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

Ramie and kenaf were traditional fiber crops in China, but their stalk after decorticating has not been used effectively. The stalk contains a lot of cellulose, and can therefore be used for the production of bioethanol. We studied the effects of different chemical pretreatment on enzymatic digestibility of ramie stalk and kenaf stalk. Ramie and kenaf stalks pretreated with alkali were chosen to produce ethanol using quasi-simultaneous saccharification and fermentation (Q-SSF) process. The results show that for the stalks pretreated with 4% NaOH and 0.02% anthraquinone-2-sulfonic acid sodium salt (AQSS) as catalyzer at 170 degrees C for 1 h, the ethanol concentration could reach 51 g/L after fermentation for 168 h at 18% of solid substrate concentration. By fed-batch to 20% of solid substrate concentration, the ethanol concentration could reach 63 g/L, 77% and 79% of the cellulose conversion could get for ramie stalk and kenaf stalk, respectively. For kenaf stalk pretreated with 5.2% NaHSO3 and 0.2% H2SO4 at 170 degrees C for 1 h, the ethanol concentration and cellulose conversion could reach to 65 g/L and 72%, respectively.
Alkalies ; Biofuels ; Biotransformation ; Boehmeria ; Cellulose ; China ; Ethanol ; chemistry ; Fermentation ; Hibiscus ; Hydrolysis ; Plant Stems ; chemistry

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.

To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.
Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; growth & development ; physiology ; ultrastructure ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Humans ; Virus Release ; Virus Replication

Microscopy, Electron, Transmission ; methods ; Negative Staining ; methods ; Viruses ; ultrastructure

Objective:To obtain stable radioresistant non-small-cell lung cancer (NSCLC) cell lines and identify the genetic pattern of conventional fractioned and hypofractionated radiotherapy. Methods:A549 NSCLC cells were treated with 6 MV of x-rays through conventional fractionated (2 Gy, 17 f) and hypofractionated irradiation (4 Gy, 7 f) to establish a radiation resistance cell model. Tumor cell radioresistance was determined using a clonogenic assay andγ-H2AX immunofluorescence staining combined with confocal microscopy. After extracting total mRNA from the cells, a whole genome expression microarray was applied to detect differential gene expression. The genes with at least a twofold increase in expression (P<0.05) were analyzed, and the pathway (Q<0.05) methods were used to further analyze the chip results. Results:After irradiating the A549 cells, two radioresistant cell lines were obtained, namely, the A549R2Gy-R and the A549R4Gy-R cell lines. The A549R2Gy-R cell line was radioresistant to the conventional fractionated irradiation, whereas the A549R4Gy-R cell line was ra-dioresistant to hypofractionated irradiation. Microarray analysis showed that the A549R2Gy-R cells exhibited 1 701 differentially expressed genes (357 upregulated, 1 344 downregulated) compared with the parental A549 cell. By contrast, the hypofractionated irradiation-resistant A549R4Gy-R cells had 944 upregulated genes and 2 602 downregulated genes compared with the A549 cells. The A549R2Gy-R cells exhibited 318 upregulated genes and 699 downregulated genes compared with the A549R4Gy-R cells. Several signaling pathways were implicated in radioresistance when conventional fractionated radiotherapy was compared with hypofractionated irradiation radiotherapy using path way-significant enrichment analysis, especially the PI3K and Erb B channel signaling pathway kinase. Conclusion:Multiple genes and signaling pathways are involved in the development of radiation resistance in NSCLC. The underlined radioresistance mechanisms under conventional and hypofractionated radiotherapy need further study and elucidation to provide new targets for drug development.

Objective To analyze the causes of time consumption in CT enhancement scanning (CTES) and CT angiography (CTA) examinations in order to optimize the procedures, and help to save time and medical costs for patients. Methods A total of 2328 outpatients and 1402 inpatients to take CTES and CTA examinations were randomly selected as the normal control group, and another 2085 outpatients and 793 inpatients who underwent the optimized procedures were randomly selected as the experimental group. The problems of time consumption and patients'satisfaction degree were analyzed. Results The major causes for time consumption in CTES and CTA examinations included taking wrong contrast medicine, forgetting to take contrast medicine, having no auxiliary examination results, waiting in the wrong line, and opening the cap of contrast medicine. The time spent for checkup for inpatients and outpatients in the control and experimental groups was (119.8±15.6) minutes and (31.5±8.6 ) minutes vs (55.2 ± 10.6) minutes and (8.4 ±2.1) minutes. The satisfaction degree of inpatients and outpatients in the control and experimental groups were 90.16%(1264/1402) and 88.66%(2064/2328) vs 98.49%(781/793) and 97.94%(2042/2085). The experimental group spent shorter time and had higher satisfaction degree than those in the control group, and the differences were statistical significantly, tinpatient=34.96, P<0.01, toutpatient=12.03, P<0.01;χ2inpatient=55.20, P<0.01,χ2outpatient=146.27, P<0.01. Conclusions After the procedures of CTES and CTA examinations are optimized, the checkup time is significantly shortened, and patients' satisfaction degree is remarkably improved.