OBJECTIVETo establish a localization ultrathin section method through which target cytopathic cells could be sectioned in situ.

METHODSLab-Tek Chamber slide system (177402) was selected as resin embedding mould. Cells infected with Human adenovirus type 5 (Ad5) or A/HN/SWL3/ 2009 (H1N1) influenza virus were embedded in situ as models. Target cytopathic cells were exposed by trimming, sectioned and observed under transmission electron microscope (TEM).

RESULTSTarget cells could be sectioned in situ and virus particles could be found easily on sections.

CONCLUSIONA localization ultrathin sectioning method was established and this technique could be applied in virus detection in cytopathic cells to improve TEM detection efficiency.

Adenovirus Infections, Human ; pathology ; virology ; Adenoviruses, Human ; physiology ; ultrastructure ; Cell Line ; Humans ; Influenza A Virus, H1N1 Subtype ; physiology ; ultrastructure ; Influenza, Human ; pathology ; virology ; Microscopy, Electron, Transmission ; Microtomy ; methods

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae. Filoviruses cause severe filovirus hemorrhagic fever (FHF) in humans, with high case fatality rates, and represent potential agents for bioterrorism and biological weapons. It is necessary to keep surveillance of filoviruses, even though there is no report of their isolation and patients in China so far. To characterize MARV morphology, the Lake Victoria marburgvirus--Leiden was stained negatively and observed under a transmission electron microscope which is one of important detection methods for filoviruses in emergencies and bioterrorism. MARV showed pleomorphism, with filamentous, rod-shaped, cobra-like, spherical, and branch-shaped particles of uniform diameter but different lengths. Pleomorphism of negatively stained MARV is summarized in this article, so as to provide useful information for possible electron microscopic identification of filoviruses in China.
Animals ; Humans ; Marburg Virus Disease ; virology ; Marburgvirus ; growth & development ; ultrastructure ; Microscopy, Electron, Transmission ; Virion ; growth & development ; ultrastructure

A simple,reliable RPHPLC method on ODS column with UV detector for deter-mination of the anticoagulant rodenticide coumatetralyl in animal tissue was developed.1,1′-Bi-(2-Naphthol)was used as an internal standard to check the process ofexperiments.The mean recoveries from the spiked rabbit liver were around 90% atthe levels of 0.4-16mg/kg.The coeffecint of variation of 5 determination was 3.2%.The results showed that this method has a satisfactory reproducibility.The methodis practical for examining poisons in poisoning cases.

Nine Rabbits were orally administrated with a single dose of 25mg/kg,100mg/kgcoumatetralyl to establish poisoning model.Poisoning sympton and pathological changes were observed.Coumatetralyl content in main internal organsand muscle was determined by RPHPLC method.The results were discussed.

Mucosal adjuvants play important roles in vaccine development. By now, the common used mucosal adjuvants can be divided into three categories: the bacterial derivatives, cytokines and chemokines, and antigen delivery systems. Progresses of the three kinds of adjuvants were reviewed to give a reference to novel vaccine research.

NSP4, as the diarrhea-related protein of rotavirus, is becoming an attractive candidate for vaccine development. To compare the immunogenicity of NSP4 from different genetic groups, we constructed eukaryotic expression plasmids comprising the NSP4 genes from four different genetic types using the pCI vector. The recombinant vectors were designated as pCI-97B6, pCI-97S36, pCI-97S34 and pCI-97SZ8, respectively. Following the conformation of the transient expression of the constructs in 293 cells, the plasmids were respectively subjected to the 5 round i. m. inoculation of BALB/c mice. The specific antibodies against NSP4 as well as the IgG1/IgG2a subclasses of immunoglobulin in mice sera were examined with indirect ELJSA after each immunization. The results showed that the immunization of plasmids expression NSP4s could elicit not only humoral but also cellular immunity, but the humoral immune response is dominant. There is a difference of immunogenecity among the NSP4 of different genetic type. Further studies were needed to focus on the relationship between the immunogenicity and protection effect.

RNA interference ( RNAi) is a process in which double-stranded RNA ( dsRNA) induces specific postranscriptional silencing of homologous transcripts. Systematic silencing of genes on a genome-wide scale using RNAi library targeting many thousands of genes provides a powerful research tool in functional genomics. RNAi libraries, which may be derived from plasmids cloning, virus packaging, PCR amplification, chemically synthesis or enzymatic digestion, have been successfully used to identify gene function, dissect signaling pathway and discover drug targets, etc. Promising progresses have been made in this field. The development, application and problems of RNAi library methodology and future prospects were reviewed.

The development of immunotoxin DT386-GMCSF, a fusion protein which bears the N-terminal 386 amino acids of diphtheria toxin and human granulocyte-macrophage colony-stimulating factor (GM-CSF) and targets the GM-CSF receptor (GM-CSFR), has provided a promising alternative therapy to the acute myeloid leukemia (AML). However, the poor expression of the protein in E.coli is still a bottleneck which limits the industrial production. To identify the critical down-regulating factors on the expression of DT386-GMCSF, a series of truncated mutants of DT386-GMCSF at the C-terminal of GM-CSF were generated and expressed in E.coli. The results showed that the encoding sequences for the L114 of the GM-CSF dramatically impact the expression of DT386-GMCSF. On this basis, a serial of mutants integrating amino acid substitutes were generated. The results revealed that the expression level of the mutant DF123GVT, which harbors the amino acids 1-123 of GM-CSF whose L114L115V116 was substituted with G114V115T116, was evidently higher than that of the DT386-GMCSF, whereas the specific cytotoxicity to blast recovered from mice injected with HL60, a cell line highly expresses the GM-CSFR, was similar. These results have provided an important basis for the future development of the immunotoxins targeting the GM-CSFR.

Objective To study the relationship between TGF-?signaling pathway and pathogenesis of gastric carcinoma.Methods The expression of TGF-?RⅠ,TGF-?RⅡand Smad4 protein was deter- mined by immunohistochemistry in normal gastric mucosa(26 cases),intestinal metaplasia(22 cases),dysplasia (20 cases)and gastric carcinoma(43 cases).Results The positive expression rate of TGF-?RⅠ,TGF-?RⅡand Smad4 decreased following the malignant degree in gastric tissues(P

Objective To analyze the sequence of microsatellite and the flanking sequence from four populations of Oncomelania hupensis. Methods We cloned 159 SSR-PCR amplification products of a commonly used primer, (CA)8RY, using O. hupensis genomie DNA as template, and sequenced 82 products Results The sequences obtained were novel O. hupensis genomic sequences but not repeat simple sequence. It was observed that 36 out of 82 clones contained microsatellites between priming sites.The flanking sequences of certain microsatellite were invariant. Both (GA/CT). and (TTAGGG/CCCAA)n were found in four populations of O. hupensis. However, (CAA)n were found only in O. hupensis from Fuqing,Fujian province and (TCTCTG), were found only in O. hupensis from Guichi,Anhui province and (GAA/TTC)n, (CAA/TTG)n, (CAT), were found only in O.hupensis from Puge,Sichuan province. Conclusion The results obtained by SSR-PCR should not be interpreted as the amplification of microsatellite loci, and analytical rules similar to those for Random Amplified Polymorphic DNA should be used. SSR-PCR could not make the most of the priority of microsatellite. It seems better to amplify the microsatellites with the primers designed on the basis of the flanking sequence.