza spread were simulated by Monte Carlo method. Results The temperature screening on border could inhibit the transmission of influenza A (H1N1) to some extent, so that after 3 months the cumulative number of cases will be reduced by 21.5% (1718 cases) and transmission speed of influenza A (H1N1) in mainland China will be delayed by about 4 days. Furthermore, taking positive measures of prevention and control could efficiently slow down the epidemic, so that after 3 months the cumulative number of cases will be reduced by 93.4%(about 90 thousand cases) and it would be delayed by about 15 days if influenza A (H1N1) spreads to the whole country. In addition, if the immigrants were able to practise quarantine measures consciously by themselves at home the effect of prevention and control against influenza A(H1N1) would be more significant. If 30%, 60% and 90% of immigrants would take quarantine measures home consciously, after 3 months the cumulative number of cases will be reduced by about 15% (about 940 cases), 34% (about 2230 cases) and 64% (about 4180 cases), respectively. Also, influenza A (H1N1) spreads to the whole country will be delayed by about 4 days, 10 days and 25 days, respectively. It is difficult to curb fully the development of the epidemic by taking existing control measures, and influenza A (H1N1) may spread to almost all provinces after about 3 months. Conclusion The effects of existing prevention and control measures were objectively assessed and the results showed the necessity and effectiveness of these measures against the transmission of influenza A (H1N1) , in the mainland of China.

Aim To explore the mechanism of Danshen decoction in the treatment of colon cancer using network pharmacology and molecular docking.Methods The active components and corresponding target proteins of Salvia miltiorrhiza, Santalum album and Amo-mum villosum in Danshen decoction were screened based on Traditional Chinese Medicine Systems Pharmacology database and analysis platform.The targets of colon cancer were searched by using Genecards database, and the common targets were selected.The network diagram of traditional Chinese medicine-active components-target-disease was constructed by using Cytoscape 3.7.0.The protein protein interaction network of common targets was constructed by using STRING database.The gene ontology(GO)and Kyoto Encyclopedia of Genes and Gnomes(KEGG)enrichment analysis were carried out based on R4.0.2.The important targets in the key pathways and the important active components in the network diagram of traditional Chinese medicine-active ingredients-target-disease network were selected for Surflex Dock.Results A total of 78 active components, 142 targets, 3 239 colon cancer targets, 105 overlapping targets and 69 corresponding active components were screened out.KEGG analysis showed that the key signaling pathway was PI3K/AKT.Luteolin and Tanshinone IIA with high correlation were selected to dock with protein kinase B(AKT1).Both active components had hydrogen bonding with AKT1.Conclusions Danshen decoction plays a positive role in colon cancer treatment.The mechanism may be related to the regulation of PI3K/AKT signaling pathway.

ObjectiveTo explore the common syndromes of patients with cerebral infarction in rural areas of eastern Henan based on latent structure model and factor analysis，and provide reference for clinical differentiation of cerebral infarction. MethodThe data samples of patients with cerebral infarction in rural areas in eastern Henan were preprocessed. With Lantern 5.0 of latent structure method and LTM-EAST algorithm of two-step latent tree analysis， the manifest variable latent structure model of related symptoms was built to interpret different latent nodes， and common syndromes of cerebral infarction were obtained via comprehensive cluster analysis. SPSS 20.0 was used for factor analysis and cluster analysis of related symptoms to infer the distribution of syndrome types. ResultThe data of 888 patients with cerebral infarction were included， involving symptoms， tongue and pulse （88 in total）. The 65 symptoms with a frequency of ≥5% were constructed into a latent structure model， and 31 latent variables were obtained. The Bayesian information criterion （BIC） score was -15 367.17. Based on professional knowledge， s6 common syndrome types were found， namely， syndrome of upward disturbance of wind-fire， Qi deficiency and blood stasis syndrome， syndrome of phlegm and blood stasis blocking collaterals， syndrome of phlegm-heat and fu-organ excess， syndrome of wind phlegm obstructing collaterals， and syndrome of stirring wind due to yin deficiency. In factor analysis， the symptoms with a frequency of >10% were selected， and 13 common factors were obtained and used for systematic cluster analysis. And 5 syndrome types were inferred： syndrome of wind phlegm obstructing collaterals， syndrome of phlegm-heat and fu-organ excess， Qi deficiency and blood stasis syndrome， syndrome of combined phlegm and blood stasis， and syndrome of yin deficiency and internal heat. According to the determination criteria of syndrome types in traditional Chinese medicine （TCM）， 6 common syndrome types of cerebral infarction were finally determined. ConclusionAccording to the severity of the disease， the common syndromes of patients with cerebral infarction in rural areas of Eastern Henan were divided into the following categories： apoplexy involving channel and collateral： syndrome of upward disturbance of wind fire， syndrome of wind phlegm obstructing collaterals， and syndrome of stirring wind due to yin deficiency. Apoplexy involving zang and fu-viscera： syndrome of phlegm-heat and fu-organ excess， and syndrome of phlegm and blood stasis blocking collaterals. Recovery period： Qi deficiency and blood stasis syndrome. This study was basically consistent with the syndrome law in TCM theory， and provided reference for further establishing syndrome diagnostic criteria of cerebral infarction.

ObjectiveTo explore the material basis and mechanism of Nardostachyos Radix et Rhizoma （NRER）-Agrimoniae Herba （AH）， the herbal pair effective in regulating the liver， invigorating Qi， and calming palpitations， in the treatment of premature ventricular contractions （PVCs） by network pharmacology and molecular docking. MethodThe chemical components and targets of NRER and AH were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） combined with relevant literature. GeneCards，Online Mendelian Inheritance in Man（OMIM），and DrugBank were used to predict the potential targets against PVCs. STRING platform was used for protein-protein interaction （PPI） analysis. Metascape platform was used for Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analysis. Cytoscape 3.8.0 was used to construct the NRER-AH component-potential target-signaling pathway network. The main target proteins underwent molecular docking to the active components of NRER-AH by AutoDock 4.2.6. ResultThe targets of nine active components in NRER-AH （such as quercetin，kaempferol，and acacetin） against PVCs mainly involved tumor necrosis factor （TNF），mitogen-activated protein kinase 1（MAPK1），and protein kinase B1（Akt1）. The potential targets were mainly enriched in 26 signaling pathways，such as pathways in cancer and the advanced glycosylation end product （AGE）-receptor of advanced glycosylation end product（RAGE） signaling pathway. The results of molecular docking showed that the majority of the active components （92.59%） of NRER-AH had good binding activities with the main target proteins TNF，MAPK1，and Akt1. ConclusionThe active components of NRER-AH can regulate cardiac ion channels，resist inflammation， and combat oxidative stress to treat PVCs through multi-target and multi-pathway interventions. They can also improve symptoms related to depression and anxiety by inhibiting monoamine oxidase activity and protecting nerves from damage. This study is expected to provide research ideas and the theoretical basis for further exploring the material basis and mechanism of NRER-AH in the treatment of PVCs.