In Silico Signature Prediction Modeling in Cytolethal Distending Toxin-Producing Escherichia coli Strains

In this study, cytolethal distending toxin (CDT) producer isolates genome were compared with genome of pathogenic and commensal Escherichia coli strains. Conserved genomic signatures among different types of CDT producer E. coli strains were assessed. It was shown that they could be used as biomarkers for research purposes and clinical diagnosis by polymerase chain reaction, or in vaccine development. cdt genes and several other genetic biomarkers were identified as signature sequences in CDT producer strains. The identified signatures include several individual phage proteins (holins, nucleases, and terminases, and transferases) and multiple members of different protein families (the lambda family, phage-integrase family, phage-tail tape protein family, putative membrane proteins, regulatory proteins, restriction-modification system proteins, tail fiber-assembly proteins, base plate-assembly proteins, and other prophage tail-related proteins). In this study, a sporadic phylogenic pattern was demonstrated in the CDT-producing strains. In conclusion, conserved signature proteins in a wide range of pathogenic bacterial strains can potentially be used in modern vaccine-design strategies.

Non-coding CK19 RNA in peripheral blood and tissue of breast cancer patients

Breast carcinoma is the major cause of cancer-related death in women. The incidence of this carcinoma is rising and there are many attempts to decrease this problem. The aim of this study was detection of full-length cytokeratin 19 [CK19] mRNA, in peripheral blood and tissue of breast cancer patients in early stage of cancer. In this study, RT-PCR [reverse transcriptase-polymerase chain reaction] technique was used for detection of CK19 mRNA in peripheral blood and tissue of breast cancer patients. Primers were established to amplify the CK19 as a tumor marker. Moreover, CYFRA 21-1 subunit of CK19 protein was measured in the serum of patients. CK19 mRNA was detected and sequenced. It is shown that the most released CK19 mRNAs in blood and tissue of cancer patients are non-coding RNA. The mutated forms of mRNA are the incomplete transcripts of protein-coding gene as a long non-coding RNA [lncRNA] that could regulate gene expression. Moreover, small non-coding RNA [ncRNA] as fragments of CK19 is mostly observed in this experiment. They may play a role in tumorogenesis and their biologic exact function in breast cancer should be further elucidated

In vivo characterization of fusion protein comprising of al subunit of shiga toxin and human GM-CSF: assessment of its immunogenicity and toxicity

Most cancer cells become resistant to anti-cancer agents. In the last few years, a new approach for targeted therapy of human cancer has been developed using immunotoxins which comprise both the cell targeting and the cell killing moieties. In the present study, the recombinant Shiga toxin Al subunit fused to human granulocyte-macrophage colony stimulating factor [Al-GM-CSF], previously produced in E. coll, was further characterized. The recombinant protein could cause 50% cytotoxicity and induced apoptosis in cells bearing GM-CSF receptors. The non-specific toxicity of the fusion protein was assessed in C57BL/6 and BALB/c mice. No mortality was observed in either group of mice, with different concentration of fusion protein. The lymphocyte proliferation assay, induction of specific IgG response and a mixed [Thl/Th2] response were observed only in BALB/c mice. The mixed response in BALB/c mice [Thl/Th2] could be explained on the basis of the two components of the fusion protein i.e. Al and GM-CSF

Immune responses of mice immunized with active recombinant shiga toxin and its derivatives

Bacterial protein toxins have been exploited as therapeutic agents and as vaccines. An issue of deserving interest is development of new generations of vaccines and immune adjuvants. In this study an active assembled recombinant Shiga toxin of Escherichia coli [rStx1] and its derivatives, recombinant A and B subunits [Stx1-A and Stx1-B], were used to immunize mice. The elicited antibody responses were compared with and without using adjuvant. Protection against intraperitoneal lethal dose of rStx challenge was observed by immunization with sublethal dose of rStx1, rStx-A and rStx-B subunits. The immunological studies on toxin subunits can be used for immunization against systemic shiga toxin mediated disease and also subunits as a vector for antigen presentation in immunotherapeutic approaches. In our experiment, while stimulation of the immune system by A and B subunits were different, both subunits produced neutralizing antibodies. Regarding B subunit the amount of specific IgG1/IgG2a antibody ratio was higher than A subunit. In addition B subunit stimulated proliferation of immune cells with IFNê production the same as rStx1, suggesting that B subunit can be used as an immunomodulator to stimulate the immune response in conjunction with other recombinant proteins

Expression of a Chimeric protein containing the catalytic domain of Shiga-like toxin and Human Granulocyte macrophage colony-stimulating Factor [hGM-CSF] in Escherichia coli and Its recognition by reciprocal antibodies

Fusion of two genes at DNA level produces a single protein, known as a chimeric protein. Immunotoxins are chimeric proteins composed of specific cell targeting and cell killing moieties. Bacterial or plant toxins are commonly used as the killing moieties of the chimeric immunotoxins. In this investigation, the catalytic domain of Shiga-like toxin [A1] was fused to human granulocyte macrophage colony stimulating factor [hGM-CSF] gene and the fused gene was then expressed using an expression vector containing arabinose promoter. The protein thus obtained could be recognized by two different ELISA system designed for detection of hGM-CSF and Shiga-toxin and reconfirmed by Western-blot. The recognition of the chimeric protein by specific antibodies could be indicative of the proper form of the protein, which justifies further steps to be taken to evaluate the potential effects of the chimeric protein

PCR-mediated expression of the human GM-CSF gene in escherichia coli

Four exons of the human genomic GM-CSF gene were assembled together using gene splicing by overlap extension [SOE] method. The resulting nucleotide sequence was cloned in the pET23a [+] expression vector under the control of strong bacteriophage T7 transcription and translation signals. The construct obtained was Transferred into the E. coli strain, BL21 [DE3] pLysS and IPTG was used for induction of GM-CSF gene and production of the target protein, one mg of protein per liter of cell culture, was obtained as revealed by ELISA