Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power

Abstract Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm

Sequences Type Analysis of Candida Albicans Isolates from Iranian Human Immunodeficiency Virus Infected Patients with Oral Candidiasis

The growing number of immunocompromised individuals has increased the incidence of infections caused by Candida species during the recent decades. Typing of C. albicans on the basis of DNA sequences at multiple loci has greatly advanced our knowledge about the epidemiology and phylogeny of candidiasis. The aim of this study was to evaluate the diversity, and genetic relationships among C. albicans isolates obtained from HIV patients in Iran. using multilocus sequence typing (MLST) method. We analyzed 25 C. albicans isolates obtained from HIV positive patients referred to Iranian Research Center for HIV/AIDS. After diagnostic test and DNA extraction C. albicans isolates were typed using the original MLST scheme explained previously include of six loci: ACC1, VPS13, GLN4, ADP1, RPN2, and SYA1. Fifty one (2.17%) nucleotide sites were found to be polymorphic; all were found to be heterozygous in at least one isolate. For the 25 clinical isolates, 22 diploid sequence types were defined by the genotypes identified from the six loci. The MLST data suggest a relatively high level of divergence in the population structure of C. albicans isolated from HIV infected patients. These findings indicate that in these patients there is a favorable context for the growth of potential pathogenic C. albicans. We found no association between fluconazole resistance, highly active antiretroviral therapy (HAART) receiving and either sequence type or group

Efficient downregulation of alb1 gene using an AMA1-based episomal expression of RNAi construct in Aspergillus fumigatus

International audienceAn episomal RNAi silencing construct containing the inducible cbhB promoter and a hairpin structure has been made to downregulate the alb1 gene in the human pathogen Aspergillus fumigatus. Transformation of fungal protoplasts resulted in a high number of transformants with an inducible silenced phenotype (white spores). Efficient downregulation of the alb1 gene using this system suggests that this approach may overcome the variable downregulation observed with integrative constructs

The Aspergillus niger annexin, anxC3.1 is constitutively expressed and is not essential for protein secretion

International audienceAn annexin, anxC3.1, was isolated and characterised from the industrially important filamentous fungus Aspergillus niger. anxC3.1 is a single copy gene encoding a 506 amino acid predicted protein which contains four annexin repeats. Disruption of the anxC3.1 gene did not lead to any visible changes in phenotype, nor in the levels of secreted protein, nor specifically in glucoamylase production, suggesting no major role in secretion. anxC3.1 expression was found to be unaltered under a variety of conditions such as increased secretion, altered nitrogen source, heat shock, and decreased Ca2+ levels, indicating that anxC3.1 is constitutively expressed. This is the first reported functional characterisation of a fungal annexin

The Effect of Planting Density and Different Nitrogen and Phosphor Application Rates on Saffron Yield

ABSTRACT Increasing the saffron yield as a source of economic income has an effective role in the economic development. Selecting a proper planting density and using nitrogen and phosphor fertilizers are important for increasing saffron yield production. This study was conducted at the Research Farm of the Islamic Azad University, Karaj branch, Iran, in a factorial experiment in the form of a randomized complete block design with three replications. Treatments included three planting densities (120 corms/4.5 m 2 , 180/4.5 and 240/4.5), application of three nitrogen fertilizer rates (50, 75 and 100 kg/ha) and application of three phosphor fertilizer rates (25, 50 and 100 kg/ha). Analysis of variance showed the significant effect of planting density and nitrogen fertilizer on the flower number, flower fresh weight and fresh weight and dry weight of stigma (P≤0.01). Phosphor fertilizers had no effect on the measured traits. Moreover, length of stigma and style were not affected by the treatments. Generally, the three-fold interaction of the highest planting density × 75 kg N/ha × 100 kg P/ha was the most effective treatment compared with the others

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran

INTRODUCTION: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity. METHODS: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran. RESULTS: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%. CONCLUSIONS : This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs