The inhibitory effect of rBCG on EB virus-positive tumours using an EB virus fusion gene.

At present, studies have found that latent Epstein-Barr virus (EBV) infection is associated with a variety of human tumours, and a vaccine is not available in this field. In this research, RT-PCR was used to obtain BZLF1 (immediately expressed early antigen Z) and LMP2 (latent membrane protein 2) cDNA from EBV. A ZLP2 fusion gene containing a linker sequence that encoded the polypeptide (Gly4Ser)3 was obtained using the sequence splicing overlap extension method. Then, ZLP2 was inserted into pMV261 cells, and the recombinant plasmid pMV-ZLP2 was transformed into BCG competent cells. After EB virus-positive tumour cell (NPRC18) cancer models were established with C57BL/6 J mice, tumour weight, tumour formation time and mouse survival conditions were analyzed, and flow cytometry was used to analyze the quantities of CD8 + and CD4 + T cells. HE staining was used to detect and analyze lymphocyte infiltration, and statistical analysis was used to analyze the immunological effect of recombinant BCG (rBCG). Compared with the control group, rBCG could significantly prolong the survival time of mice, slow tumour growth and delay tumour formation time. Recombinant BCG exhibits an obvious immune effect in mice and an inhibitory effect on EBV-positive cancer.Key points• AZLP2 fusion gene with BZLF1 and LMP2 of EB virus was constructed.• ZLP2 fusion gene was expressed with rBCG.• rBCG with ZLP2 has an obvious effect on EBV-positive cancer.

Pontixanthobacter rizhaonensis sp. nov., a marine bacterium isolated from surface seawater of the Yellow Sea, and proposal of Pseudopontixanthobacter gen. nov., Pseudopontixanthobacter confluentis comb. nov. and Pseudopontixanthobacter sediminis comb. nov.

A bacterial strain designated RZ02T was isolated from surface seawater collected from the Yellow Sea in PR China and characterized by polyphasic taxonomy. Cells of strain RZ02T were Gram-stain-negative, aerobic, non-motile, catalase- and oxidase-positive rods forming ochre-pigmented colonies. Growth occurred at 7-36 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.0) and with 1-5 % (optimum, 2 %) NaCl. The major cellular fatty acids (>10 %) of strain RZ02T were summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and C16 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol and sphingoglycolipid. The genome size of strain RZ02T was 2.79 Mbp with a G+C content of 55.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RZ02T was mostly related to Pontixanthobacter luteolus SW-109T and Pontixanthobacter aestiaquae HDW-31T (97.3 and 97.1% sequence similarity, respectively), and formed a phyletic lineage with members of the genus Pontixanthobacter. The phylogenetic analysis based on the up-to-date bacterial core gene sequences confirmed that strain RZ02T clustered within the genus Pontixanthobacter. The average nucleotide identity and in silico DNA-DNA hybridization values between strain RZ02T and P. luteolus SW-109T and P. aestiaquae HDW-31T were 72.8 and 72.9 % and 18.7 and 18.5%, respectively. Based on these evidences, strain RZ02T is proposed to represent a novel species of the genus Pontixanthobacter under the name Pontixanthobacter rizhaonensis sp. nov. The type strain is RZ02T (=KCTC 62828T=MCCC 1K04521T). In addition, based on the results of whole genome analyses, proposals of Pseudopontixanthobacter gen. nov., Pseudopontixanthobacter confluentis comb. nov. and Pseudopontixanthobacter sediminis comb. nov. are also included.

Reclassification of Algibacter wandonensis as a Later Heterotypic Synonym of Algibacter lectus Based on Whole-Genome Sequence Analysis.

The genus Algibacter belongs to the family Flavobacteriaceae of the Bacteroidetes, and all members of this genus were isolated from marine environments. Among the Algibacter species, two members, Algibacter lectus KMM 3902T and Algibacter wandonensis WS-MY22T, were isolated from green algae and sediment around a brown algae respectively. The 16S rRNA gene sequences of these two type strains possess 99.4% sequence similarity. In this study, further studies were undertaken to clarify the taxonomic assignments of the two species. Whole-genome sequence analysis showed that the similarities for other phylogenetic markers are also very high (i.e. 99.9% for gyrB, 99.6% for recA and 99.9% for rpoD). Average nucleotide identity, average amino acids identity and digital DNA-DNA hybridization value between A. lectus KMM 3902T and A. wandonensis WS-MY22T are 98.3%, 98.6% and 89.4% respectively, all clearly exceed suggested species delineation thresholds. Furthermore, phylogenetic trees based on sequences of 16S rRNA gene and up-to-date bacterial core gene set (UBCG) consisting of 92 genes provided additional evidence that A. lectus KMM 3902T and A. wandonensis WS-MY22T are very closely related. In addition, a review of their profiles indicated that A. lectus KMM 3902T and A. wandonensis WS-MY22T did not present pronounced differences at phenotypic and chemotaxonomic levels. Based on these evidence, we propose that A. wandonensis should be reclassified as later heterotypic synonyms of A. lectus.

Whole genome analysis calls for a taxonomic rearrangement of the genus Colwellia.

Microbial taxonomy is the foundation of microbiology and rapid advancements in DNA sequencing technologies are providing new approaches to address prevailing questions in this field. The family Colwelliaceae, which currently comprises four genera, is a diverse and globally abundant group of Gamaproteobacteria. Based on 14 publically available genomes of bacteria strains labeled as members of the family Colwelliaceae, phylogenomic analyses were conducted to revisiting the taxonomic status of this family both in the genus and species level. Using genome-based phylogeny as a primary guideline and genome-based similarity indexes including average amino acid identity, percentage of conserved proteins, average nucleotide identity, and the digital DNA-DNA hybridization as supplements, the following taxonomic proposals were proposed: Colwellia polaris, Colwellia beringensis, Colwellia sediminilitoris, Colwellia aestuarii, Colwellia chukchiensis and Colwellia mytili should be reclassified into the novel genus Cognaticolwellia; Colwellia agarivorans should be reclassified into the novel genus Pseudocolwellia. Our results constitute a solid framework for current and future taxonomic decisions within this family, which will be helpful for avoiding confusion with ecological and evolutionary interpretations in subsequent studies.

Litorilituus lipolyticus sp. nov., isolated from intertidal sand of the Yellow Sea in China, and emended description of Colwellia asteriadis.

A Gram-stain negative, rod-shaped, aerobic, oxidase-positive and catalase-weakly positive bacterial strain with polar or subpolar flagellum, designated RZ04T, was isolated from an intertidal sand sample collected from a coastal area of the Yellow Sea, China. The organism was observed to grow optimally at 25 °C and pH 6.5-7.0 with 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain RZ04T was closely related to Colwellia asteriadis (similarity 96.9%) and Litorilituus sediminis (similarity 96.8%), and 94.4-96.4% sequence similarities to other type strains of species of the genera belonged to the family Colwelliaceae. The dominant fatty acids of strain RZ04T were determined to be C17:1ω8c, C15:1ω8c, C16:0 and summed feature 3 (C16:1ω6c and/or C16:1ω7c), and the predominant isoprenoid quinone was determined to be quinone 8 (Q-8). Phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid and four unidentified lipids were determined to be the major constituents of the polar lipids. The genome of strain RZ04T is 4.14 Mbp with a G + C content of 37.4 mol%. A total of 3631 genes are predicted, with 3531 protein-coding genes, 75 RNA genes and 25 pseudogenes. Based on phenotypic, genotypic and phylogenetic analysis, strain RZ04T is considered to represent a novel species in the genus Litorilituus, for which the name Litorilituus lipolyticus is proposed. The type strain is RZ04T (= MCCC 1K03616T = KCTC 62835T). An emended description of Colwellia asteriadis is also provided.

EB virus-positive tumors are inhibited by rBCG expressing hGM-CSF and LMP2A.

For the development of safe and effective EBV (Epstein-Barr virus) vaccines, the Ag85A signal peptide from M. tuberculosis H37Rv was used to construct a recombinant secretory BCG (Bacillus Chalmette-Guérin) plasmid. The Ag85A gene, fused to the EBV LMP2A (latent membrane protein) and hGM-CSF (human granulocyte/macrophage colony-stimulating factor) genes, was inserted into the pMV261 vector (secretory BCG plasmid). The expression levels of the hGM-CSF and LMP2A proteins in rBCG (recombinant BCG) were measured by Western blot analysis. Humoral immunity, cellular immunity, and antitumor effects were determined by a series of experiments. The recombinant pMVGCA plasmid effectively expressed GCA (hGM-CSF and LMP2A fusion protein) in BCG after transformation, and the rBCG proteins were recognized by antibodies against hGM-CSF and LMP2A. Six weeks after immunization, the maximum dose of rBCG resulted in antibody titers of 1:19,800 (hGM-CSF antibody) and 1:21,800 (LMP2A antibody). When the effector:target ratio was 40:1, specific lysis was maximal and approximately two times stronger than that in mice immunized with the control. Tumorigenicity was lower in the rBCG treatment group, with a tumor inhibition rate of 0.81 ± 0.09 compared with the control groups. EB virus-positive tumors are inhibited by rBCG expressing an hGM-CSF and LMP2A fusion protein.

Flavivirga rizhaonensis sp. nov., a marine bacterium isolated from intertidal sand.

A bacterial strain designated RZ03T was isolated from an intertidal sand sample from the Yellow Sea in China and characterised using a polyphasic taxonomic approach. Cells of strain RZ03T were observed to be Gram-stain negative, aerobic, and oxidase and catalase positive rods showing gliding motility and forming yellow colonies. Growth was found to occur at 7-30 °C (optimum, 25 °C), at pH 5.5-9.5 (optimum, pH 6.5-7.0) and with 0.5-5% NaCl (optimum, 1.5-2%). Phylogenetic analysis based on 16S rRNA gene sequences indicates that strain RZ03T clusters within members of the genus Flavivirga of the family Flavobacteriaceae and is closely related to the type strains Flavivirga amylovorans JCM 17112T and Flavivirga jejuensis JCM 17113T (97.9% and 97.5% similarity, respectively). The predominant cellular fatty acids are iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and iso-C15:0 3-OH and the major respiratory quinone is MK-6. Polar lipids include phosphatidylethanolamine, three unidentified aminolipids, an unidentified phospholipid and four unidentified lipids. The genome of strain RZ03T is 4.88 Mbp with a G+C content of 32.2 mol%. A total of 4152 genes are predicted, with 4052 protein-coding genes, 51 RNA genes and 49 pseudogenes. This polyphasic study suggests that strain RZ03T represents a novel species in the genus Flavivirga, for which the name Flavivirga rizhaonensis is proposed. The type strain is RZ03T(= KCTC 62833T = MCCC 1K03615T).

MiR-1298 affects cell proliferation and apoptosis in C6 cells by targeting SET domain containing 7.

Our previous high-throughput sequencing indicated that rno-miR-1298 was down-regulated in ischemia-reperfusion model of rat. However, little is known about the function and molecular mechanism of rno-miR-1298 in rat tumor cell. In this study, rno-miR-1298 was detected to be significantly down-regulated in rat tumor C6 cells. Moreover, overexpression of rno-miR-1298 obviously inhibited the proliferation and induced apoptosis in C6 cells. SET domain containing 7 (SETD 7) was identified to be a target of rno-miR-1298 using bioinformatics and luciferase reporter assays. Overexpression of rno-miR-1298 markedly reduced the expression of SETD 7 at protein level. Knockdown of SETD 7 also suppressed proliferation and promoted apoptosis in C6 cells. It was indicated that rno-miR-1298 affected cell proliferation and apoptosis of rat tumor cells by targeting SETD 7. Thus, the newly identified miR-1298/SETD 7 expands the elaboration of the mechanisms of the development and progression of tumors and may provide therapeutic target for tumors of nervous system.

Anti-tumour research of recombinant BCG using BZLF1 and hGM-CSF fusion genes.

The random primer Oligo(dT)15 was used in RT-PCR to obtain cDNA from the human granulocyte macrophage colony stimulating factor (hGM-CSF) gene and the Epstein-Barr virus (EBV) gene BZLF1. Then, the sequence splicing overlap extension method was used to obtain a GCBF fusion gene containing a linker sequence that encoded the polypeptide (Gly4Ser)3. The GCBF fusion gene was inserted into pMV261, which was then transformed into competent E. coli DH5 alpha cells, and positive cells were selected based on kanamycin resistance on LB plates. The recombinant plasmid pMVBZLF1 was extracted from E. coli, and BCG (Bacillus Calmette-Guérin) was transformed into competent cells. According to the RT-PCR results, the target genes hGM-CSF and BZLF1 were 461bp and 788bp in size, which was in agreement with the expected values. Construction of the recombinant plasmid by double enzyme digestion, amplification, sequencing and Western blotting confirmed that the GCBF fusion gene (1204bp) was correctly inserted into pMV261, successfully transformed into BCG competent cells, and properly expressed. After mice were injected with rBCG (recombinant BCG), antibody levels were detected using ELISA, and spleen cells were obtained and the killing rates of specific CTLs by rBCG were detected using a CTL assay kit. Then, the influence of rBCG on tumour cells was analysed in C57BL/6 mice. We found that rBCG-secreting cytokines hybridized with hGM-CSF and BZLF1 antibodies and that the rBCG vaccine stimulated antibody production in C57BL/6 mice. The specific cytotoxic effects of the spleen cells from the rBCG group on EB virus-positive tumour cells was significantly different from the cytotoxic effects of the control group cells (P<0.01). CD8+ T and CD4+ T lymphocytes were detected in the tumour tissues of the rBCG group mice by flow cytometry, indicating that CD8+ T and CD4+ T lymphocytes infiltrated into the tumour tissue in the mice. Morphological observations of the tumour sections from the rBCG-immunized mice showed the infiltration of lymphocytes into the tumour tissues. The average rBCG tumour volume was less than the average tumour volume of the control group. Thus, rBCG may inhibit the growth of EB virus-positive tumour cells in mice.

Construction of a recombinant-BCG containing the LMP2A and BZLF1 genes and its significance in the Epstein-Barr virus positive gastric carcinoma.

The signal peptide Ag85B of Bacillus Chalmette-Guerin (BCG) was used to construct a recombinant plasmid of BCG. The BCG-Ag85B gene and fused EBV LMP2A and BZLF1 genes were amplified and successively inserted into the Escherichia coli-BCG shuttle-vector pMV261. The recombinant plasmids were then amplified in E. coli DH5α and transformed into competent BCG. The expression of BZLF1 and LMP2A fusion proteins in recombinant-BCG (rBCG) was shown by Western blot. After the injection of recombinant-BCG into mice, antibodies against the fusion protein BZLF1 and LMP2A were measured by ELISA, and the cellular immune effects were determined by the lactate dehydrogenate (LDH) release assays. The results confirmed that the cloned genes of BCG-Ag85B and Z2A were correctly inserted into the vector pMV261. The recombinant plasmid pMVZ2A expressed Z2A in BCG effectively after transformation. The rBCG proteins were recognized by the BZLF1 (LMP2A) antibody. An ELISA demonstrated that rBCG could stimulate the generation of antibody against the fusion protein. The fusion gene was constructed successfully, and the rBCG induced humoral and cellular immune response in mice.

[Experimental study on cross-resistance of Culex pipiens pallens to 3 kinds of chemical pesticides].

OBJECTIVE: To understand the cross-resistance of Culex pipiens pallens to common pesticides, so as to provide the evidence for improving the application of chemical pesticides. METHODS: The IV instar larvae of DDVP-resistant, propoxur-resistant and cypermethrin-resistant strains as well as the sensitive strain of Culex pipiens pallens were collected to detect the resistance to DDVP, propoxur and cypermethrin based on the WHO bioassay method. RESULTS: The resistance coefficients of DDVP-resistant strain to DDVP, propoxur and cypermethrin were 14.47, 8.96 and 207.27 respectively. The resistance coefficients of propoxur-resistant strain to DDVP, propoxur and cypermethrin were 3.27, 6.93 and 8.65 respectively. The resistance coefficients of cypermethrin-resistant strain to DDVP, propoxur and cypermethrin were 2.93, 1.61 and 501.11 respectively. CONCLUSION: The resistance and cross-resistance could be generated during the long-term application of a single kind of chemical pesticide, and we should pay more attention to the varieties and dosages of them.

[DNA extraction from old bones].

OBJECTIVE: To explore an efficient method for extracting DNA from old bones. METHODS: Using an organic combined with and Microcon 100 to extract and purify DNA. RESULTS: The extracted DNA was successfully genotyped by using florescence labeling STR multiplex amplification. CONCLUSION: This method will be useful for forensic scientists in identification of DNA from old bones.