Hydrogen-water ameliorates radiation-induced gastrointestinal toxicity via MyD88’s effects on the gut microbiota

Although radiation therapy is a cornerstone of modern management of malignancies, various side effects are inevitably linked to abdominal and pelvic cancer after radiotherapy. Radiation-mediated gastrointestinal (GI) toxicity impairs the life quality of cancer survivors and even shortens their lifespan. Hydrogen has been shown to protect against tissue injuries caused by oxidative stress and excessive inflammation, but its effect on radiation-induced intestinal injury was previously unknown. In the present study, we found that oral gavage with hydrogen-water increased the survival rate and body weight of mice exposed to total abdominal irradiation (TAI); oral gavage with hydrogen-water was also associated with an improvement in GI tract function and the epithelial integrity of the small intestine. Mechanistically, microarray analysis revealed that hydrogen-water administration upregulated miR-1968-5p levels, thus resulting in parallel downregulation of MyD88 expression in the small intestine after TAI exposure. Additionally, high-throughput sequencing showed that hydrogen-water oral gavage resulted in retention of the TAI-shifted intestinal bacterial composition in mice. Collectively, our findings suggested that hydrogen-water might be used as a potential therapeutic to alleviate intestinal injury induced by radiotherapy for abdominal and pelvic cancer in preclinical settings.

Protective Effect of S-isopentenyl-L-cysteine against DNA Damage in Irradiated Mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the protective effect of S-isopentenyl-L-cysteine,a new cysteine derivative,on DNA damage induced by radiation by using acute radiation injury animal models.</p><p><b>METHODS</b>Forty ICR mice were randomly divided into five groups:the control group,1.0Gy gamma irradiation group,1.0Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,7.2Gy gamma irradiation group,and 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,with 8 mice in each group.The comet assay and bone marrow polychromatic micronucleus experiments were performed to evaluate the double-strand DNA breaks in ICR mice exposed to 1.0 and 7.2Gy gamma-ray, respectively.</p><p><b>RESULTS</b>The tail DNA percentage,tail length,tail moment,and olive tail moment of peripheral blood lymphocytes in 7.2Gy gamma irradiation group were significantly higher than that of the control group (P<0.01).And it was also observed that above experimental indexes of 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group was significantly less than that of 7.2Gy gamma irradiation group (P<0.05). In addition,the micronucleus rate of 1.0Gy gamma irradiation group and 7.2Gy gamma irradiation group were both significantly higher than in the control group (P<0.01). In addition,in mice given S-isopentenyl-L-cysteine before irradiation,the micronucleus rate of ICR mice exposed to 1.0 and 7.2Gy gamma-ray decreased from (39.5000 ± 3.3141)‰ to (28.1667±4.1345)‰ (P=0.033) and from (76.5000 ± 4.6242)‰ to (22.8333 ± 3.6553)‰(P=0.000),respectively. The bone marrow polychromatic micronucleus experiment indicated that the value of polychromatic erythrocyte (PCE)/normochromatic erythrocyte(NCE) of ICR mice exposed to 1.0 and 7.2Gy gamma-ray was less than the control group(P<0.05). Meanwhile,after irradiating by certain dose,the value of PCE/NCE in mice given S-isopentenyl-L-cysteine before irradiation was significantly higher than the corresponding groups (P<0.05).</p><p><b>CONCLUSION</b>S-isopentenyl-L-cysteine has a good protective effect against DNA damage induced by radiation.</p>

Construction and immunogenicity of recombinant bacteriophage T7 vaccine expressing M2e peptides of avian influenza virus / 病毒学报

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.

Influence of epitope A modification and N-linked glycosylated site mutation of PRRSV NJ-a strain ORF5 gene on the ability to induce neutralizing antibodies and T cell proliferation response / 生物工程学报

To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTf method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.

Study on the Immune Efficiency for General Vaccine Against Avian Influenza Virus Using Human Mycobacterium Tuberculosis hsp70 as the Carrier for Peptide Epitopes / 中国生物工程杂志

M2e gene of three copies for H5N1 subtype AIV was synthesized and fused with human mycobacterium tuberculosis hsp70 gene.The fused gene was cloned into the prokaryotic expression vector to get pET-3M2e and pET-3M2e-hsp70.Recombinant protein r3M2e and r3M2e-hsp70 were successfully expressed induced with IPTG and purified with Ni2+-NTA collumn.Following that,the immunity of the recombinant protein was analysized with Western blot.20-day-old AIV non-immunized chickens were vaccined with r3M2e and r3M2e hsp70,at the same time,Trx and KLH-M2e inoculated chickens were served as vector and positive controls.Two weeks after the primary vaccination,every group was boosted with the same vaccine as in the primary vaccination.The humoral immunity of the vaccined chickens was evaluated with antibody detection against M2e,cytopathic suppression test,and indirect fluorescence assay.The cellular immunity was estimated according to lymphocyte subtype analysis with flow cytometry and M2e specific cytokine detection.Four weeks after the boost vaccine,all groups were challenged with 100EID50 AIV of H9N2 subtype,and the virus from swabs was detected with Real-time PCR.Results indicated that r3M2e hsp70 vaccined chicken developed the better humoral and cellular immune response,also,made a better performance compared with r3M2e vaccined group in virus challenge.

Conjunctive Use of Various Adjuvant and Fusion Protein Which Composed of M2e and N P Genes of Avian Influenza Virus,and the Influence on Immunogenicity / 中国生物工程杂志

Based on the gene sequence of AIV matrix protein 2(M2) and two cytotoxic T-lymphocyte epitopes derived from nucleoprotein,the prokaryotic expression vector pET-3M2e-NP1-NP2 was constructed.The target gene was expressed in the solvable form in BL21(DE3) when induced with 1.0 mmol/L IPTG and Western blot analysis showed that the expression product had good immunogencity.Purified fusion protein was mixed with various amount of adjuvant,including Freund's,Vash oil and chitosan,and then immunized 20-day-old chicken by intramuscular injection and boosted 3weeks later.Blood samples were collected weekly following the primary vaccination.The anti-M2e antibody was detected with ELISA method with the synthesized peptide as coated antigen;the neutralizing ability of anti-serum was evaluated on MDCK cell line and chick embryo,the CD4+ and CD8+ T lymphocyte amounts in peripheral blood of immunized chicken was measured by flow cytometry.Results showed that the fusion protein can induce immunological reaction,and the antibody can bind with the viral M2 protein that expressed on the surface of MDCK cells.Flow cytometry result showed that CD4+ and CD8+ T lymphocyte in peripheral blood increased obviously following immunization(P

Characterization of murine leukemia virus recombinants that express H5N1 subtype avian influenza virus hemagglutinin glycoproteins / 生物工程学报

One highly pathogenic strain of avian influenza virus (AIV) was isolated from goose in China recently, designated as F-3. In order to study the viral entry mechanisms, the hemagglutinin (HA) gene of H5N1 subtype AIV isolate was amplified by RT-PCR, and then cloned into pGEM-T vector and sequenced. The sequencing result has logging in GenBank, the accession number was AY639405. The HA gene of F-3 had a complete open reading frame (ORE) and composed of 1707 nucleotides, coding for 568 amino acids. The deduced amino acid sequence at the cleavage site of the HA protein was RKKR GLF, matched to the characteristic of virulent avian influenza strain. The HA gene were subcloned into pcDNA3, so the plasmid pcDNA-HA can express the HA glycoprotein. Co-transfected pcDNA-HA, pHIT60 (include Murine Leukemia Virus structural genes, namely gag and pol) and pHIT111 (retroviral vector genome,containing LacZ as a reporter) into 293T cells. The retroviral supernatant were harvested 48 hours post-transfection, filtered through 0.45 micromol/L filter. The supernatant were used to analysis the characteristic of the pseudotyping virions by Western blotting and infection test. Western blotting revealed the HA glycoproteins can be expressed on the virions, indicated the glycoproteins were incorporated onto the retroviral virions. Infection test were performed on 293T, NIH3T3 and COS-7, all the three kinds of cells infected were lacZ positive, indicating viral entry, and revealed the pseudotype virions of MuLV-HA were infectious. So the pseudotype system of MuLV particles with AIV Hemagglutinin proteins were setted up and it can be used to study the entry of avian influenza virus isolated from goose in China.