Amino acid mutation in Plasmodium vivax dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes in Hormozgan Province, southern Iran.

Molecular analysis of antifolate resistance-associated genes-dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) of Plasmodium vivax is important in predicting the emergence of drug resistance to sulphadoxine-pyrimethamine (SP). The present study aimed to determine the polymorphism of dhfr and dhps genes in P. vivax field isolates. Samples from 80 microscopically diagnosed vivax malaria cases were collected from endemic areas of malaria in Hormozgan Province of Iran, from June 2010 to November 2015. The two sets of codons at position 33, 57, 58, 117, 173 of dhfr and 382, 383, and 553 of dhps genes were analysed by direct sequencing of PCR products. The majority of the isolates (70%) harboured a wild-type allele for P. vivax dhfr (Pvdhfr) and P. vivax dhps (Pvdhps). Mutations were detected in three codons of Pvdhfr (P33L, S58R and S117N) and single codon in Pvdhps (A383G). Novel mutations that have not been identified previously at codon 459 (D459A) of Pvdhps were also observed. The high prevalence of point mutation as well as the rising triple mutation of Pvdhfr and Pvdhps genotypes necessitate change in programmes and guidelines to eliminate P. vivax in future.

Genetic Diversity of Dihydropteroate synthetase Gene (dhps) of Plasmodium vivax in Hormozgan Province, Iran.

BACKGROUND: The present study was formulated in order to determine polymorphism of dihydropteroate synthetase gene (dhps) of Plasmodium vivax (P. vivax) in Hormozgan Province, southern Iran and mutations at codons 382, 383, 512, 553, and 585 associated with resistance of P. vivax to sulfadoxine. METHOD: One-hundred eighteen isolates of P. vivax were prepared within 2007-2008 to determine dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene. The isolates were determined in the study of genetic diversity of dihydropteroate synthetase gene (dhps) of P. vivax. The study was performed via PCR test and nucleotide sequencing. RESULTS: Of 118 blood samples infected by P. vivax, 46 and 72 samples belonged to Minab and Jask, respectively. No mutation was detected at 5 target codons. However, among these 118 samples, three isolates (2.54%) were found to have a mutation at the new codon 421. CONCLUSION: Since mutation was detected in dihydrofolate reductase (Pvdhfr) gene in the same samples but no mutation was found at five main codons of Pvdhps gene, it can be concluded that P. vivax, considering their mutations in Pvdhfr, is still susceptible to sulfadoxine and therefore, to fansidar in Hormozgan Province, Southern Iran.

Expression of recombinant human coagulation factor VII by the Lizard Leishmania expression system.

The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.

Dunaliella as an attractive candidate for molecular farming.

Pharmaceutical recombinant proteins are widely used in human healthcare. At present, several protein expression systems are available to generate therapeutic proteins. These conventional systems have distinct advantages and disadvantages in protein yielding; in terms of ease of manipulation, the time required from gene transformation to protein purification, cost of production and scaling-up capitalization, proper folding and stability of active proteins. Depending on the research goal and priorities, a special system may be selected for protein expression. However, considering the limited variety of organisms currently used and their usage restrictions, there are still much more pharmaceutical proteins waiting to be economically and efficiently produced. Distinguished biological and technical features of microalgae Dunaliella such as inexpensive medium requirement, fast growth rate, the ease of manipulation, easy scaling up procedure, facility of milking in bioreactors and the ability of post-translational modifications make this microorganism an attractive candidate for molecular farming.

Kaposi's sarcoma herpesvirus load in biopsies of cutaneous and oral Kaposi's sarcoma lesions.

OBJECTIVES: To evaluate human herpesvirus 8/Kaposi's sarcoma associated herpesvirus (HHV-8/KSHV) viral load in diagnostic, (formalin fixed, paraffinised) biopsies and patient serum during tumour progression of oral and cutaneous AIDS-related Kaposi's sarcoma (AKS), and endemic Kaposi's sarcoma (EKS) by a sensitive and specific quantitative real time polymerase chain reaction (qRT-PCR) assay. STUDY DESIGN: Eighty six biopsies of both AKS (oral and cutaneous AKS, 68) and EKS (cutaneous EKS, 18) were evaluated by qRT-PCR and immunohistochemistry (IHC). The viral load in human tumour tissue and serum of some individual patients were compared. RESULTS: Higher viral load as well as frequency of latency-associated nuclear antigen (LANA)+ tumour spindle cells (SC) and number of LANA granules per SC was found in oral AKS compared to cutaneous AKS. Although few cases were available, serum viral load appeared to decrease compared to tumour tissue during KS progression. CONCLUSIONS: The higher viral load in oral rather than cutaneous AKS is consistent with the well recognised reservoir function of the oral mucosa. Decrease of serum HHV-8 load during KS progression may indicate decreased virus release and/or increased virus clearance.