Phage display technology for fabricating a recombinant monoclonal ScFv antibody against tetanus toxin.

Tetanus is a specific infectious disease, often associated with lower immunization in developing countries and catastrophic events (such as earthquakes). Millions of people, especially children, die every year from tetanus disease. Therefore, it is necessary to devise a rapid and sensitive detection method for tetanus toxin to ensure an early diagnosis and clinical treatment of tetanus. The current study looks at developing a novel, high specific, low-cost, and sensitive ScFv antibody. It is capable of tetanus detection immunoassays in clinical diagnosis, suspicious foods, and water monitoring. For this regard, a high-quality phage display antibody library (8.7 × 107 PFU/ml) was constructed. Tetanus-specific antibodies with high affinity retrieved from libraries. After phage rescue and four rounds of biopanning, clone screening was performed by phage ELISA. Recombinant antibodies expressed from the AC8 clone showed the highest affinity for tetanus. SDS-PAGE and western blotting confirmed the presence of a high-quality, pure ScFv band at 32 kDa. ELISA was used to determine the affinity value, estimated to be around 10-8 M. The results suggest that the proposed detection method by ScFv antibodies is an alternative diagnostic tool enabling rapid and specific detection of the tetanus toxin.

Evaluation of cold atmospheric-pressure plasma against burn wound infections and gene silencing.

BACKGROUND AND OBJECTIVES: Non-thermal atmospheric-pressure plasma or cold plasma is defined as an ionized gas. This study aimed to investigate the effect of cold plasma on Pseudomonas aeruginosa strains. Also, the expression level of the alp virulence gene before and after treatment with cold plasma was compared with the Housekeeping gene gyrA. MATERIALS AND METHODS: P. aeruginosa isolates recovered from hospitalized burn patients at Shahid Motahari Burns Hospital, Tehran, Iran. The Kirby Bauer disk diffusion method was used to determine the antimicrobial susceptibility test. Then, the antibacterial effect of atmospheric non-thermal plasma was evaluated on P. aeruginosa in as in vitro and in vivo studies at different times on Muller Hinton agar and in mouse model (treated by plasma every day/ 90 sec). The histopathological study was evaluated by Hematoxylin-Eosin staining. Data were analyzed using SPSS software by the Chi-square test and Pvalues less than 0.05 considered as statistically significant. RESULTS: Results indicated that non-thermal atmospheric plasma inhibited the growth of P. aeruginosa. The non-thermal helium plasma accelerates wound healing for 6 days. Results showed that cold plasma decreased virulence gene expression alp after treatment. Therefore, cold plasma can be suggested as a complementary therapeutic protocol to reduce bacterial infection and accelerate wound healing and reduce the expression of virulence genes of pathogens. CONCLUSION: Cold plasma showed pathogen inhibitory properties of P. aeruginosa and virulence alkaline protease and wound healing properties in animal models, so this inexpensive and suitable method can be presented to the medical community to disinfect burn wounds and improve wound healing.

Interaction of human oral cancer and the expression of virulence genes of dental pathogenic bacteria.

Oral squamous cell carcinoma (OSCC) are one of the major causes of cancer morbidity and mortality worldwide. Dental microbiome has been considered as inducing agents in oral carcinogenesis. Therefore, the objective of this study was to investigate the interaction of the gene expression of the dental microbiome and OSCC patients. A cross-sectional study was designed by recruiting confirmed OSCC patients attending the University hospital during October 2018 and July 2019. The dental bacteria were isolated and confirmed by PCR technique. The expression of host and bacterial virulence genes was determined using qPCR. This study shows that 54% of T. forsythia found to be the most predominant organisms in 30 positive cases, followed by 34% of Campylobacter rectus and 29% of Prevotella intermedia. The expression of mRNA levels of bspA, csxA, fadA and interpain A in the OSCC- bacteria positive cases was significantly higher than the control group (P < 0.001). It was further found that interpainA, csxA, fadA, and bspA genes have the potential effects on the cellular gene expression in OSCC patients. A significant correlation was seen between expression patterns of CXCL10, DIAPH1, NCLN and MMP9 genes with interpain A, fadA, and bspA involved in OSCC cases The results indicate that the species specific bacteria may play a role in triggering chronic inflammation in OSCC patients. Therefore, alteration in the gene expression through the dental microbiome could be used as an alternative target in the clinical practice to detect OSCC.

Rapid and Sensitive Detection of Staphylococcal Enterotoxin B by Recombinant Nanobody Using Phage Display Technology.

Staphylococcal enterotoxin B, from Staphylococcus aureus (S. aureus), is one of the most potent bacterial superantigens with profound toxic effects on the immune system. It is associated with food poisoning, toxic shock, atopic dermatitis, asthma, and nasal polyps in humans. The current diagnostic methods for staphylococcal enterotoxin are mainly based on traditional monoclonal antibodies which hardly meet the requirements for clinical applications, and hybridoma clones lose their ability to secrete antibodies during time. The present study investigates the development of a novel, highly specific, low-cost, and sensitive nanobody capable of being used in immunoassays for Staphylococcal enterotoxin B (SEB) detection in suspicious foods. For this purpose, Camelus dromedarius was immunized against SEB toxin. After obtaining acceptable titration, a high-quality phage display nanobody library (4 × 1010 PFU/ml) was constructed. High-affinity SEB-specific nanobodies were retrieved from constructed libraries. After phage rescue and five round of biopanning, clone screening was performed by phage ELISA. Recombinant nanobodies which were expressed from C7 and C21 clone showed the highest affinity for SEB. The presence of high quality and pure nanobody band at ~ 15 kDa was confirmed by SDS-PAGE and western blotting. The affinity constant which was measured by ELISA was calculated to be around 10-9 M. The results suggest that the proposed detection method by nanobodies is an alternative diagnostic tool enabling a rapid, inexpensive, and specific detection of the SEB.

DISC1 gene polymorphisms and the risk of schizophrenia in an Iranian population: A preliminary study.

BACKGROUND: Schizophrenia, schizoaffective disorder, and bipolar illness are common psychological disorders with high heritability and variable phenotypes. The disrupted in schizophrenia 1 ( DISC1) gene, on chromosome 1q42, has an essential role in neurite outgrowth and cell signaling. The purpose of this study was to investigate the association of three single-nucleotide polymorphisms (SNPs; rs6675281, rs2255340, and rs2738864) with schizophrenia disorder. These three SNPs were chosen as they had been used in most of the previous studies. METHODS: In a case-control study of Iranian population for the first time 778 blood samples were collected including, 402 schizophrenic patients and 376 healthy controls. Genomic DNA was extracted from peripheral blood using DNA extraction kit (BioFlux Co). The genotypes of rs6675281, rs2255340, and rs2738864 were detected by nested allele-specific multiplex polymersae chain reaction (PCR). RESULTS: Our data revealed that the three SNPs are significantly associated with schizophrenia (rs2255349 C>T: confidence interval (CI), 2.115 to 3.268; P = 0.0000 OR: 2.629; rs2738864 C>T: CI, 1.538 to 2.339; P = 0.0000 OR: 1.897; rs6675281 C>T: CI, 2.788 to 4.662; P = 0.0009241 OR: 3.605). Through applying the expectation-maximization (EM) algorithm, we calculated the haplotype frequency, and finally performed haplotype analysis with Bonferroni correction and data preprocessing methods and the results showed rs66875281 to have the highest association. DISCUSSION: Our findings primarily showed that DISC1 gene polymorphisms contribute to schizophrenia risk and have a significant association with this disorder among Iranian population. The strategy was found to be easy, rapid, specific, and consistent for the co-occurring detection of the DISC1 polymorphisms. We could finally confirm that the polymorphisms are related to schizophrenia studied in Iranian population.

Development a rapid and accurate multiplex real time PCR method for the detection Chlamydia trachomatis and Mycoplasma hominis.

BACKGROUND: Sexually transmitted diseases easily spread among sexually active people and often have no symptoms. Rapid and accurate method for detecting these infections are necessary in early stages. The traditional detection methods of them are difficult and time-consuming. METHODS: In this study, multiplex real time PCR was optimized for rapid identification of Chlamydia trachomatis and Mycoplasma hominis in a single tube and was performed with our designed primers. The sensitivity test was carried out to designed primers with diluted genomic DNA. To defined the specificity, non STD bacteria were used as DNA template. RESULTS: This study indicated that the developed multiplex real time PCR can be an effective alternative procedure to the conventional methods for rapid and accurate identification of C Chlamydia trachomatis and Mycoplasma hominis. Multiplex real-time PCR Results of them were checked with melting curves. The sensitivity of our designed primer by multiplex real time PCR for Chlamydia trachomatis and Mycoplasma hominis were 4.78×1010 and 8.35×1010 , respectively, Which the primers did not amplify any product from a non-STD species. CONCLUSIONS: Multiplex real time PCR by our new primers and analysis of melting curves were successfully usable for rapid and accurate detection of Chlamydia trachomatis and Mycoplasma hominis. This assay instead of traditional culture method, has considerable potential to be rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous and direct detection.

Construction of human anti-tetanus single-chain variable fragment applying SYMPLEX technology.

BACKGROUND: Human monoclonal antibodies are important molecules in clinical research. Current Limitations of mAb technologies namely instability of immortalized B-cell line and probability of forming unusual VH-VL pairs in phage-display method led to mAbs technology based on single plasma cell called ``SYMPLEX''. OBJECTIVE: In this method, cognate VH and VL fragments generated from individual antibody genes exactly the same as natural ones. METHODS: PBMCs of whole blood of an immunized candidate was used as a resource of rearranged Ab genes. Then flow-cytometric screening was performed to isolate VH and VL from PBMCs. Various VH and VLκ were amplified by six pairs of primers. Overlap Extension PCR was accomplished to link VH and Vκ regions. ScFv inserted into T-vector and its sequence was determined and eventually analyzed by using blast analysis tools. RESULTS: Electrophoresis results indicated that VH and VL fragments were separately amplified by PCR with a length of about 400bp and linked through OE-PCR. Hence, ScFv, which was approximately 800bp in size, was constructed then sequencing and BLASTn results of the ScFv fragment consequently proved the accuracy. CONCLUSION: Results showed 88% similarity to available sequences in mentioned databank. ScFv was ultimately inserted into expression vector for producing recombinant human anti-tetanus mAb.

Draft Genome Sequence of Kocuria rhizophila RF, a Radiation-Resistant Soil Isolate.

Kocuria rhizophila RF, a soil isolate from Iran, is a radiation-resistant bacterium. Only a limited amount of genomic information for radiation-resistant bacteria is currently available. Here, we report the draft genome sequence of this bacterium, providing knowledge to aid in the discovery of the genomic basis of its resistance to radiation.

Construction of scFv library of human antibodies against tetanus toxin.

BACKGROUND: Over the last three decades, monoclonal antibodies have become powerful therapeutics and diagnostics tools. Progress in the antibody engineering, and the appearance of great selection technology such as phage display has made human antibodies production possible against antigens with high affinities. OBJECTIVE: The purpose of this study was to construct an immune antibody library from a vaccinated donor against tetanus toxin. METHODS: A blood sample was drawn from the donor who was vaccinated with tetanus toxoid. PBMC were isolated by using ficoll. After RNA extraction and cDNA synthesis. In order to amplify VH and VL regions, two uniplex PCRs were performed and put considering NcoI & HindIII, MluI & NotI restriction sites respectively for their cloning. These amplicons were cloned to pSEX81 vector and transformed to E. coli XL1blue strain. Eventually, recombinant plasmids were extracted and sequenced. RESULTS: The result showed acceptable similarity between antibody gene library nucleotide sequences and the antibody genes were deposited in this database. CONCLUSION: In this study, the VH and VL genes human antibody library was constructed and confirmed by using DNA sequencing and alignment of sequences with blast database.

Detection of toxigenic Vibrio cholerae with new multiplex PCR.

OBJECTIVE: Vibrio cholerae (V. cholerae) is the etiological agent of epidemic cholera. In recent years, cholera has become endemic in different regions of the world. Traditional culture and microscopic methods are considered the standard for the diagnosis of V. cholerae. However, these methods require days for confirmation. Therefore, molecular methods that may be used for the sensitive, accurate, and rapid analysis of V. cholerae are urgently needed. MATERIALS AND METHODS: In the present investigation, a multiplex PCR assay was developed for the detection of virulence- and toxigenic-associated (VTA) genes (ctxA, tcpA and ompW). To evaluate PCR specificity, other bacteria from the Enterobacteriaceae family (Salmonella typhi, Shigella dysenteriae, and enterotoxigenic Escherichia coli) and Aeromonas hydrophila were examined. The assay sensitivity was evaluated using colony counting and genome dilution methods. RESULT: Our results showed that this PCR-based method represents an ideal tool for the rapid detection of VTA genes due to its simplicity, cost effectiveness, and accuracy. This multiplex PCR method can be used to determine the presence of VTA genes and can therefore distinguish V. cholerae bacteria from other Vibrio species and bacteria. This method can detect 10-100 CFU V. cholerae and 8.5-85 pg genomic DNA. DISCUSSION: This multiplex PCR method has higher sensitivity and specificity than other methods. The proposed test provides an appropriate and sensitive tool for detecting the presence of toxigenic and pathogenic V. cholerae.

Site-directed mutagenesis in bacteriorhodopsin mutants and their characterization for bioelectrical and biotechnological equipment.

Bacteriorhodopsin (BR) mutagenesis plays an important role in the development of BR-based materials and tools with enhanced optical and electrical properties. Previously reported protocols for generating BR mutations are inefficient for the preparation and purification of mutant proteins. Therefore, a series of BR mutations were generated by using improved methods, which are described in further detail. The functional activity of the recombinant proteins was confirmed by spectroscopic and electrochemical assays. Modified proteins with different wavelengths and activities form a foundation for color-sensitive sensors and can be utilized to produce unique bioelectrical and biotechnological tools and materials. The proton-pumping activity of the generated mutant D85E was normal, indicating that the mutant could be used in light batteries. However, mutants D85Q and D85N were almost inactive; and D85N had a prolonged M state, suggesting that it could be utilized in light memories.

Evaluation of mammalian codon usage of fimH in DNA vaccine design.

Uropathogenic Escherichia coli (UPEC) bacteria are the principal cause of urinary tract infections (UTI). Because these bacteria propagate intracellularly, the cellular immune response is an important factor in UTIs. Therefore, we designed a genetic construct to induce a cellular immune response. In order to develop a genetic construct that induces strong cellular immunity against this pathogen, we used the fimH synthetic gene according to mammalian codon usage, and the gene expression was compared with wild type codon usage. Initially, we designed two constructs, pVAX/fimH mam and pVAX/fimH wt, which contain mammalian and wild type codon usage, respectively. The Cos-7 cell line was transfected separately with a complex of pVAX/fimH mam-ExGene 500 poly cationic polymer and pVAX/fimH wt-ExGene 500 poly cationic polymer. Expression of the fimH gene in both constructs in COS7 cells was confirmed by RT-PCR, SDS-PAGE, and Western blotting. Both of the pVAX/fimH cassettes expressed inserted fimH genes (mam and wt) in Cos-7 cells. Our results suggest that codon optimization successfully expressed the fimH gene because the fimH gene with mammalian codon usage is compatible with the eukaryotic expression system. Therefore, mammalian codon usage could be appropriate in a pVAX/fimH construct as a DNA vaccine.

Assessing the role of the RND efflux pump in metronidazole resistance of Helicobacter pylori by RT-PCR assay.

INTRODUCTION: Metronidazole is a significant antibiotic used for eradication of Helicobacter pylori infections and it is of notice that metronidazole-resistant clinical isolates have been found in high rates worldwide. While the RND family of efflux pumps plays a central role in drug resistance among Gram-negative bacteria, this is questionable for H. pylori. METHODOLOGY: To understand whether TolC homologues of RND pumps contribute to metronidazole resistance in H. pylori isolates, expression of four TolC homologous genes of five resistant clinical isolates exposed to varying concentrations of metronidazole were evaluated by RT-PCR and transcriptional analysis. RESULTS: The results indicate that excess amounts of metronidazole are able to increase the expression level of these genes at the transcriptional stage. CONCLUSIONS: Therefore, it may be hypothesized that use of metronidazole in H. pyori infection can induce metronidazole resistance. Furthermore, the RND family of efflux pumps may contribute to metronidazole resistance in clinical isolates of H. pylori.