Clinical factors associated with composition of lung microbiota and important taxa predicting clinical prognosis in patients with severe community-acquired pneumonia / 医学前沿

Few studies have described the key features and prognostic roles of lung microbiota in patients with severe community-acquired pneumonia (SCAP). We prospectively enrolled consecutive SCAP patients admitted to ICU. Bronchoscopy was performed at bedside within 48 h of ICU admission, and 16S rRNA gene sequencing was applied to the collected bronchoalveolar lavage fluid. The primary outcome was clinical improvements defined as a decrease of 2 categories and above on a 7-category ordinal scale within 14 days following bronchoscopy. Sixty-seven patients were included. Multivariable permutational multivariate analysis of variance found that positive bacteria lab test results had the strongest independent association with lung microbiota (R2 = 0.033; P = 0.018), followed by acute kidney injury (AKI; R2 = 0.032; P = 0.011) and plasma MIP-1β level (R2 = 0.027; P = 0.044). Random forest identified that the families Prevotellaceae, Moraxellaceae, and Staphylococcaceae were the biomarkers related to the positive bacteria lab test results. Multivariable Cox regression showed that the increase in α-diversity and the abundance of the families Prevotellaceae and Actinomycetaceae were associated with clinical improvements. The positive bacteria lab test results, AKI, and plasma MIP-1β level were associated with patients' lung microbiota composition on ICU admission. The families Prevotellaceae and Actinomycetaceae on admission predicted clinical improvements.

A autophagy induced by non-stuctural protein 2B of human rhinovirus / 中华实验和临床病毒学杂志

Objective@#To study the intracellular location and autophagosome production of rhinovirus 16 2B protein using miniSOG labeling technique.@*Methods@#2B was fused with miniSOG and flag tags to construct pcDNA3.1-2B-miniSOG-flag plasmid, which was used to transfect HEK293 cells, LC3 protein was detected by western blot. The transfected cells were fixed, stained with DAB through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Localization of 2B-miniSOG protein in cells and ultrastructural changes of cells were observed under electron microscope.@*Results@#2B-miniSOG protein glows green under a fluorescence microscopy. Green flourescence coold be observed in the cells expressing 2B-miniSOG protein.LC-II protein increased in the cells transfected with pcDNA3.1-2B-miniSOG-flag. Under electron microscopy it was observed that 2B-miniSOG protein was located in the mitochondria, and a large number of vesicular structures appeared in the cytoplasm. Both autophagosomes and autophagic lysosomes can be observed.@*Conclusions@#Non-structural protein 2B of HRV16 can induce autophagy.

Intracellular location of severe fever with thrombocytopenia syndrome bunyavirus NP protein with correlative light and electron microscopy based on miniSOG / 中华实验和临床病毒学杂志

Objective@#To study the intracellular location and characteristic of SFTSV NP protein in different phases using mini singlet oxygen generator (miniSOG) labeling technique.@*Methods@#MiniSOG is a recently-invented genetically-encoded tag for EM. MiniSOG-fused SFTSV NP (NPSOG) gene was cloned by PCR, and inserted into pcDNA3.0 plasmid to form pTPL-NPSOG, which was used to transfect 293 cells. The transfected cells of different phases were fixed in 2.5% glutaraldehyde in situ, stained with DAB through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Intracellular location and characteristic of SFTSV NP protein in different phases were studied by observing the sections under transmission electron microscope.@*Results@#After transfecting the plasmid with NPSOG to 293 cells, NP protein was expressed in cytoplasm and peri nucleus, and gradually aggregated, which connected with endoplasmic reticulum and Golgi apparatus to form larger volume and irregular inclusion bodies in cytoplasm. No obvious subcellular structure changes were found.@*Conclusions@#The SFTSV nucleoprotein can be expressed separately to form inclusion bodies without the assistance of other viral proteins. The formation of inclusion bodies requires the directional movement and aggregation of a certain number of NP proteins, which may involve the interaction of NP protein and host organelles during this period.

Study of intracellular location of adenovirus protein IX with correlative light and electron microscopy based on miniSOG / 中华实验和临床病毒学杂志

Objective@#To establish a method of correlative light and electron microscopy (CLEM) to study the intracellular location of adenovirus protein IX.@*Methods@#MiniSOG (mini singlet oxygen generator) is a recently-invented genetically-encoded tag for CLEM. MiniSOG-fused adenovirus IX gene (IXSOG) was cloned by PCR, and inserted into pcDNA3 plasmid to form pTPL-IXSOG, which was used to transfect 293 cells. IXSOG expressing cells could be distinguished under fluorescence microscope due to the emission of green fluorescence of miniSOG. The transfected cells were fixed in 2.5% glutaraldehyde in situ, stained with diaminobenzidine(DAB) through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Intracellular location of IXSOG was studied by observing the sections under transmission electron microscope.@*Results@#Eukaryotic expression plasmid carrying IXSOG fusion gene was constructed. IXSOG expressing cells were selected for DAB photooxidation and preparation of ultrathin sections. IXSOG fusion mainly formed punctate aggregations or inclusions in the nucleus.@*Conclusions@#The correlative light and electron microscopy method based on miniSOG was successfully established, and it could be used to study the intracellular localization of viral proteins.

Development of human embryonic stem cell platforms for human health-safety evaluation / 北京大学学报(医学版)

SUMMARY Thehumanembryonicstemcells(hESCs)serveasaself-renewable,genetically-healthy, pluripotent and single source of all body cells,tissues and organs.Therefore,it is considered as the good standard for all human stem cells by US,Europe and international authorities.In this study,the standard and healthy human mesenchymal progenitors,ligament tissues,cardiomyocytes,keratinocytes,primary neurons,fibroblasts,and salivary serous cells were differentiated from hESCs.The human cellular health-safety of NaF,retinoic acid,5-fluorouracil,dexamethasone,penicillin G,adriamycin,lead ace-tate PbAc,bisphenol A-biglycidyl methacrylate (Bis-GMA)were evaluated selectively on the standar-dized platforms of hESCs,hESCs-derived cardiomyocytes,keratinocytes,primary neurons,and fibro-blasts.The evaluations were compared with those on the currently most adopted cellular platforms.Parti-cularly,the sensitivity difference of PM2.5 toxicity on standardized and healthy hESCs derived fibroblasts, currently adopted immortalized human bronchial epithelial cells Beas-2B and human umbilical vein endo-thelial cells (HUVECs)were evaluated.The results showed that the standardized hESCs cellular plat-forms provided more sensitivity and accuracy for human cellular health-safety evaluation.

Preparation of Recombinant Human Adenoviruses Labeled with miniSOG / 病毒学报

We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.

Expert consensus on induction of human embryonic stem cells into tenocytes / 浙江大学学报·医学版

Embryonic stem cells have unlimited proliferative capacity, which may provide a source of tendon stem/progenitor cells for tissue engineering. Experts of International Science and Technology Collaborative Program of Ministry of Science and Technology have developed a protocol consensus on differentiation of human embryonic stem cells into the tendon cells. The consensus recommends a protocol of two-step generation of human embryonic stem cells into tendon cells: the human embryonic stem cells are first differentiated into mesenchymal stem cells on different material surfaces; then with the scaffold-free tissue engineering tendon formed by high-density planting, the mesenchymal stem cells are induced into tendon cells under static or dynamic mechanical stimulation in vivo and in vitro. Tissue engineering tendon established in vitro by the protocol can be used as a model in toxicological analysis and safety evaluation of tendon-relevant small molecule compounds, medical materials and drugs.

Google Flu Trends--the initial application of big data in public health / 中华预防医学杂志

Google Flu Trends (GFT) was the first application of big data in the public health field. GFT was open online in 2009 and attracted worldwide attention immediately. However, GFT failed catching the 2009 pandemic H1N1 and kept overestimating the intensity of influenza-like illness in the 2012-2014 season in the United States. GFT model has been updated for three times since 2009, making its prediction bias controlled. Here, we summarized the mechanism GFT worked, the strategy GFT used to update, and its influence on public health.

Rescue and Amplification of Recombinant Human Adenovirus Type 41 in 293 Cells / 病毒学报

Human adenovirus type 41 (HAdV-41) is considered to be a "fastidious adenovirus". E1-deleted HAdV-41 cannot be rescued or amplified in 293 cells. To propagate recombinant HAdV-41 in 293 cells, the backbone plasmid pAdbone41 was reconstructed. That is, the E3 coding sequence of HAdV-41 was deleted and replaced with the HAdV-5 E4orf6 gene; and the E1A enhancer of HAdV-5 was inserted upstream of the E4 promoter of HAdV-41. Novel adenoviral plasmid pAd41E4EE-GFP was generated by homologous recombination of the shuttle plasmid pSh41-GFP with the modified backbone plasmid in the Escherichia coli BJ5183 strain. Adenovirus HAdV-41-E4EE-GFP was rescued by transfecting 293 cells with linearized pAd41E4EE-GFP. After seven rounds of propagation, viruses were purified by the CsCl ultracentrifugation method. HAdV-41-E4EE-GFP in 1.0 ml with a particle titer of 8 x 10(10) vp/mL was obtained which had a particle-to-infectious ratio of 50 : 1. The genome of HAdV-41-E4EE-GFP was confirmed by restriction analyses and polymerase chain reaction. These results showed that a novel HAdV-41 vector system was established in which recombinant HAdV-41 could be constructed and packaged in 293 cells.

Clinical application and research of autologous chondrocyte implant / 中华骨科杂志

ObjectiveTo investigate and evaluate the result and the possibility of the clinical application of autologous chondrocyte implant (ACI).MethodsFrom November 2007 to June 2009,6 cases of knee articular cartilage defect were treated with ACI,including 2 males and 4 females with an average age of 39.5 years (range,19-55).All the defects were located on the condyles of femur with a mean size of 7.3 cm2 (range,3.8-11.6).ACI comprises a two-stage procedure:chondrocytes are first harvested from the non-load bearing area of the joint,expand in vitro to acquire enough cells,and then the chondrocytes are implanted.The defect of cartilage were covered with bone membrane and fixed with sutures and fibrin albumen glue.Lysholm score system,International Knee Documentation Committee (IKDC) grading system,and MRI were used to evaluate the effect of ACI,6 and 12 months post-operatively.ResultsAll the patients were followed up.The clinical outcomes of the 6 and 12 months follow-ups demonstrated increased of clinical scores.The MRI follow-up showed good filling of the defect with tissue having the imaging appearance of cartilage in all patients.Only one patient suffered adhesion,because she refused to finish rehabilitation exercises as our treatment advises.ConclusionAs the clinical effect of ACI for knee cartilage defect is satisfied,the ACI may be a good choice for treating knee cartilage defect in future.It is very important to control the indications strictly and guarantee to finish the post-operative rehabilitation exercises.

Clinical analysis of acute cerebral vascular accident related factors of patients with nosocomial infection / 中国基层医药

Objective To explore acute cerebral vascular accident factors in patients with nosocomial infection. Methods Clinical data with 680 cases with acute cerebral vascular accident were retrospectively analyzed,and the patients with hospital flu infected as the observation group, selected in accordance with 1 ∶ 1 over the same period without cerebrovascular accident combined hospital patients feel as control group. The patient age,gender,state of consciousness , invasive operation, dehydrating agent application time, whether use of antibiotics and other differences were compared . Results In 680 cases of patients with acute cerebral vascular accident, there were 90 cases of hospital infection; two groups gender, dehydrating agent application time had no difference(P ＞ 0. 05 ); the observation group compared with the control group older, unconscious, to implement invasive operation to prevent high proportion of antibiotics(P ＜0. 05), hospital infection-related factors. Conclusion Acute cerebral vascular accident patients should have a higher incidence of nosocomial infection, and be related with many factors. Taking corresponding measures against the relevant factors could prevent hospital infection.

Preparation of human parvovirus B19 virus-like particles / 生物工程学报

The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.