Design and Production of a Novel Recombinant Chimeric IL2-Omp31 Antigen against Brucella Infection.

Brucellosis is a zoonotic disease in human and animals. Brucella melitensis is one of the most pathogenic species of Brucella in goat and sheep. Omp31 is an outer membrane protein of Brucella that acts as an immunogenic protein. Cytokines are glycoproteins with low molecular weight that play the role of an immune adjuvant and regulate immune responses. Interleukin-2 is one of the most important cytokines, which are secreted by the white blood cells and involved in T cell immune responses. In the present study, a chimeric Omp31-Interleukin2 recombinant protein was generated by means of genetic engineering techniques. This chimeric coding sequence was amplified by using specific primers and using Splicing Overlap Extension (SOE) PCR technique. The fusion of the two mentioned proteins was accomplished using a rigid linker. The generated chimeric IL2-Omp31 fragment was TA cloned, and then subcloned into pEt22b vector as an expression vector. The chimeric protein was successfully expressed in E. coli BL21 (DE3) and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and also Western-blotting analysis. Finally, in order to assess the antigenic features of the recombinant chimeric IL2-Opm31 protein, its secondary structure and antigenicity were predicted in silico.

Serum levels of 9α,11ß-PGF2 and apolipoprotein A1 achieve high predictive power as biomarkers of anaphylaxis.

Anaphylaxis is a life-threatening hypersensitivity reaction. To identify biomarkers for the condition, we assessed serum levels of apolipoprotein (Apo)A and ApoE. We found a reduction of both lipoproteins in anaphylactic mice as well as in orally challenged food allergic patients. We then compared patients after acute anaphylaxis with several control groups (nonallergic, history of allergen-triggered anaphylaxis, acute cardiovascular/febrile reactions). In this unpaired setting, ApoE levels were unaltered, while ApoA1 was reduced in the anaphylactic group. Although unable to discriminate between anaphylaxis and cardiovascular/febrile reactions, ROC curve analysis revealed a reasonably high area under the curve (AUC) of 0.91 for ApoA1. Serum 9α,11ß-PGF2 , recently identified as a suitable biomarker for anaphylaxis, outperformed ApoA1 with AUC=0.95. Intriguingly however its power further increased upon combination of both mediators reaching AUC=1. Our data suggest that ApoA1 combined with 9α,11ß-PGF2 represents a useful composite biomarker of anaphylaxis, achieving superior diagnostic power over either factor alone.

Production and characterization of egg yolk antibody (IgY) against recombinant VP8-S2 antigen.

Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. VP8 subunit of rotavirus is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Studies showed that immunoglobulin of egg yolk (IgY) from immunized hens has been identified to be a convenient source for specific antibodies for using in immunotherapy and immunodiagnostic to limit the infections. In this study, chimeric VP8-S2 gene was designed using by computational techniques. The chimeric VP8-S2 gene was cloned and sub-cloned into pGH and pET32a (+) vectors. Then, recombinant pET32a-VP8-S2 vector was transferred into E. coli BL21 CodonPlus (DE3). The expressed protein was purified by Ni-NTA chromatography column. Hens were immunized with the purified VP8-S2 protein three times. IgY was purified from egg yolks using polyethylene glycol precipitation method. Activity and specificity of anti-VP8-S2 IgY were detected by dot-blotting, Western-blotting and indirect ELISA. We obtained anti-VP8-S2 IgY by immunizing hens with the recombinant VP8-S2 protein. The anti-VP8-S2 IgY was showed to bind specifically to the chimeric VP8-S2 protein by dot-blotting, Western-blotting analyses and indirect ELISA. The result of this study indicated that such construction can be useful to investigate as candidates for development of detection methods for simultaneous diagnosis of both infections. Specific IgY against the recombinant VP8-S2 could be recommended as a candidate for passive immunization against bovine rotavirus and bovine coronavirus.

Lentiviral mediated overexpression of Insulin like Growth Factor-1 in mouse myoblast.

Insulin-like growth factor gene (IGF-1) is one of the most important growth factors that plays a key role in the proliferation and differentiation of muscle cells. IGF-1 is also a radial stimulant in muscle hypertrophy in mammals. In our study, we constructed a lentiviral vector inducing an overexpression of IGF-1 in order to study the regulating mechanisms of this gene. The IGF-1 gene was cloned into the lentiviral shuttle plasmid pCDH-cGFP and the recombinant lentiviral vector was transducted into myoblast C2C12 cell line. The overexpression of IGF-1 was confirmed by RT-PCR and western blotting for IGF-1 receptor gene. Additionally, chemiluminescence results also showed that the concentration of IGF-1 in the transduced cells significantly increased compared to the control group. The results of our study suggests that constructed recombinant lentiviral vector can potentially be used for regulating the expression of IGF-1 in myoblast C2C12 cells.

The suitability of some blood gas and biochemical parameters as diagnostic tools or early indicators of ascites syndrome in broiler sire lines.

In recent few years, there have been some attempts to find a reliable indicator trait as a selection criterion against susceptibility to ascites syndrome (AS). Blood parameters were of great interest as they could be measured in live animals without implementing an ascites-inducing challenge (AIC). In this work, the suitability of some blood parameters was evaluated for diagnosing AS-susceptible chicks in later steps of the disease in trial 1 as well as their early predictive ability in trial 2. In the first trial, one hundred 1-day-old chicks from two pure broiler lines namely S1 and S2 and, in the second trial, 226 1-day-old chicks from line S2 were subjected to AIC. Saline drinking water (1200 mg/l) and lower-than-standard ambient temperatures were the implemented AICs in trials 1 and 2 respectively. The blood parameters including pH, partial pressure of O2 (pO2 ), partial pressure of CO2 (pCO2 ), bicarbonate ion concentration (BIC), percentage of haematocrit (HCT) and saturated haemoglobin (SaO2 ) were measured twice per each bird at days 28 and 35 in trial 1 and once in trial 2 at day 21. The results of the first trial revealed that in line S2 some of the blood parameters differed significantly between the ascitic and non-ascitic groups following exposure to AIC. In this line, the incidence of AS was accompanied by a lower pO2 , SaO2 and BIC, while with higher pCO2 and HCT values. In the second trial, however, although almost all of the parameters showed meaningful differences between the ascitic and non-ascitic broilers, only mean difference of BIC parameter was statistically significant. The general conclusion of this study is that the blood parameters can somewhat have diagnostic ability in the condition in which the AIC is already present, whereas the results did not approve their usefulness as early predictors of AS.

BosFinder: a novel pre-microRNA gene prediction algorithm in Bos taurus.

MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression transcriptionally (transcriptional activation or inactivation) and/or post-transcriptionally (translation inhibition or degradation of their target mRNAs). This phenomenon has significant roles in growth and developmental processes in plants and animals. Bos taurus is one of the most important livestock animals, having great importance in food and economical sciences and industries. However, limited information is available on Bos taurus constituent miRNAs because its whole genome assembly has been only recently published. Therefore, computational methods have been essential tools in miRNA gene prediction and discovery. Among these, machine-learning-based approaches are used to characterize genome scale pre-miRNAs from expressed sequence tags (ESTs). In this study, a support vector machine model was used to classify 33 structural and thermodynamic features of pre-miRNA genes. Public bovine EST data were obtained from different tissues in various developmental stages. A new algorithm, called BosFinder, was developed to identify and annotate the whole genome's derived pre-miRNAs. We found 18 776 highly potential pre-miRNA sequences. This is the first genome survey report of Bos taurus based on a machine-learning method for pre-miRNA gene finding. The bosfinder program is freely available at http://lbb.ut.ac.ir/Download/LBBsoft/BosFinder/.

Cloning, Nucleotide Sequencing and Bioinformatics Study of NcSRS2 Gene, an Immunogen from Iranian Isolate of Neospora caninum.

BACKGROUND: Neosporosis is caused by an obligate intracellular parasitic protozoa Neospora caninum which infect variety of hosts. NcSRS2 is an immuno-dominant antigen of N. caninum which is considered as one of the most promising targets for a recombinant or DNA vaccine against neosporosis. As no study has been carried out to identify the molecular structure of N. caninum in Iran, as first step, we prepared a scheme to identify this gene in this parasite in Iran. METHODS: Tachyzoite total RNA was extracted and cDNA was synthesized and NcSRS2 gene was amplified using cDNA as template. Then the PCR product was cloned into pTZ57R/T vector and transformed into E. coli (DH5α strain). Finally, the recombinant plasmid was extracted from transformed E. coli and sequenced. Bioinformatics analysis also carried out. RESULTS: The PCR product of NcSRS2 gene was sequenced and recorded in GenBank. The deduced amino acid sequence of NcSRS2 in current study was compared with other N. caninum NcSRS2 and showed some identities and differences. CONCLUSION: NcSRS2 gene of N. caninum successfully cloned in pTZ57R/T. Recombinant plasmid was confirmed by sequencing, colony PCR and enzymatic digestion. It is ready to express recombinant protein for further studies.

Impact of milk components on recovery of viral RNA from MS2 bacteriophage.

Noroviruses are responsible for approximately 44 % of outbreaks involving dairy products for which causative agents are reported. Recovery of viruses from milk and dairy products is a difficult task. The role of different components of milk in the recovery of viral RNA was evaluated in this study. Four model milk formulations (A-D) were prepared by mixing different combinations of lactose, whey protein, casein, and fat in water. Each model formulation was spiked with five concentrations of bacteriophage MS2. The phenol-guanidine thiocyanate-chloroform protocol was used for extracting viral RNA from the model milk formulations and then extracted RNA was measured by a nanodrop spectrophotometer in ng/µl. The results showed that casein and whey protein had the highest negative impact on RNA yield, especially when the number of MS2 was less than 1.3 pfu/ml. The highest RNA recovery was obtained from the model milk formulation containing all four components; lactose, whey protein, casein, and fat. The amount of extracted RNA was closely correlated with the dry matter content of each formulation and the spiked concentration of coliphage using response surface modeling (R²:0.93). It was determined that milk fat is the most effective component in facilitating RNA extraction and the highest RNA yield can be achieved via elimination of whey protein and casein from milk by centrifugation at 40,000×g for 60 min. To achieve the highest viral RNA recovery efficiency by the proposed method, milk fat must be recombined with the supernatant of the centrifuged sample and then homogenized before performing the extraction protocol.

Computational modelling of long bone fractures fixed with locking plates - How can the risk of implant failure be reduced?

BACKGROUND AND PURPOSE: The Locking Compression Plate (LCP) is part of a new plate generation requiring an adapted surgical technique and new thinking about commonly used concepts of internal fixation using plates. Knowledge of the fixation stability provided by these new plates is very limited and clarification is still necessary to determine how the mechanical stability and the risk of implant failure can best be controlled. METHODS: Upon validation, a finite element model of an LCP attached to a cylinder was developed to simulate and analyse the biomechanics of a transverse long bone fracture fixed with a locking plate. Of special interest were the factors influencing the mechanical conditions at the fracture site, the control of interfragmentary movement and implant failure. RESULTS: Several factors were shown to influence stability in compression. Increasing translation and/or fracture angle post fixation reduced construct stability. Axial stiffness was also influenced by the working length and plate-bone distance. The fracture gap had no effect on the construct stability when no bone contact occurred during loading. Stress analysis of the LCP demonstrated that the maximum Von Mises stresses were found in the innermost screws at the screw-head junction. INTERPRETATION: For the clinical use of the LCP as a locked internal fixator in fractures with an interfragmentary gap of 1 mm, at least two to four plate holes near the fracture gap should be omitted to allow fracture motion and bone contact to occur. This will also achieve a larger area of stress distribution on the plate and reduce the likelihood of fatigue failure due to cyclic loading.

Loss of heterozygosity on chromosome 5 in Iranian esophageal cancer patients.

There is a high incidence of esophageal squamous cell carcinoma (ESCC) in Iran. Non-functionality of some tumor suppressor genes has been reported in esophageal cancer. Loss of heterozygosity on chromosome 5 has also been reported in esophageal carcinomas. We assessed loss of heterozygosity along a region of the long arm of chromosome 5 (5q), from 5q23.1 to 5q23.2, by PCR amplifying DNA fragments of tumor tissues from patients with ESCC and their corresponding normal samples. The PCR products were electrophoresed on 6% non-denaturing polyacrylamide gels, and band intensity was shown by silver staining. Of 40 patients with ESCC, 27, 25 and 36% of informative cases showed allelic losses at microsatellite markers D5S1384, D5S1478 and D5S1505, respectively. Two of the 40 patients studied had microsatellite instability at marker D5S1384. Based on the fact that loss of heterozygosity with more than 22% incidence for a specific marker cannot be regarded as a random event, we add support to previous reports concerning the presence of tumor suppressor genes in this chromosome region and that they affect esophageal cancer development. According to the data in NCBI UniSTS, the PCR product size of human DNA with primers of the D5S1505 marker ranges from 243 to 275 bp, containing about 20 repeats of the TAGA tetranucleotide, while the amplicon size of one allele of one of our cases was 207 bp, with about 10 repeats of the TAGA tetranucleotide, which would be the shortest sequence reported so far.

Single nucleotide polymorphisms and mRNA expression of VEGF-A in papillary thyroid carcinoma: potential markers for aggressive phenotypes.

BACKGROUND AND OBJECTIVES: Polymorphisms of the VEGF gene are known to affect the biological behaviour of cancers but have seldom been studied in thyroid cancer. The aim of the current study is to evaluate the prevalence and relevance of VEGF-A polymorphisms and mRNA expression in papillary thyroid carcinoma (PTC). MATERIALS AND METHODS: Genomic DNA and total RNA were isolated from paraffin-embedded tissue from 91 PTC (51 conventional PTC and 40 follicular variant) and 78 control thyroid tissues. Three DNA polymorphisms (+936C > T, +405C > G and -141A > C) in the 3' and 5' untranslated region (3'-UTR, 5'-UTR) of VEGF-A were studied using PCR and RFLP. Also, the mRNA expression of VEGF-A in these tissues was studied by real-time PCR. RESULTS: Distribution of polymorphisms in the 5'-UTR (VEGF-VEGF -141A > C and +405C > G) and 3'-UTR (VEGF +936C > T) were all significantly different in PTC and benign thyroid tissue (p = 0.0001, 0.001 and 0.028 respectively). The VEGF -141 C allele was more common in PTC with lymph node metastases (p = 0.026). VEGF + 405 Galleles andVEGF +936 CC genotype were more common in PTC of advanced pathological staging (p = 0.018 and 0.017 respectively). Also, increased expression of VEGF-A mRNA was noted in PTC compared to control (p = 0.009). Within the group of patients with conventional PTC, those with lymph nodal metastases had a higher level of VEGF-A mRNA expression than other patients (p = 0.0003). CONCLUSION: These findings suggest that VEGF polymorphisms and mRNA expression may predict the aggressiveness behaviour of thyroid cancer.

Impact of milk components in recovery of the MS2 bacteriophage as an indicator of enteric viruses.

The objective of this study was to characterize the role of milk components in the recovery of viral particles from raw milk. For such characterization, four model milk formulations (A-D) were constituted by mixing different combinations of lactose, whey protein, casein, and fat into water. Each model formulation was spiked with six concentrations of bacteriophage MS2. The soluble and insoluble components of each model milk formulation were separated by centrifugation at 40,000 x g and viruses were enumerated in each supernatant fluid and pellet by the double agar layer (DAL) method. When samples were spiked with MS2 at concentrations lower than 4.8 x 10(5) pfu/ml, milk components did not significantly impact the overall recovery. However, the impact of milk components was measurable at higher concentrations. In general, higher numbers of MS2 were recovered from supernatant fluids of model milk formulations containing no fat. The highest number of viral particles were recovered from the pellet of model C (lactose+whey protein+casein). The recovery efficiency of MS2 was correlated with the dry matter contents of each model milk formulation and the initial spiking concentration of coliphage using response surface modeling.

Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system.

A recently introduced histologic fixative (Universal Molecular Fixative [UMFIX]) has been shown to preserve macromolecules in tissue at ambient temperature. When UMFIX-exposed tissues are processed by a formalin-free, microwave-assisted rapid processing system, the resulting paraffin blocks retain good histomorphology and intact nucleic acids suitable for expression microarray analysis. Because UMFIX may be used as an alternative to formalin, the authors set out to study the effect of this new fixation and processing system on immunohistochemistry (IHC) by analyzing a range of human neoplastic and non-neoplastic specimens. Parallel slices from surgically removed specimens were fixed in formalin and UMFIX and processed in a rapid microwave-assisted tissue processor. IHC was performed following routine procedures. The staining for those antibodies that normally required antigen retrieval was carried out with and without that step. The intensity and pattern of reactions were compared in 144 tissue samples fixed by the two methods using 70 monoclonal and polyclonal antibodies. The intensity of IHC reactions for most cytoplasmic antigens was generally equal or stronger in UMFIX tissues. This was particularly true with intermediate filaments and HercepTest, where the antigen retrieval step became unnecessary. Conversely, there was a decrease in the intensity of reactions for HepPar1, bcl-2, and three nuclear antigens (Ki-67, TTF-1, and estrogen receptor). Increasing their exposure times optimized the sensitivity of the latter four antibodies. The study shows that IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens. There is an enhancement of the sensitivity of few antibodies in UMFIX-exposed tissue, rendering antigen retrieval unnecessary. This increased sensitivity may be due to the effect of eliminating formalin from fixation and processing or the microwave energy.

Antimicrobial activity of UMFix tissue fixative.

AIMS: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms. The UMFix solution was compared with 10% neutral buffered formalin. METHODS: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin. RESULTS: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses. Bacterial spores were resistant to killing by UMFix. All organisms were killed by the 10% neutral buffered formalin preparation. CONCLUSIONS: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species. The methanol content of the fixative was responsible for the killing effect of this fixative. No killing was seen when polyethylene glycol was used alone.

Infliximab monotherapy in psoriasis: a case of rapid clinical and histological response.

BACKGROUND: Psoriasis is a common chronic relapsing, inflammatory, hyperproliferative skin disorder with genetic predisposition. There is currently no experimental model for psoriasis and the pathogenesis is not fully understood. Psoriatic plaques have been shown to contain increased levels of cytokines, including tumor necrosis factor alpha (TNF-alpha). Anti-tumor necrosis factor therapy with infliximab has been shown to be highly effective in recalcitrant psoriasis. METHODS: We evaluated the efficacy and timeline of histological changes in a psoriatic plaque following infliximab infusion. A patient with severe recalcitrant plaque psoriasis was clinically and histologically assessed for improvement. RESULTS: We found rapid clinical improvement with infliximab accompanied by histopathological changes. The earliest effects were seen on neutrophils and lymphocytes whereas keratinocyte normalization was not evident at the early stages. CONCLUSION: Infliximab is not only an effective agent in the treatment of psoriasis but appears to have a very rapid onset of action.

Cell cycle arrest and autoschizis in a human bladder carcinoma cell line following Vitamin C and Vitamin K3 treatment.

Exponentially growing cultures of human bladder tumor cells (T24) were treated with Vitamin C (VC) alone, Vitamin K(3) (VK(3)) alone, or with a VC:VK(3) combination for 1, 2, or 4hr. Flow cytometry of T24 cells exposed to the vitamins for 1h revealed a growth arrested population and a population undergoing cell death. Cells in G(1) during vitamin treatment arrested in G(1) while those in S phase progressed through S phase and arrested in G(2)/M. DNA synthesis decreased to 14 to 21% of control levels which agreed with the percent of cells in S phase during treatment. Annexin V labeling demonstrated the majority of the cells died by autoschizis, but necrosis and apoptosis also were observed. Catalase treatment abrogated both cell cycle arrest and cell death which implicated hydrogen peroxide (H(2)O(2)) in these processes. Redox cycling of VC and VK(3) increased H(2)O(2) production and decreased cellular thiol levels and DNA content, while increasing intracellular Ca(2+) levels and lipid peroxidation. Feulgen staining of treated cells revealed a time-dependent decrease in tumor cell DNA, while electrophoresis revealed a spread pattern. These results suggest that Ca(2+) disregulation activates at least one DNase which degrades tumor cell DNA and induces tumor cell death.

Topical application of imiquimod 5% cream to keloids alters expression genes associated with apoptosis.

Keloids are benign mesenchymal tumours, usually present at and extending beyond the margins of sites of previous injury. It is reported that keloids display aberrant expression of apoptotic genes: TGFB1 is activated, whereas caspase 8 and 3 are not, thus indicating a block upstream in the apoptosis cascade in keloids. Interferon-alpha 2b normalizes the excessive synthesis of collagen, glycosaminoglycans and collagenase by keloidal fibroblasts, reduces recurrences following keloid excision, and enhances the expression of native p53 and apoptosis. Imiquimod, a rapid and potent inducer of interferons locally at the site of application to the skin, reduces recurrences following keloid excision and alters gene expression of markers of apoptosis in basal cell carcinoma cells. We investigated the effects with respect to the expression of apoptotic genes in keloidal tissue compared with nontreated controls of imiquimod 5% cream applied topically to keloids. Total RNA was extracted from excised keloidal tissue, cDNA probes synthesized and then hybridized to gene-specific cDNA fragments spotted on membranes. The expression levels of 96 genes involved in apoptosis, relative to cyclophilin expression, were compared in the imiquimod-treated and untreated groups. The mean ratio of expression, relative to cyclophilin of caspase 3 and DFFA were significantly enhanced. Caspase 3 was significantly downregulated and DFFA was significantly upregulated in the group of imiquimod-treated keloids (P < 0.05) compared with the untreated group of keloids. Although imiquimod is capable of altering the expression of these markers of apoptosis in keloids, their role, if any, in the therapeutic response of keloids to imiquimod requires further investigation.

Prevalence and risk factors for hepatitis B virus infections among STD patients in northeast region of Iran.

BACKGROUND: In many parts of the world, sexual transmission of hepatitis B virus plays a major role in acquisition of infections. In Northeast region of Iran the prevalence rate and risk factors influencing this type of transmission was not investigated. Therefore, the concurrence of hepatitis B virus (HBV) and STDs was studied to determine the prevalence and risk factors of sexual transmission of hepatitis B virus. MATERIAL/METHODS: This study was carried out among 1500 attendances to the laboratories for STDs examination between 1998 and 2000. Those who were positive for STDs (syphilis & gonorrhea) were examined for HBV infection by determination of hepatitis B surface antigen (HBsAg). The data was analyzed and compared to the normal population. RESULTS: The prevalence of STD in this population was 4.66% for syphilis and 6% for gonorrhea. Among this population the seroprevalence of HBsAg was 10% in women and 14.2% in men (mean seroprevalence of HBsAg was 13.13%). The concurrence of hepatitis B virus and syphilis was 14.28% which was slightly higher than concurrence for gonorrhea (12.22%). CONCLUSIONS: The prevalence of HBV in our patient population was high, exceeding the national estimates. This population also represents a high-risk group in Northeast of Iran. Further, our data indicates that such high prevalence is significantly more evident in patients with low socioeconomic status.