Development of a Novel HTLV-1 Protease: Human Fcγ1 Recombinant Fusion Molecule in the CHO Eukaryotic Expression System.

Human T-cell leukaemia virus type 1 (HTLV-1) is the causative agent of two life-threatening diseases, adult T cell leukaemia/lymphoma (ATLL), and HTLV-1-associated myelopathy/tropical spastic (HAM/TSP). HTLV-1 protease (HTLV-1-PR) is an aspartic protease that represents a promising target for therapeutic purposes like human immunodeficiency virus-PR inhibitors (HIV-PR). Therefore, in this study, the human Fc fusion recombinant-PR (HTLV-1-PR:hFcγ1) was designed and expressed for two applications, finding a blocking substrate as a potential therapeutic or a potential subunit peptide vaccine. The PCR amplified DNA sequences encoding the HTLV-1-PR from the MT2-cell line using specific primers with restriction enzyme sites of Not1 and Xba1. The construct was then cloned to pTZ57R/T TA plasmid and, after confirming the PR sequence, subcloned into the pDR2ΔEF1α Fc-expression vector to create pDR2ΔEF1α.HTLV-1-PR:hFcγ1. The integrity of recombinant DNA was confirmed by sequencing to ensure that the engineered construct was in the frame. The recombinant fusion protein was then produced in the Chinese hamster ovary cell (CHO) system and was purified from its supernatant using HiTrap-rPA column affinity chromatography. Then, the immunofluorescence assay (IFA) co-localisation method showed that HTLV-1-PR:hFc recombinant fusion protein has appropriate folding as it binds to the anti-Fcγ antibody; the Fcγ1 tag participates to have HTLV-1-PR:hFcγ1 as a dimeric secretory protein. The development and production of HTLV-1-PR can be used to find a blocking substrate as a potential therapeutic molecule and apply it in an animal model to assess its immunogenicity and potential protection against HTLV-1 infection.

Investigation of the rpoB Mutations Causing Rifampin Resistance by Rapid Screening in Mycobacterium Tuberculosis in North-East of Iran.

BACKGROUND AND OBJECTIVES: The incidence of rifampin-resistant strains of Mycobacterium tuberculosis has attracted more attention than the tuberculosis infection due to laborious treatment and control. Recognizing the Mycobacterium tuberculosis genotypes involving in drug resistance via multiplex PCR, a simple and rapid genotyping method, is an emergency for better treatment and control of tuberculosis. This study was designed to specify the frequency of rifampin-resistant strains of Mycobacterium tuberculosis isolated from patients by multiplex allele-specific Polymerase Chain Reaction assay (MAS-PCR). METHODS: In this study, 88 Mycobacterium tuberculosis positive samples were included from Qaem Hospital, Mashhad. MAS-PCR was used to detect the rifampin resistance associated mutations in rpoB gene. RESULTS: Mutations in three codons of rpoB gene causing rifampin resistance were detected in 51 isolates (58.96%). The detected mutations in codons 531, 526, and 516 were 55.68%, 38.63%, and 13.63%, respectively. The simultaneous mutations were detected in 11 isolates (12.50%) in codons 531, 526 and 516, in 21 isolates (23.86%) in codons 531 and 526, and in one isolate (1.13%) in codons 526 and 516. CONCLUSION: According to the results of this study, the frequency of rifampin-resistant strains of Mycobacterium tuberculosis isolated from Khorasan province patients (North-East of Iran) was high. The developed MAS-PCR assay can be used for rapid detection in clinical diagnostic laboratories in areas with high prevalence of multidrug-resistant Mycobacterium tuberculosis strains. In this respect, MAS-PCR is simple, rapid, and highly sensitive method for drug susceptibility tests for detecting multidrug-resistant Mycobacterium tuberculosis.

Comparison of the prevalence of enteroviruses in blood samples of patients with and without unstable angina.

BACKGROUND: Although the role of enteroviruses has been proved in heart diseases, extensive information is not available on the association between enteroviruses and unstable angina. In the present study, the authors compared the prevalence of enteroviruses in patients with and without unstable angina. METHODS: Blood samples were taken from 51 patients with unstable angina and 55 patients without unstable angina or myocardial infarction that were admitted to Imam Reza and Ghaem hospitals (Mashhad, northeast of Iran). Reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers for the detection of the enteroviruses in blood samples of study subjects. RESULTS: Patients with and without unstable angina were similar in age with mean ± standard deviation of 62.6 ± 12.8 and 59.7 ± 12.7 years, respectively (P = 0.243) and there were no differences in gender in these two groups (P = 0.174). Prevalence of the enteroviruses in patients with unstable angina was higher only in 66-80 years age group compared to the control group (patients without unstable angina, P = 0.032). There was a higher prevalence of enterovirus RNA positivity in the blood samples of women with unstable angina (75.9%) than those without unstable angina (41.7%, P = 0.011), however, no significant difference was observed in men (P = 0.983). CONCLUSION: Our data showed that enteroviral RNA positivity was higher in patients with unstable angina compared to those without unstable angina. However, the differences between the two groups were not statistically significant.