mohammad reza bassami

Aptamers are oligonucleotides that can be easily synthesized and bind to their targets with high affinity and specificity. Several aptamers specific to soluble factors of coagulation cascade have been produced, however, aptamers specific to platelet cell membrane molecules have not been reported yet. We aimed to discover DNA aptamers that specifically bind to human platelets. The cell-SELEX method was used for aptamer discovery. Synthetic 79 nucleotides length single-strand oligonucleotides were used as a library. Ultra-pure platelets were prepared using differential centrifugation steps and magnetic-bead-assisted removal of contaminating cells. The FITC-labeled forward primer was used for amplification of the selected oligonucleotides by PCR, and Lambda exonuclease was used for digestion of the lagging strand. After 12 rounds of cell-SELEX, selected oligos were amplified and cloned to pTG19-T vector, transfected into E. coli  (TOP10) and sequenced. Sequences of aptamers from 200 individual positive colonies were aligned and seven clusters were identified. Representative aptamers were amplified and their affinity, specificity, and digestibility of their targets were evaluated. Interferences of the aptamers to two platelet function tests were also investigated. Affinity (KD) of the representative aptamers were between 109 and 340 nM. Trypsin exposure of the platelets completely abolished the binding of the 7 aptamers to the targets. The binding of the four aptamers fully protected their target molecules from digestion. No one of the aptamers changed the parameters of the platelet function tests. Seven aptamers specific to platelets were identified and characterized. These aptamers may have potentially diverse applications in the diagnosis or treatment of platelet disorders.

The apply of aptamers as a new generation’s way to probe diagnostic for the de-tection of target molecules has gained ground. Aptamers can be used as alternatives to diagnos-tic antibodies for detection of blood groups due to their unique features. This study was aimed to produce DNA diagnostic aptamer detecting the antigen of A1 blood group using the Cell-Selex method.

DNA aptamer was isolated against A1 RBC antigen after ten stages of Cell-Selex and amplification by an asymmetric polymerase chain reaction. The progress of the stages of selection was evaluated using flow cytometry analysis, which the DNA aptamer isolated from the tenth cycle with an affinity of 70% fluorescent intensity, was selected from four positive colonies followed by determination of the sequences and secondary structures.

The aptameric sequence obtained from C4 cloning was calculated with the highest binding affinity to A1 antigen having an apparent dissociation constant (Kd value) of at least 29.5 ± 4.3 Pmol, which was introduced as the selected aptamer-based on ΔG obtained from a colony of C4 equal to –13.13.

The aptamer obtained from using Cell-Selex method could be used as an example for the development of diagnostic tools such as biosen-sors for detecting A1 blood group antigens.

Mycoplasma is one of the most important pathogens of respiratory system in poultry. The aim of the present study was to identify Mycoplasma spp. isolated from pigeons. Sixteen pooled samples were provided and cultured on PPLO medium and finally the DNA was extracted from the resulting single colonies. Through 16S rRNA gene amplification Mycoplasma genus has been detected. Overall, 31% (5 out of 16) of pooled samples were positive which were identified as Mycoplasma cloumborale and Mycoplasma gallinaceum. Screening of large numbers of pigeons for known poultry pathogenic mycoplasmas will be required to establish the role of pigeons in the spread and maintenance of these organisms in the environment

Adjuvants are an essential component of modern vaccines. An adjuvant is an entity added to a vaccine formulation to ensure that robust immunity to the antigen is inoculcated. The adjuvant is typically vital for the efficacy of vaccines using subunit (pepdids, proteins and virus like particles) and DNA antigens. Furthermore, these components are used to reach the current new goals of preventing and/ or treating chronic infectious diseases and cancers. This review focuses on formulation aspects of adjuvants, safety considerations, progress in understanding their mechanisms of action and also their side effects with using 97 articles are acceceble in pubmed central and google scholar indexing which published during 1980-2016. Adjuvants can be broadly divided into two classes, based on their principal mechanisms of action; the first class are vaccine delivery systems that generally particulate and mainly function to target associated antigens into antigen presenting cells. The others are immunostimulatory adjuvants that predominantly derived from pathogens and often represent pathogen associated molecular patterns which activate cells of the innate immune system. Adjuvants induce cellular and humoral responses, in particular neutralizing antibodies that able to inhibit the binding of pathogens to their cellular receptors. Efficient Th1-immunity-inducing adjuvants are highly in demand. The adjuvants promote good cell-mediated immunity against subunit vaccines that have low immunogenicity themselves. However, attempts to develop a new generation of adjuvants, which are essential for new vaccines, is important, but their use is limited because, little is known about their mechanisms of action and health risks.

Reverse cholesterol transport (RCT) is a crucial procedure for preventing Atherosclerosis, and ATP-binding cassette 1 (ABCA1) is a key factor in it.
The purpose of this study was to examine the effects of two months of a cardiac rehabilitation program on ABCA1 gene expression and other indices of RCT in Coronary artery bypass grafting (CABG) patients.
In this quasi-experimental study, 24 CABG patients were assigned to the cardiac rehabilitation (n = 12) and control (n = 12) groups. The CR group performed two months of cardiac rehabilitation program while the CON group was asked to stay sedentary at home. 48 hours before and after starting the program, 10 cc of blood was sampled for q-RT PCR and biochemical analysis.
ABCA1 expression and plasma HDL-C concentration elevated in the CR group following two months of rehabilitation program compared to the CON group (F = 23.66, P = 0.0002 and F = 5.52, P = 0.034, respectively). In addition, in the CR group, there was no significant change in plasma LDL-C and triglyceride (F = 1.89, P = 0.191 and F = 1.61, P = 0.213, respectively).
Based on our findings, it can be concluded that two months of CR program elevate lymphocyte ABCA1 mRNA expression coinciding with increased levels of plasma HDL-C. The present results also indicated that the CR program must be seriously considered for CABG patients to improve reverse cholesterol transport and hence, attain the cardiovascular benefits.

The aim of the present study was to analyze antibiotic susceptibility profileand genetic diversity of L. monocytogenes, isolated from chicken carcasses using RAPD-PCR.
In this study, 26 isolates of Listeria monocytogenes recovered from chicken carcasses were used. Antibiotic susceptibility of these isolates were examined for eleven antimicrobial agents and RAPD-PCR with use of three different primers were applied to detect genetic diversity of the isolates.
Some of the isolates showed multidrug resistance to commonly used antibiotics. Totally, 26 L. monocytogenes isolates had 16 different antibiogram patterns. Isolates of A and B clusters showed 7 and 6 different pattern, respectively. 12 Isolates of cluster C showed 7 different pattern.
Some of isolates had multidrug resistance to antibiotics. This may raise a public health hazard. RAPD-PCR showed that L. monocytogenes isolated from chicken carcasses had low genetic diversity and there was no clonalrelationship between the isolates.

Introduction Glutamine (Gln), a semi-essential or conditionally essential amino acid, is an abundant amino acid in plasma and skeletal muscle. It is the main energy substrate for cells that undergo intense replication, such as enterocytes, lymphocytes, macrophages, neutrophils and kidney cells and plays an important role in their function and homeostasis. Apart from providing nitrogen for protein synthesis, Gln is a precursor for nucleic acids, nucleotides, hexose amines, the nitric oxide precursor arginine (Arg), and the major antioxidant-glutathione. It plays a central role in nitrogen transport between tissues, specifically from muscle to gut, kidney, and liver. In addition to its role as a gluconeogenic substrate in the liver, kidney, and intestine, Gln is involved in the renal handling of ammonia, serving as a regulator of acid base homeostasis. So the aim of this study was to evaluate the effect of nutrient dilution and L- glutamine (Gln) supplementation on growth performance, intestine morphology and immune response of broilers during starter (0 to 10 days), growth (11 to 24 days) and finisher (25 to 42 days) periods.
Materials and methods A total of 320 one-day-old male Ross 308 broiler chicks were randomly assigned to eight treatments with 4 replicates and 10 chicks per each. In this study two levels of nutrient dilution (Ross 308 broiler nutrition recommendation and 5% diluted) and 4 levels of Gln supplementation (0, 0.5, 1 and 1.5%) were used in a completely randomized design as factorial arrangement 2×4. Growth performance was measured periodically. In order to investigate jejenual histomorphology such as villus height, depth of crypt, villus height to depth of crypt ratio, villus width, muscle layer thickness and epithelium thickness, on day 42 after 4 h fasting, one bird per each replicate was randomly selected, slaughtered and 1 cm of middle section of jejenum was cut. Cellular immune response was assessed in 40-d-old chick using the in vivo cutaneous basophilic hypersensitivity response lectin phytohaemagglutinin (PHA-P) and humoral immune response was evaluated by injection of 1 ml of 10 % suspension of sheep red blood cell (SRBC) on day 18. Primary immune response was measured after 6 (24 –day-old chick) and 12 (30 –day-old chick) days of the injection and secondary immune response was assessed on day 36 and 42 experiment.
Results and Discussion The results indicated that nutrient dilution and Gln supplementation significantly improved feed conversion ratio (FCR) in grower and finisher periods. Gln supplementation increased relative weights of jejunum, small intestine, thymus and bursa of fabricius. The nutrient dilution and Gln significantly affected villi height and crypt depth of jejunum. Gln is an important oxidative fuel for rapidly proliferating cells such as those of the gastrointestinal tract and immune system, reticulocytes, fibroblast. To study humoral immunity, the highest primary and secondary antibody response against Sheep red blood cell (SRBC) was seen in diets containing 1.5% Gln and the lowest was seen in control (without Gln supplementation). In cellular immunity determination, 24 h after subcutaneous injection of Phytohemagglutinin-P (PHA-P) revealed that Gln supplementation increased toe web thickness. Gln is known to modulate immune function. Glutamine is utilized at a high rate by cells of the immune system in culture and is required to support optimal lymphocyte proliferation and production of cytokines by lymphocytes and macrophages. More recently, Gln has also been shown to have anti-inflammatory effects, modulating cytokine production, both in vitro and in vivo, possibly through decreasing a major transcription factor regulating immune and inflammatory responses. In addition, it has been demonstrated that glutamine can modulate immune response by T cell activation. Therefore the increased toe web thickness after PHA-P injection can be explained by increasing T cell proliferation.
Conclusion The results of present study revealed that formulation of diets with Ross 308 nutrient recommendation and 0.5% Gln supplementation improved growth performance and enhancement of immune system function was observed in chicks fed diet with 1% Gln supplementation and Ross 308 nutrient recommendation.

Listeria monocytogenes is an important food borne pathogen with a high case fatality rate. Among 13 different serotypes of this bacterium, 4 serotypes (1/2a, 1/2b, 1/2c, and 4b) were considered as the main cause of listeriosis outbreaks. The aim of this study is to identify the major serotypes and virulence genes in Listeria monocytegenes isolated from chicken carcasses which were collected from different supermarkets and butcheries in Mashhad. Among the 80 isolated Listeria spp., most of them were identified as L. monocytogenes (36 out of 80). Most of the L. monocytogenes isolates belonged to serogroup llb (52.77%) which contains 1/2b and 3b serotypes. The second and third major serogroups were IVa (27.77%) and IIa (16.66%). Serogroup IVb (2.77%) which contains 4b serotype was the fourth major isolate. In order to differentiate serotype 1/2a and 3a from 1/2c, amplification of flaA gene was used. L. monocytogenesis isolates were also examined for the presence of inlC, inlJ and hlyA virulence genes. 26 out of 36 isolates were positive for inlC, and inlJ, whereas hlyA gene was detected in 32 isolates. Chicken carcasses may act as a source of infectious listeriosis for humans living in this area.

The effect of ethanolic extract of Iranian green propolis, rosemary and earthworm meal to determine on performance, immune system and blood metabolites of Japanese quail, using 160 mixed-sex quail chicks in a completely randomized design by four treatments (including ethanolic extract of propolis 0.1%, rosemary leaves meal 0.5%, earthworm meal 0.5% and control) and four replicates of 10 birds in each replication for 42 days. Earthworm meal improved the performance (P

Newcastle is a significant avian disease continuing to cause considerable loss. Developments in genetic engineering have led to plant-based platforms for human and animal vaccine production. Recombinant vaccine production in hairy root systems have several advantages over stable expression in whole plants, including high growth rates, ready genetic manipulations, high levels of recombinant protein production, and the potential for bioreactor culture. In an attempt to develop a recombinant vaccine in hairy roots, the sequences encoding fusion (F) and haemagglutinin-neuraminidase (HN) epitopes of Newcastle disease virus were cloned in pBI121 expression vector which was then transferred into leaf disks of tobacco (Nicotiana tabaccum) ''Turkish'' cultivar by means of Agrobacterium rhizogenes. Hairy roots developed on MS medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Incorporation of the heterologous gene in the genome of hairy roots was confirmed by PCR. Expression analyses were performed by real-time PCR at transcription level and by dot-blot and ELISA assays at translation level, all confirming the expression of the heterologous gene and production of the recombinant protein.

IgY is the homologue of immunoglobulin G in mammals and is composed of four polypeptide chains (two light and two heavy chains). The IgY light chain has 2 domains (variable and constant) in chickens. The constant regions possess class and subclass markers of the immunoglobulin and the variables region is involved in antigen recognition and binding. The aim of this research was to reveal the genetic and phylogenetic analysis of the light chain of immunoglobulin Y transcripts in Khorasan native chickens. For this purpose, 20 spleen samples were collected from the chickens for RNA extraction and cDNA synthesis. The IgY light chain with a length of 764 bp was amplified by polymerase chain reaction using specific primers. After sequencing, mutation investigation was performed in the constant and variable regions. The results demonstrated that CDR3 and constant regions had the highest variation. Phylogenetic analysis showed that the coding gene of IgY in Khorasan native chickens had the highest similarity with French leghorn and United kingdom chickens. The constant region possessed four alpha helix structures compared to the variable region which had no alpha helix structure according to amino acid sequencing analysis and active site determination. Altogether, this study demonstrated for the first time the phylogenic basis of genetic diversity in Khorasan native chickens.

Ozone is a strong oxidant and a potent disinfecting agent. There are numerous application areas of ozone in food industry. While there are many investigations on the application of ozone in food industry, relatively little information is available on the potential of ozone to reduce microbial populations in fresh-cut fruits and vegetables and the visual quality of lettuce. In this study, ozone water was applied at five concentrations (0.6, 0.8, 1.2, 1.6 and 2 ppm) for four exposure times (1, 3, 5 and 10 min) on natural microflora and E. coli O157:H7 and Salmonella inoculated of lettuce. The reduction in the total bacterial count, yeast and mold, total coliform, as well as lactic acid bacteria counts were examined. The promising results indicated the efficacy of ozone water to reduce the microbial populations on lettuce. In the best condition, the inoculated samples of E. coli O157:H7 (ATCC 35150, NCTC 12900), Salmonella typhi morium (ATCC 14028, NCTC 12023) and Salmonella enterica subsp, enterica (PTCC 1709) on lettuce were decreased further than 2 log10 cfu/g. The results showed the population of aerobic mesophilic bacteria, yeast/ moulds, total coliforms and lactic acid bacteria were decreased to 1.54, 0.94, 1.94 and 1.35 log10 cfu/g respectively. The method of ozone generation, type of application, as well as the optimal exposure time and concentration of ozone as an antimicrobial agent on lettuce is mentioned in detail.

Nowadays, production of recombinant proteins in eukaryotes is gaining good deal of attention. Transgenic chicken as a eukaryotic system has a high potential for producing recombinant proteins. Post-translational changes, especially glycosylation, are characteristic of the eukaryotic proteins. In practice we need to choose a proper expressing host when considering over-expression of a recombinant protein. Chickens are among the well-considered candidates for such application. Production of transgenic chickens could be achieved in different ways, including application of primordial germ cells. Primordial germ cells are progenitor of sperm and ovum. These cells are round, with a big nucleus and a cytoplasm with lipid and glycogen particles. The first step for having transgenic chickens is isolation and culture of the primordial gem cells. In the present study, these cells were isolated by centrifugation method in presence of ficoll and using magnetic cell sorting, and were cultured in optimal culture medium. These cells were finally characterized with defined methods, like Periodic acid-schiff staining, alkaline phosphates activity assessment, and antibody staining.

We investigated the microbiological quality of raw vegetables in Mashhad, Iran. In order to document the incidence of indicator and pathogenic microorganisms in this area. ninety eghit raw vegetable samples collected during 7 months were analyzed. All samples were examined for the presence of aerobic mesophilic bacteria, coliforms, Enterobacteriaceae, Escherichia coli, Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus, yeast and mould. Our data showed that the incidence levels of aerobic mesophilic bacteria was less than 107 cfu/g on 83% raw vegetables and 100% of raw vegetables were contained Entrobacteriace and total coliforms, and their range varied from 1 log to 6 log cfu/g. The mean counts of Lactic acid bacteria and yeasts and moulds were 4.1 log cfu/g and 4.5 log cfu/g, respectively, and the incidence levels of yeasts and moulds in 83% of raw vegetables was less than 5 log cfu/g. Our results were similar with other studies that determined microbial levels on raw vegetables. 12.7% of samples raw vegetables contained E.coli, but only 4.1% were higher than ≥ 2 log cfu/g. The incidence levels for E.coli O157:H7 and Salmonella on raw vegetables were 6% and 12.7% respectively. 94.9% of raw vegetables were contaminated with S. aureus and 7.8% of them were coagulase positive.

Avian Intestinal Spirochaetosis (AIS) is an intestinal infection caused by anaerobic spirochaetes of the genus Brachyspira, including B.pilosocoli. The purpose of this study was isolation and identification of B.pilosocoli from laying hen flocks, located in Mashhad suburb, KhorasanRazavi province, Iran, and investigating the frequency of the infection. One hundred and eighty cloacal swab samples from 18 randomly selected flocks (10 samples/flock) were cultured anaerobically on selective agar and confirmed as intestinal spirochaete by itsspirichaetal form using phase contrast microscopy. Then, the samples were subjected to PCR amplification followed by DNA sequencing. A total of 24 samples from 8 flocks were selected as suspected cases by culture and phase contrast microscopy. Upon PCR amplification by specific primers, only 9 cultures belonged to 3 flocks appeared to be B.pilosicoli. Sequence analysis of the amplicons confirmed the identity of all isolated ones. Based on the results obtained, it was concluded that B.pilosicoli might be strongly involved in AIS among laying hen flocks of this geographical region. The results could also be considered as an indicator for large scale investigation into the true prevalence of the infection. This study is the first report of infection in laying hens flocks of Iran.

Bovine viral diarrhoea virus (BVDV) is an important pathogen of dairy cattle. In this study, bulk milk samples representing a total of 4105 milking cows, from 18 dairy cattle herds in the suburb of Mashhad- Iran, were tested for presence of BVDV by the use of a nested reverse transcription polymerase chain reaction (Nested RT- PCR) assay. Non of the cows in the herds had been vaccinated against BVDV. RNA was extracted from somatic cell pellets of bulk milk tank samples. Oligonucleotide primers were selected based on the 5 untranslated region of the BVD virus genome. BVD virus was detected in 2 (11.1%) out of 18 samples, representing 742 lactating cows. These results indicate that nested RT-PCR analysis of bulk milk samples may provide a rapid and sensitive screening method for the detection of BVDV infections in non-vaccinated dairy cattle herds.