Hepatitis E virus seroprevalence in haemodialysis patients in Zanjan province, Islamic Republic of Iran

Hepatitis E virus [HEV] infection is the most common form of acute hepatitis in adults in endemic regions of Asia. In a descriptive, cross-sectional study, anti-HEV antibody was measured in patients referred to the 2 haemodialysis centres in Zanjan city, Islamic Republic of Iran. Among 93 patients with chronic renal failure, mean age 57.0 [SD 18.5] years, antibodies against HEV were positive in 25 [26.9%] patients. HEV infection was not significantly associated with sex, age, educational level, residence or water source. The rate of HEV positivity was higher in patients with > 1 than

[Standardization of the molecular method for detection of the ent D in staphylococcus aureus isolated from human infections: and sequence determination]

Staphylococcal enterotoxin D as a supper antigen is produced by infected samples of human and animal sources. The aim of this study was to standardize the detection methods for the Staphylococcus strain producing enterotoxin D. A PCR method was set up for detection of enterotoxin D gene [ent D] in Staphylococcus aureus samplesisolated from the human subjects [310 strains isolated from clinical samples]. The specific PCR-product [a band about 700 bp] was purified and sent off for DNA sequencing. Blast analysis showed a 99% identity with the standard gene sequence from Genebank. The ability to produce enterotoxin D by all strains carrying ent D was analyzed by using an ELISA kit. The results of this study show that the PCR method has been well set up. There were two PCR products obtained by the primer pair, one at 700 bp and another at 1400 bp. Both bands were gel purified and sent for DNA sequencing. The results, based on the alignment with the standard ent D sequences from GenBank, suggest that ent D is contained within the 700-bp product. Production of the entrotoxin D in the positive strains was confirmed by ELISA. Based on the available information, coagulase positive Staphylococcus aureus strains are recorded in clinical samples. However, there is no routine method available to analyze the ability of the bacterial strains for producingtoxins including enterotoxin D. This study represents a simple, fast, and standard method for verification of the bacteria enterotoxin D and the strains producing it

[ relationship between perceived stress and the top five heart disease characteristics in patients with myocardial infarction]

Stress and stressful situations can be a prelude to fatal diseases such as cardiovascular diseases and hypertension. The aim of this study was to investigate the relationship between perceived stress with five major characteristics of the heart disease in patients with myocardial infarction. In this cross sectional descriptive-analytical study that was conducted from May 2005 till October 2009, a total of 3,200 patients with myocardial infarction, from cardiovascular care unit of Gha'em and Imam Reza hospital, Mashhad, were randomly selected. A demographic questionnaire, an instrument for recording laboratory and electrocardiograph finding, and the Perceived Stress Scale were used for data collection. The results of this study show that while 35% of all cases suffer from moderate stress, 65% percent of them suffer from high level of stress. The level of perceived stress in different categories of variables such as gender, educational level, hypertension, history of hypertension, depression, cigarette smoking, exercising, job, level of incoming, location of living, and family history of cardiovascular disease was significantly different from each other. Considering the high levels of stress among patients with myocardial infarction, design and implementation of interventions for identifying stressors, as well as their management seem to be crucial

[Comparison of the PCR and LAMP techniques in the diagnosis of salmonella infection]

There are several techniques for the diagnosing of salmonella infectious. Several molecular methods such as PCR and hybridization assay have recently been used for the detection of this bacterium. However, these methods require precision instruments for amplification and complex procedures, which are the major obstacles to the widespread use of these methods in relatively small scale clinical laboratories, clinics and the filed laboratories. Recently, a new, rapid and sensitive technique called loop-mediated isothermal amplification [LAMP] was developed. In this study we used 7 different strains of salmonella to compare the PCR with LAMP method. For PCR test we used thermocycler, but The LAMP reaction can be conducted under isothermal conditions by using only one type of enzyme and four primers recognizing six distinct regions. The most important merit of this method is that no denaturation of the DNA template is required, so, technique is simple and no need to thermocycler machine and several temperatures cycles. Conventional PCR method for the detection of Salmonella with standard thermocylcer takes 3 hrs but, with LAMP method we were able to amplify and detect the salmonella in very simple thermal block made in IRAN. After Optimization of the process it was possible to rapidly detect and identify Salmonella typhi bacteria within 90 minutes. This method was also 100 times more sensitive comparing to the PCR method. According to the results, comparing LAMP isothermal amplification method for detection and identification of Salmonella with conventional PCR we have been able to determine the simplicity, speed [3 times] and the superior sensitivity [100 times] of the LAMP to PCR method. This Method is more simple, faster and cheaper [10 times]. Another advantage is independence to cycle's temperature and thermo-cycling and replacement with one thermo block which is very simple, inexpensive and made in inside the country

[Correlation of null Btk expression and gene noncoding mutations in XLA patients]

X-linked agammaglobulinemia [XLA] is a primary immunodeficiency disorder characterized by recurrent bacterial infections, profound lack of serum antibodies and reduced circulating B lymphocytes. Mutations in Bruton's tyrosine kinase gene [BTK] result in XLA. It is shown that absence of Btk protein expression may be accompanied by no mutations in coding regions in some cases, instead alterations in conserved regulatory domains of promoter and the first intron of BTK gene maybe occurred The aim of this study was evaluation of Btk expression and mutation analysis in coding and regulatory regions of the gene. In this study, eleven XLA patients were enrolled. Btk expression was analyzed by western immunoblotting method. Mutation analysis was carried out in eight patients. In three cases, PCR of the regulatory regions was performed with designed primers, followed by sequencing. According to western blot, normal Btk expression in three patients and null expression in eight others was observed. Mutation analysis showed two novel BTK mutations in two patients [1038-1040 delAGG and IVS8-2delA]. No coding or regulatory region mutations were found in three cases with null Btk expression. Based on these results, three cases with null expression and had no coding or regulatory region mutations are interesting. It is possible that some rare regulatory defects may have been occurred, other than conventional sites. This must be taken into account for future investgations

[Cloning, expression and purification of snap-25 proterin]

Clostridial neurotoxin inhibits neurotransmitter release by selective and specific intracellular proteolysis of synaptosomal associated protein of 25KDa [SNAP-25], synaptobrevin/VAMP-2 and syntaxin. SNAP-25 is one of the components that forms docking complex in synaptic ends. This protein is subtrate for botulinum neurotoxins types A,C, and E. Each of these toxin serotypes specifically cleaves SNAP-25 in a particular position and thereby block docking and synaptic vesicle membrane fusion and finally prevents neurotransmitter exocytosis and transition of neurotic signals. Recombinant production of SNAP-25 in the laboratory can be used as a subtrate for the detection of clostridium botulinum types A, and E neurotoxins. In order to use the protein as a subtrate for detection of different types of clostridium neurotoxins in-vitro the protein was produced by recombinant technique. The cDNA from SNAP-25 was synthesized from total RNA purified from frozen Rattus norvegicus brain. and amplified by RT-PCR The amplified fragment was cloned into pET32a expression vector. The identity of recombinant protein was confirmed by Western blot using specific antibody and finally the recombinant protein was purified through an affinity column chromatography [Ni-NTA]. The optimum conditions of expression of SNAP-25 were found to be IPTG[1mM] and incubation at 37°C for 5 hours. The recombinant protein was isolated and purified using Ni-NTA column with imidazole at a concentration of 25OmM. Using enterokinase to cut the fision at 37°C comparatively yielded better results than room temperature. The protein retained its structure during the purification process being suitable for cutting and further tests. The purified protein we obtained can be used as subtrate for detection of clostridium botulinum types A, and E toxins

[Immunological characterization of light chain recombinant protein of clostridium botulinum type A]

Botulinum neurotoxin type A, structurally consists of a 50KD light chain and a 100 KD heavy chain linked by a disulfide bond. The protein can further be divided into three functional domains of which catalytic domain corresponds to the light chain. In this research we aimed to produce recombinant catalytic domain in order to obtain a protective protein. Bacteria were grown in anaerobic conditions and genomic DNA was extracted by alkaline method. Following the gene coding a set of primers was designed and the catalytic domain was amplified through PCR. The PCR product was then cloned into three expression vectors namely pRSETA, pET28a and pET32a. The expressed protein was analyzed on SDS-PAGE and confirmed by ELISA and western blotting and then purified by affinity chromatography. In this research the maximum expression was obtained at 0.5 mM IPTG,OD[600]:0.6 and 15 hours of induction at 30§C. The protein so expressed was purified by affinity coulmn chromatography .The antibody raised against recombinant protein could protect the rats by 100 LD[50]. Though the expression of AT- rich genes in E.coli system is low, we could obtain an appropriate level of expression in this study. The purification of recombinant protein in the early stages was elusive in the extreme by affinity chromatography due to the weak binding of histidine N-terminal to the column, howerer 90% purification was achieved through modification of the technique. The antibody produced against this domain was less protective compared to that of binding domain