Journal of Pathobiology Reaearch
Volume:23 Issue: 1, 2020

Candida species are normal flora in the human body and are the main cause of hospital infections in people with underlying disease. The purpose of the present study was comparison of the conventional and molecular laboratory methods in identifying different species of Candida to find the optimal method.

60 Candida isolates were obtained from oropharyngeal candidiasis patients and recognized using laboratory routine methods including germ tube production, culture on CHROMagar medium, API 20 C AUX kit and molecular ITS sequencing method. The concordances between the methods compared to the molecular method were measured by the kappa coefficient using SPSS 16.0 software.

Out of 60 Candida isolates, 10 isolates (16.6%) in germ tube formation test, 8 isolates (13.33%) in the API test, and 9 isolates (15%) in the culture on CHROMagar medium have different results in comparison to the ITS sequencing method. Germ tube (k= 0.90) and culture on CHROMagar medium methods (k= 0.66) showed the highest and the lowest agreement with the molecular method in the identification of Candida albicans species respectively.

The results showed that conventional methods alone cannot diagnose all Candida species and molecular methods such as ITS sequencing can be used to confirm the identification.

Coccidiosis is one of the most common infectious diseases in poultry that causes huge economic losses. Glycyrrhiza glabra is a medicinal plant that used traditionally. The aim of the present study was to evaluate the effect of aqueous and alcoholic extracts of this plant on the Emeria tenella oocysts in vitro.

The unsporulated oocysts were obtained by inoculation of 14th day-old broiler chicks with 75,000 oocysts. To obtain sporulated oocysts, 9g of feces samples was soaked in 2% potassium bichromate and incubated at 27°C for 72h. Aqueous and alcoholic extracts of Glycyrrhiza glabra in concentrations of 1, 2, and 5% were prepared and oocysts were exposed to these extracts for 48 hours. Thereafter, the number of sporulated and unsporulated oocysts were counted at 1, 12, 24, and 48 hours. In order to prevent any error, the experiments were repeated three times.

Both extracts of Glycyrrhiza glabra in all tested concentrations cause a significant reduction in the number of sporulated and unsporulated Eimeria tenella oocysts compared with control (p<0.05). The rate of inhibitory effect of extracts had a direct relationship with exposure time, and inhibition was continuously increased over time.

Alcoholic extracts of Glycyrrhiza glabra had a better effect than aqueous extracts. 5% alcoholic extract had the best effect. However, further studies are needed to find the best dose for the most anticoccidial effects and also to show its effects on other species of Eimeria and in animal models.

The soil is one of the important sources of Acanthamoeba human infection. Regarding the increasing number of Acanthamoeba keratitis cases in recent years in Iran, more attention to investigating this amoeba now is made. The present study aimed to identify the genotypes of Acanthamoeba in Varamin City using polymerase chain reaction (PCR) technique.

Totally, 18 samples of soil were collected from 12 parks in Varamin City. The samples were filtered using 0.45µm nitrocellulose membrane filters. Then, they were cultured in non-nutrient agar medium 1.5% enriched with killed cultured bacteria Escherchia coli. Genomic DNA was extracted by DNG-plusTM solution and PCR amplification was performed using specific primers to amplify a 500bp 18S rRNA. Sequencing analysis and BLAST search were done for genotype identification of positive samples.

Out of 18 soil samples, 6 isolates (33.3%) were found to be positive for Acanthamoeba in medium by microscopic observation meanwhile 4 isolates (22.2%) were confirmed by Acanthamoeba genus-specific primers. Genotype identification was revealed that all samples belonged to T4 type.

Considering this result, the soil of parks is possible be a risk factor for people, particularly children in this area. Therefore, more attention of public health authorities is recommended.

Interleukin 15 (IL-15) is a cytokine that, due to its physiological activity, can play a role in anti-cancer therapies. It shows positive effect on Natural Killer cells differentiation, proliferation, activation, and surveillance and also on surveillance of memory CD8+ T cells. However, the expression and purification yield of recombinant IL-15 is low. So, it worth improving the production conditions of this useful protein. Therefore in the present study, cloning and expression of human IL-15 are reported in E. coli, strain Rosetta (DE3) which is different from previous bacterial hosts BL21 strain. This strain (Rosetta DE3) has the potential to use codons rarely used by bacteria and consequently, the expressed protein can be more similar to the human protein. First, the human IL-15 coding sequence was synthetized, and then the sequence was cloned into pET28a plasmid. Confirming the accuracy of the final construct was done by colony PCR, restriction analysis (which was done using BamHI and XhoI restriction enzymes and the expected 391bp band was observed) and sequencing. Then, the recombinant construct was transformed into competent E. coli Rosetta (DE3) bacteria. Expression was done in OD600 of 0.6 in the presence of 1mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG). Protein characterization was done by SDS-PAGE and then by western blotting using a specific commercial anti-IL-15 antibody. The 15kDa band on the gel and blot, showed the presence of IL-15. Densitometry by Fiji software determined 37% production yield. It is expected a suitable function from this produced recombinant cytokine due to expression in E. coli Rosetta (DE3) in future studies.

No definitive treatment or effective vaccine has been suggested against the East Respiratory Syndrome Coronavirus (MERS-CoV) virus which indicates the growing importance of the study of this virus. Amongst all MERS proteins, glycoprotein S has always been the main candidate for vaccine research against this virus, due to its function and structure. The aim of the present study was to investigate the structural, functional, and immunological properties of S protein using bioinformatics software that paves the way for designing an effective vaccine against this virus.

35 glycoprotein S sequences of MERS were obtained from Genbank and amino acid changes were investigated. In addition, sequences were analyzed by various software for post-translational changes. Five types of software were used to evaluate the immunologic and allergenic properties. Finally, different structural aspects of this protein were predicted by SOPMA software.

The highest prevalence substitutions were found in amino acids of 95, 123, and 696 and the results indicate that there are four B-cell epitopes in glycoprotein S, and this protein has been affected by post-translational changes, including glycosylation and phosphorylation. This protein has no allergenic properties and the majority of its structure contains Alpha helix.

Glycoprotein S, especially in the RBD region of S1, has a high potential to induce the host immune system and the other features mentioned protein make it appropriate for the production of recombinant protein, including stability in host cells. Therefore, the use of glycoprotein S, especially S1, is recommended as a suitable candidate for vaccine design.

In recent years, carbon nanotubes have attracted the attention of many researchers because of their unique properties. In the present study, carbon nanotubes were coated using PEI. Then, their ability to gene delivery to E. coli cells was examined.

Nanotube- PEI nanoparticles were synthesized by the reaction between amine groups of PEI and carboxyl groups of nanotubes. In order to prepare the appropriate DNA vector for delivering to E. coli cells, the Gus A gene was transferred from pBI121 to PUC18 vector (pUC-Gus). Nanotube-PEI/DNA complexes were prepared by combining different mass ratios of nanotube-PEI (0.5, 1, and 2 w/w%) with the fixed amount of DNA. To the transformation of E. coli, the appropriate amount of nanotube-PEI/DNA complexes was added to E. coli cells under stirring at 37°C for 7h. The transformation efficiency of E. coli was determined by colony counting on LB agar supplemented with Ampicillin. Moreover, Gus staining assay was used to confirm the function of the plasmid. Determination of cytotoxicity of nanotube-PEI was performed using MTT assay at 6, 24, and 72 hours intervals at different concentrations of nanotube-PEI (10, 100, and 500μg/ml).

The nanotube-PEI was synthesized successfully. Nanotube- PEI nanoparticles have a great ability to protect DNA from enzymatic digestion. The percentage of E. coli cells viability was decreased by increasing both the concentration of nanotube-PEI nanoparticles and also the duration of incubation. The results of the agarose gel electrophoresis of plasmid extracted from E. coli and digested using EcoRI enzyme showed that the pUC-Gus plasmid has been successfully transfected by nanotube-PEI nanoparticles to E. coli bacterial cells.

Cationic carbon nanotubes have a high ability to gene transfer to E. coli.

The aim of the present study was to design and express an anti-HER2 single chain variable antibody fragment in E. coli BL21 (DE3) and evaluate its efficiency in recognition of HER2 protein.

An approximately 746bp encoding gene fragment was cloned into pET28a and the recombinant protein was expressed in E. coli BL21 (DE3) strain. Following protein purification by affinity chromatography, western blotting and ELISA were used to evaluate the efficiency of anti-HER2 scFv against HER2 protein.

E. coli can express the anti-HER2 scFv molecule possessing appropriate function and can detect this protein on the surface of breast cancer cells.

This antibody fragment can be used in laboratory diagnostic methods for HER2 diagnostic approaches. Potential capability of this protein in immunohistochemical and imaging approaches against HER2 should be considered.