mohammad-javad motamedi

Escherichia coli (E. coli) O157:H7 is an intestinal pathogen of humans and animals, which causes serious gastrointestinal, urinary tract infection and hemolytic uremic syndrome. Connecting to the host cell is important in pathogenesis. EspA, Intimin and Tir proteins (EIT) are the most important bacterial features in the process of binding. These antigens can be very useful in detecting these bacteria. The aim of this study was to produce recombinant EspA, Intimin and Tir proteins (rEIT) to detect pathogenic E. coli O157:H7 by means of ELISA method.

The eit recombinant gene was expressed using IPTG in E. coli BL21 (DE3) and evaluated by western blotting. The purified rEIT protein was injected to rabbits and mice subcutaneously. Purified antibody was evaluated using indirect, competitive and sandwich ELISA confirming the precise detection of E. coli O157: H7.

Indirect, competitive and sandwich ELISA specifically detected E. coli O157:H7 and each methods had the ability to identify more than 104, 104, 103 bacteria. The specificity of this method was evaluated by Entroheamoragic E. coli, enterotoxygenic E. coli, Klebsiella pneumoniae, Vibrio cholera and Acinetobacter.

These methods are the fastest, most accurate and cost effective methods for diagnosis of E. coli O157: H7, comparing to the conventional methods.

One of the most causes of diarrhea disease is Escherichia coli enterotoxigenic (ETEC). The first stage of the disease is the binding of ETEC to small intestinal epithelial cells by colonization factors. ETEC then produces Heat-labile enterotoxin (LT) and heat-stable (ST) enterotoxins. The yncE gene potentially encodes a protein with sequence similarity to a pyrroloquinoline quinone containing periplasmic oxidase. The YncE gene is conserved in various strains of E. coli. The protein encoded by the YncE gene is present in the membrane structure of the ETEC. The YncE gene can be considered as a novel protective vaccine candidate. The aim of the present study was to investigate herbal vaccines as a solution to health problems, especially in developing countries.

In the present study, the expression of the YncE protein in tobacco hairy roots and its immunogenicity in mice were investigated. The YncE gene was cloned downstream of the CaMV-35S promoter in the binary expression vector, pBI121-YncE, by using different strains of the Agrobacterium rhizogenes (A4, MSU, 15834) and LBA4404. Three groups of mice including edible, edible-injection, and control were examined. ELISA method was used to determination of IgG and IgA in Feces and Serum.

The amount of the YncE protein was estimated approximately 0.9% of the Total Soluble Protein (TSP) in the transgenic hairy roots. The results indicate that the recombinant YncE protein produced in the transgenic hairy roots tobacco was able to stimulate the immune response in BALB/c mice. Also, it can be stated that recombinant YncE protein is an effective immunogen. The results implied the potential of transgenic tobacco hairy roots–based expression for oral-injection and oral delivery of YncE protein. The antibody titer showed that the immune system was well stimulated.

The plant-based vaccine recombinant YncE protein could both humoral and mucosal the immune response, effectively. Therefore, the YncE antigen was able to stimulate the immune system of mice and produce antibodies. Actually, it appears to be a suitable vaccine candidate for ETEC-induced diarrhea.

Scar contracture is the major complication of neck burn, which impairs the organs functions. Scar contracture causes limiting the range of neck motion, organ malfunction and some damages to the patient’s esthetic and his appearance. Since the studies in this field are not developed well, it is difficult to treat and manage these patients. The principal approach is scar releasing surgery followed by reconstructive surgery. There are many methods of reconstructive surgeries, each patient has his own specific methods, and it depends on surgeons’ decision. Split thickness skin graft (STSG), full-thickness skin graft (FTSG), free flaps, local flaps, V-Y plasty, and V-M plasty are the most common methods used for reconstructive surgeries. In this study, modified free-tension skin grafting was used as the reconstructive procedure, which had shorter treatment period and better treatment outcomes in terms of esthetic and range of motion than the traditional methods.

Atherosclerosis is the main cause of cardiovascular disease (CVD) which has a key role in the development of coronary artery disease (CAD). Based on clinical studies, HSP60 is the only HSP that can cause atherosclerosis. In this paper, the expression level of HSP60 and the pathogenic degree of its cloned part was investigated in atherosclerosis condition.

 After the designation of the specific primers for HSP60, PCR was done by the Pfu enzyme. Subsequently, the PCR products were cloned into a prokaryotic expression vector pET-28a. The resultant recombinant vector was transferred in BL21 and purified. Purification of protein was done by the Nickel affinity column. After confirmation of Western blotting and HSP60 protein purification, purified protein concentration was measured by the Bradford method, and purity was analyzed by SDS PAGE 12%. New Zealand rabbits were tested as an animal model. At the next step, the recombinant protein was injected into the animal model that was on a fatty diet.

The prokaryotic expression plasmid pET28a-hps60 was successfully constructed, the HSP60 protein was expressed and purified in Escherichia coli BL21 (DE3). We found that the rabbit that was receiving the recombinant vaccine with the fatty diet showed a lower amount of fat deposition at the media endothelial level than the rabbit which received only the fatty diet. 

Taking recombinant protein concomitant with a fatty diet, causes betterment of atherosclerosis via decreasing aggregation of cholesterol and thickness of the endothelial media.

Clostridium perfringensis an anaerobic, spore-forming, and pathogenic bacterium that causes intestinal diseases in humans and animals. In these cases, therapeutic intervention is challenging; because the disease progresses much rapidly. This bacterium can produce 5 main toxins (alpha, beta, epsilon, iota, and a type of enterotoxin) among which the epsilon toxin (ETX) is used for bioterrorism. This toxin can be prevented by immunization with specific immunogenic vaccines. In the present research, we aimed at developing a recombinant chitosan-based nano-vaccine against ETX of C. perfringens and evaluate its effects on the antibody titration against epsilon toxin in BALB/c mice as the vaccine model.

The etx gene from C. perfringens type D was cloned and expressed in E. coli. After analysis by SDS-PAGE and western blotting, the expressed products were purified, and the obtained proteins were used for immunization in mice as a chitosan nanoparticle containing recombinant, purified ETX, and protein.

The results of ELISA showed that IgA antibody serum level increased sufficiently using recombinant protein with nanoparticle as an oral and injectable formulation. IgG antibody titers increased significantly after administrating the recombinant proteins with nanoparticles through both oral delivery and intravenous injection.

In conclusion, the recombinant ETX is suggested as a good candidate for vaccine production against diseases caused by ETX of C. perfringens type D.

Diarrhea is the second most common cause of under-5 mortality. The most important strains of Entrotoxigenic Escherichia coli causing Lt and St toxin cause diarrhea and Entrohemoragic E.coli causing Shiga-like toxin secretion. Chlorine enterotoxin B subunit (Ctx) plays a key role in the development of diarrhea in Vibrio cholerae. More specific antibodies could be developed to counter these toxins by combining the CtxB, LtB and StB (LSC) epitopes and the production of trivalent vaccine. The aim of this study was to cloning lsc gene into pcDNA3.1 to design a vaccine DNA.

The lsc gene sequence was transferred to pcDNA3.1(+) vector after primer design and amplification by PCR. The pcDNA3.1(+) vector and the PCR product were digested using HindIII and EcoRI enzymes. Cloning of lsc gene was performed in pcDNA3.1(+) vector and PCR. The clones were digested enzymatically. To ensure expression of lsc gene, it was transferred to HEK-293T cell and confirmed by Western blotting.

The lsc gene was confirmed by PCR and cloning in pcDNA3.1(+) vector using enzymatic digestion and a fragment length of 933 bp was detected and confirmed. Transfection kit was then transferred to HEK-293T cell and expression of the recombinant protein was confirmed by Western blotting and the protein was 39 kDa.

The results of the chimeric gene are well expressed in the cell line and confirmed by Western blotting that can be a good candidate for the fight against bacterial infection.

Enterotoxigenic E. coli (ETEC) is one of the major causes of watery diarrhea outbreaks in children under five years of age and passengers in developing countries. The pathogenicity of ETEC is due to the secretion of Colonization factors (Cfs) and two enterotoxins including heat-labile (Lt) and heat-stable (St). Although diarrhea is considered as one of the causes of mortality in developing countries, no approved vaccine is available for the disease caused by ETEC. Accordingly, the objective of the present study was to design a vaccine candidate containing Sta toxin which accounts for 30-75% of ETEC species.

A chimeric construct consisting of two Sta toxoid connected together by two linkers was designed. After expression and purification by Ni-NTA column, western blotting was performed to confirm the protein. The 2Sta protein was administered to BALB/c mice via injection and the serum and fecal antibodies titer was evaluated by the ELISA test.

The recombinant 2Sta protein was expressed in an insoluble form (inclusion body) and the 20 kDa band was observed on the SDS-PAGE 12%. The results of the ELISA test suggested that IgG and IgA antibodies had enhanced compared to the control group.

The Sta protein which was produced in most ETEC species can be induced in the immune system and raised the serum and fecal antibodies. Using this candidate subunit vaccine could actually protect bacterial infection.

Caused by bacterial, viral, and parasitic pathogens, diarrhea is the second leading cause of death among children under five. Two strains of E. coli, namely Enterotoxigenic, ETEC and Enterohemorrhagic EHEC are the most important causes of this disease in developing countries. EHEC is a major causative agent of bloody diarrhea and hemorrhagic uremic syndrome, while ETEC is the most important cause of diarrhea in neonates and travelers.

To evaluate the immunologic properties of a subunit vaccine candidate comprising the main immunogenic epitopes from these two bacterial strains.

The construct comprised of LTB and CfaB antigens from ETEC, and Intimin and Stx2B antigens from EHEC, was designed, analyzed and synthesized using bioinformatics methods. The chimeric gene was sub-cloned in the expression vector and expressed in E. coli host. The purified chimera protein was injected subcutaneously into the experimental animals. The production of specific antibodies was confirmed by immunological methods, and the protection capacity was evaluated by the challenge of immunized mice with the pathogenic bacteria.

Chimeric recombinant protein was able to increase IgG titer. Neutralization assay indicated that the antibodies generated against LtB moiety were able to neutralize ETEC toxin. In animal challenge study, all non-immune mice died within 3 days after the injection of toxin, but all immunized mice survived from Stx toxin.

The immunity to both ETEC and EHEC bacteria is significant, and this structure can be considered as a candidate for vaccine production against these bacterial strains.

Among all types of malignant diseases, breast cancer has worldwide importance because of the high mortality rate in women aged fewer than 50 in the developing countries. Identification of immunogenic antigens and the generation of specific antibodies against cancer cells are the most successful strategies for early detection and effective treatment of breast cancer. In the current study, a chimeric protein consisting of two specific surface antigens, MUC1 and HER2, were used for the production of chitosan nanoparticles and evaluated as a vaccine candidate. The pET-28a expression vector harboring the HER2-MUC1 gene was constructed. Expression of the protein in E. coli BL21 (DE3) was induced using IPTG. The recombinant HER2-MUC1 (HM) protein was purified using a Ni-NTA column and confirmed by western blotting. Chitosan nanoparticles containing the target protein were prepared and the lymphocytes viability was evaluated using MTT assay. The expression of the recombinant protein with molecular weight of 40 kDa was confirmed using SDS-PAGE and Western blotting. The electric charge and the size of the nanoparticles were determined and verified by a Zeta Sizer device. The evaluation of IgG and IgA titration suggested that inducing humoral and mucosal immune responses by administering nanoparticles containing the chimeric protein. Analysis of cell-mediated immunity showed that the chimeric HM protein could induce specific splenocyte proliferation in immunized mice. It seems that HM nanoparticles can be utilized as a vaccine candidate for inducing the cellular and humoral immune response against breast cancer.

In general, gene therapy is the transfer of a genetic material to treat a disease, or at least to improve the clinical status of a patient. One way gene therapy works is to turn viruses into genetic vectors that carry the gene of interest to the target cells. Based on the genome’s nature, these vectors are divided into RNA-based or DNA-based viral vectors. Most RNA-based vectors are derived from simple retroviruses, such as the murine leukemia virus. One major drawback of these viruses is that they are not transferred to non-dividing cells (post-mitotic cells). This problem can be solved by using new retroviral vectors derived from lentiviruses, such as the human immunodeficiency virus (HIV). DNA-based vectors originate from adeno-viruses and adeno-associated viruses (AAVs). An example of gene deletion due to gene therapy is the deletion of the human CCR5 gene in T cells (which control HIV infection). Although available vector systems have the ability to transfer genes to living cells (in the human body), an ideal vector for gene delivery has not yet been found. Therefore, the current viral vectors should be used with great caution in human cases. Moreover, the development of new vectors is necessary.

Newcastle disease virus (NDV) is a dangerous viral disease, infecting a broad range of birds, and has a fatal effect on the poultry industries. The attachment and consequently fusion of the virus to the host cell membrane is directed by the two superficial glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) which is considered as the important targets for the poultry immune response. The principal goal of this investigation was to realize the potential efficacy of the E. coli expression system for the production of the multi-epitopic HN, and F proteins with respect to the ability for the stimulation of the immune system and production of the cross-reactive antibodies in mice. The recombinant HN and F (rHN, rF) have accumulated almost 40% of the total bacterial proteins. The presence of rHN and rF proteins recognized by the Western blotting with specific anti-HN, anti-F, anti-Newcastle B1, and anti-poly 6x His-tag antibodies. Furthermore, both rHN and rF have shown the specific reactivity against the Newcastle B1 antiserum as a standard strain. The ELISA analysis showed that the higher dilutions of the antibody against Newcastle B1 could react with the as least quantity as 100 ng of the purified rHN, and rF. Cross-reactivity analysis of the sera from the mice immunized with Newcastle B1 in two time points indicated that the raise of anti-Newcastle B1, anti-HN and anti-F antibodies peaked at 28 days post immunization (dpi). Moreover, temporal variation in IgG titration between both time points was significant at 5% probability level. The results provided valuable information about the cross-reactivity patterns and biological activity of the multi-epitopic proteins compared to the NDV standard strain which was determined by the Western blotting and ELISA.

Background Suicide is one of the important health problems. In Iran, like most countries in the world, suicide has considerably increased in recent decades and its reduction is one of the key goals of the health system. This study aimed to identify the risk factors for suicide to provide appropriate strategies and planning of practitioners for better prevention and treatment of people at risk.
Methods & Materials This is a desriptive correlational study. The study population comprised all suicide attempts in 2011. To this end, 545 records were examined in proportional stratified manner. The device of collecting data was a checklist, including age, sex, history of suicide attempt,etc. The collected data were analyzed by descriptive tests using SPSS19.
Results Based on the results, 36.7% of the subjects who attempted sucide were men and 63.3% were women. The majority of suicide attempters were single (53.9%), aged 14-24 years (54.9%) and were city residents (90.1%). Suicide rates were highest in winter (30.6%). Among suicide attempters, 74.3% had used drugs. The most common reasons of attempting for suicide among married persons were marital problems.
Conclusion According to the findings, promotion of healthy marriage, premarital consultation, social and mental support with regard to social problems, promotion of correct urbanization culture, correct use of medication, expansion of hope and recreation among people, especially the young could be helpful and instrumental in prevention of Suicide.