Optimized Signal Peptide for Secretory Expression of Human Recombinant Somatropin in E. coli.

Purpose: The human somatropin is a single-chain polypeptide with a pivotal role in various biological processes. Although Escherichia coli is considered as a preferred host for the production of human somatropin, the high expression of this protein in E. coli results in the accumulation of protein as inclusion bodies. Periplasmic expression using signal peptides could be used to overcome the formation of inclusion bodies; still, the efficiency of each of the signal peptides in periplasmic transportation is varied and often is protein specific. The present study aimed to use in silico analysis to identify an appropriate signal peptide for the periplasmic expression of human somatropin in E. coli. Methods: A library containing 90 prokaryotic and eukaryotic signal peptides were collected from the signal peptide database, and each signal's characteristics and efficiency in connection with the target protein were analyzed by different software. The prediction of the secretory pathway and the cleavage position was determined by the signalP5 server. Physicochemical properties, including molecular weight, instability index, gravity, and aliphatic index, were investigated by ProtParam software. Results: The results of the present study showed that among all the signal peptides studied, five signal peptides ynfB, sfaS, lolA, glnH, and malE displayed high scores for periplasmic expression of human somatropin in E. coli, respectively. Conclusion: In conclusion, the results indicated that in-silico analysis could be used for the identification of suitable signal peptides for the periplasmic expression of proteins. Further laboratory studies can evaluate the accuracy of the results of in silico analysis.

Optimizing ozone dose and contact time for removal of antibiotic-resistant P. aeruginosa, A. baumannii, E. coli, and associated resistant genes in effluent of an activated sludge process in a municipal WWTP.

This study aimed to investigate the impact of ozonation on inactivation of antibiotic-resistant bacteria (ARB) including E. coli, P. aeruginosa, and A. baumannii, as well as on removal of 16S-rRNA gene and their associated antibiotic-resistant genes (ARGs) indigenously present in effluent of municipal wastewater treatment plant. The Chick-Watson model was used to describe bacterial inactivation rates at specific ozone doses. Maximum reduction of total cultivable A. baumannii, E. coli, and P. aeruginosa were found to be 7.6, 7.1, and 4.7 log, respectively, with the highest ozone dose of 0.48 gO3/gCOD at 12 min contact time. According to the study results, complete inactivation of ARB and bacterial regrowth was not observed after 72 h incubation. The culture methods overestimated the performance of disinfection processes and propidium monoazide combined with qPCR, and showed the presence of viable but non-culturable bacteria after ozonation. ARGs were more persistent to ozone than ARB. The results of this study highlighted the significance of specific ozone dose and contact time in ozonation process considering the bacterial species and associated ARGs as well as the wastewater physicochemical characteristics, in order to help diminish the entrance of the biological microcontaminants into the environment.

Effect of MA01 rhamnolipid on cell viability and expression of quorum-sensing (QS) genes involved in biofilm formation by methicillin-resistant Staphylococcus aureus.

A group of biosurfactants, called rhamnolipids, have been shown to have antibacterial and antibiofilm activity against multidrug-resistant bacteria. Here, we examined the effect of rhamnolipid biosurfactants extracted from Pseudomonas aeruginosa MA01 on cell growth/viability, biofilm formation, and membrane permeability of methicillin-resistant Staphylococcus aureus (MRSA) ATCC6538 bacterial cells. The results obtained from flow cytometry analysis showed that by increasing the concentration of rhamnolipid from 30 to 120 mg/mL, the cell viability decreased by about 70%, and the cell membrane permeability increased by approximately 20%. In fact, increasing rhamnolipid concentration was directly related to cell membrane permeability and inversely related to cell survival. Microtiter plate biofilm assay and laser scanning confocal microscopy analysis revealed that rhamnolipid, at a concentration of 60 mg/mL, exerts a reducing effect on the biofilm formation of Staphylococcus aureus. Real-time PCR analysis for monitoring the relative changes in the expression of agrA, agrC, icaA, and icaD genes involved in biofilm formation and related to the quorum-sensing pathway after treatment with rhamnolipid indicated a reduced expression level of these genes, as well as sortase A gene. The results of the present study deepen our knowledge regarding the use of microbial natural products as promising candidates for therapeutic applications.

Characterization of antibiotic resistance genes and bacteria in a municipal water resource recovery facility.

Municipal water resource recovery facilities (WRRFs) are important sources of antibiotic-resistant bacteria and genes (ARB and ARGs). In this study, antibiotic-resistant total heterotrophic bacteria (THBR ) counts (CFU/ml) cultivated from influent, effluent of activated sludge process, and outflow of disinfection unit of an urban WRRF were investigated for the presence of 16, 32, 64, and 128 µg/ml of nine antibiotics. The isolates of Pseudomonas spp., Acinetobacter spp., and Escherichia coli obtained from effluent of activated sludge process were subjected for molecular identification by detecting the 16S rRNA gene sequences. Additionally, using the polymerase chain reaction method (PCR), the isolates were investigated for the presence of blaSHV , blaTEM , blaCTX-M , blaVIM , sul1, and qnrS genes. According to the results, the abundance of THBR counts was not significantly reduced by the biological treatment except for cefixime and sulfamethoxazole; it also increased for some antibiotics after disinfection unit. The average removal efficiency of THBR resistant to ciprofloxacin, sulfamethoxazole, and ceftazidime were 7.9 ± 1.7%, 41.8 ± 2.1%, and 14.4 ± 6.2%, respectively. Also, all the tested isolates were resistant to at least four antibiotics. For all antibiotics, the resistance ratio (THBR /THB) significantly increased in the effluent and after chlorination unit. Among 12 resistant isolates, blaTEM and sul1 genes were the most frequently detected ones involved in 92% and 83% of the isolates, respectively. Both blaTEM and sul1 genes were found in 100% of E. coli, and 83% and 67% of Pseudomonas spp. isolates, respectively. Further efforts are necessary to limit the transmission of ARB and ARGs from WRRFs into the environment and prevent human health threats. PRACTITIONER POINTS: The ratio of resistance significantly increased after biological treatment. Up to 40% of heterotrophic bacteria in the effluent was antibiotic resistant. blaTEM and sul1 genes were more prevalent (92%) in all isolates of bacteria. Both blaTEM and sul1 genes were found in 100% of E. coli isolates. Pseudomonas spp. holds blaTEM and sul1 genes in 83% and 67% of isolates, respectively.

Antibiotic resistance and antibiotic-resistance genes of Pseudomonas spp. and Escherichia coli isolated from untreated hospital wastewater.

Hospitals are considered an important factor in the spread of antibiotic-resistant bacteria (ARBs) and antibiotic-resistance genes (ARGs). The purpose of this research was to characterize the microbial populations in hospital wastewater and investigated the prevalence of ß-lactamase, SulІ and QnrS resistance genes. In the first step, culture method was used to isolate Pseudomonas aeruginosa and Escherichia coli. In the next step, accurate identification of isolated bacteria was carried out using the polymerase chain reaction (PCR) method, then the resistance of the bacteria at different concentrations of antibiotics (8-128 µg/mL) was examined. Finally the ARGs were detected using the PCR method. The averages of heterotrophic plate count (HPC) and ARB concentration in wastewater samples were 1.8 × 108 and 4.3 × 106 CFU/100 mL, respectively. The highest resistance rates were found for sulfamethoxazole and the highest resistance rates in the ß-lactamase group were for ceftazidime, while highest sensitivity was for gentamicin and there was no isolate that was sensitive to the studied antibiotics. SulІ and QnrS were the highest and lowest abundance of all ARGs in samples respectively and blaSHV was the highest ß-lactam resistance gene. Our results indicated an increase in the resistance of identified bacteria to several antibiotics. So it can be concluded that numerous antibiotic-resistant pathogens and vast numbers of ARGs exist in the human body so that their release from hospitals without effective treatment can cause many dangers to the environment and human health.

Redox Regulation in Diazotrophic Bacteria in Interaction with Plants.

Plants interact with a large number of microorganisms that greatly influence their growth and health. Among the beneficial microorganisms, rhizosphere bacteria known as Plant Growth Promoting Bacteria increase plant fitness by producing compounds such as phytohormones or by carrying out symbioses that enhance nutrient acquisition. Nitrogen-fixing bacteria, either as endophytes or as endosymbionts, specifically improve the growth and development of plants by supplying them with nitrogen, a key macro-element. Survival and proliferation of these bacteria require their adaptation to the rhizosphere and host plant, which are particular ecological environments. This adaptation highly depends on bacteria response to the Reactive Oxygen Species (ROS), associated to abiotic stresses or produced by host plants, which determine the outcome of the plant-bacteria interaction. This paper reviews the different antioxidant defense mechanisms identified in diazotrophic bacteria, focusing on their involvement in coping with the changing conditions encountered during interaction with plant partners.

Co-Administration of Vadimezan and Recombinant Coagulase-NGR Inhibits Growth of Melanoma Tumor in Mice.

Purpose: Tumor vascular targeting appeared as an appealing approach to fight cancer, though, the results from the clinical trials and drugs in the market were proved otherwise. The promise of anti-angiogenic therapy as the leading tumor vascular targeting strategy was negatively affected with the discovery that tumor vascularization can occur non-angiogenic mechanisms such as co-option. An additional strategy is induction of tumor vascular infarction and ischemia. Methods: Such that we used truncated coagulase (tCoa) coupled to tumor endothelial targeting moieties to produce tCoa-NGR fusion proteins. We showed that tCoa-NGR can bypass coagulation cascade to induce selective vascular thrombosis and infarction of mild and highly proliferative solid tumors in mice. Moreover, combination therapy can be used to improve the potential of cancer vascular targeting modalities. Herein, we report combination of tCoa-NGR with vascular disrupting agent (VDA), vadimezan. Results: Our results show that synergistic work of these two agents can significantly suppress growth of B16-F10 melanoma tumors in C57/BL6 mice. Conclusion: For the first time, we used the simultaneous benefits of two strategies for inducing thrombosis and destruction of tumor vasculature as spatial co-operation. The tCoa-NGR induce thrombosis which reduces blood flow in the peripheral tumor region. And combined with the action of DMXAA, which target inner tumor mass, growth and proliferation of melanoma tumors can be significantly suppressed.

Performance evaluation of ozonation for removal of antibiotic-resistant Escherichia coli and Pseudomonas aeruginosa and genes from hospital wastewater.

The performance of ozonation for the removal of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) using Escherichia coli and Pseudomonas aeruginosa carrying ARGs from hospital wastewaters was evaluated in this study. Bacterial inactivation was determined using plate count methods and real time PCR for ARG damage (Sul1, blatem, blactx, blavim and qnrS). The reduction rate of bacterial cells and ARGs was increased by different amounts of transferred ozone dose from 11 to 45 mg/L. The concentration of 108 cfu/ml bacteria was reduced  to an acceptable level by ozone treatment after a 5 min contact time,  Although the removal rate was much higher for concentrations of 106 cfu/ml and 104 cfu/ml bacteria. Overall, the tendency of gene reduction by ozonation from more to less was 16S rRNA > sul1 > blatem > blactx > qnrS > blavim. Given that plasmid-borne ARGs can potentially be transferred to other bacteria even after the disinfection process, our results can provide important insights into the fate of ARGs during hospital wastewater ozonation.

Haplotype and linkage disequilibrium of TP53-WRAP53 locus in Iranian-Azeri women with breast cancer.

Among the cancer susceptibility genes, TP53 is one of the crucial genes involved in cell cycle regulations and, therefore, it greatly affects breast cancer initiation and progression. In addition, WRAP53-a natural antisense transcript-regulates TP53 transcription and, as a protein, modulates the normal cell cycle, which results in breast cancer susceptibility. In this study, we aimed to analyze a haplotype comprising four SNPs, including rs1042522, rs17878362, rs2287499, and rs2287498, which are located at 5' regions of the TP53 and WRAP53 genes, in 118 patients and 110 healthy controls of the Iranian-Azeri population. In silico studies were conducted using the SIFT, Polyphen2, Fanthmm, RNAsnp, and SNP&GO online servers. Linkage disequilibrium (LD) and D' for each combination of the markers were calculated via the Haploview program. Our results showed that the GA1CC haplotype was the most frequent in the studied population. Additionally, no significant LD between any pairwise haplotypes was observed. The GA1CC and CA2GC haplotypes were significantly associated with breast cancer susceptibility. Moreover, the in silico analysis revealed the negative effects of rs2287499 and rs1042522 on WRAP53 and P53, respectively. In conclusion, the CA1GC haplotype was strongly identified as a breast cancer risk factor, and the GA1CC haplotype was assumed to be a protective factor against breast cancer risk. Hence, these markers may potentially be used as molecular prognostic and predictive biomarkers for breast cancer.

Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha.

The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of diseases such as Crohn's disease, psoriasis, psoriatic arthritis, and rheumatoid arthritis, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some inflammatory diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α. Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized single chain fragment variable mAb against human TNF-α (hD2). The subsequent cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GST-hD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1.03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in-vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies.

NGR (Asn-Gly-Arg)-targeted delivery of coagulase to tumor vasculature arrests cancer cell growth.

Induction of selective thrombosis and infarction in tumor-feeding vessels represents an attractive strategy to combat cancer. Here we took advantage of the unique coagulation properties of staphylocoagulase and genetically engineered it to generate a new fusion protein with novel anti-cancer properties. This novel bi-functional protein consists of truncated coagulase (tCoa) and an NGR (GNGRAHA) motif that recognizes CD13 and αvß3 integrin receptors, targeting it to tumor endothelial cells. Herein, we report that tCoa coupled by its C-terminus to an NGR sequence retained its normal binding activity with prothrombin and avß3 integrins, as confirmed in silico and in vitro. Moreover, in vivo biodistribution studies demonstrated selective accumulation of FITC-labeled tCoa-NGR fusion proteins at the site of subcutaneously implanted PC3 tumor xenografts in nude mice. Notably, systemic administration of tCoa-NGR to mice bearing 4T1 mouse mammary xenografts or PC3 human prostate tumors resulted in a significant reduction in tumor growth. These anti-tumor effects were accompanied by massive thrombotic occlusion of small and large tumor vessels, tumor infarction and tumor cell death. From these findings, we propose tCoa-NGR mediated tumor infarction as a novel and promising anti-cancer strategy targeting both CD13 and integrin αvß3 positive tumor neovasculature.

RGD delivery of truncated coagulase to tumor vasculature affords local thrombotic activity to induce infarction of tumors in mice.

Induction of thrombosis in tumor vasculature represents an appealing strategy for combating cancer. Herein, we combined unique intrinsic coagulation properties of staphylocoagulase with new acquired functional potentials introduced by genetic engineering, to generate a novel bi-functional fusion protein consisting of truncated coagulase (tCoa) bearing an RGD motif on its C-terminus for cancer therapy. We demonstrated that free coagulase failed to elicit any significant thrombotic activity. Conversely, RGD delivery of coagulase retained coagulase activity and afforded favorable interaction of fusion proteins with prothrombin and αvß3 endothelial cell receptors, as verified by in silico, in vitro, and in vivo experiments. Although free coagulase elicited robust coagulase activity in vitro, only targeted coagulase (tCoa-RGD) was capable of producing extensive thrombosis, and subsequent infarction and massive necrosis of CT26 mouse colon, 4T1 mouse mammary and SKOV3 human ovarian tumors in mice. Additionally, systemic injections of lower doses of tCoa-RGD produced striking tumor growth inhibition of CT26, 4T1 and SKOV3 solid tumors in animals. Altogether, the nontoxic nature, unique shortcut mechanism, minimal effective dose, wide therapeutic window, efficient induction of thrombosis, local effects and susceptibility of human blood to coagulase suggest tCoa-RGD fusion proteins as a novel and promising anticancer therapy for human trials.

Comprehensive proteomic analysis of canola leaf inoculated with a plant growth-promoting bacterium, Pseudomonas fluorescens, under salt stress.

Plant growth-promoting bacteria can improve the tolerance of canola to salt stress. To better understand the effects of plant growth-promoting bacterium on the protein profiles of canola under salt stress condition, proteomics was performed. Salt-sensitive (Sarigol) and -tolerant (Hyola308) canola cultivars were inoculated with Pseudomonas fluorescens FY32, and the protein profiles of canola leaves were compared using a PEG-fractionation method. Cluster analysis of canola cultivars based on a stress tolerance index of several morphological parameters was used to confirm that Sarigol and Hyola308 were salt-sensitive and -tolerant cultivars, respectively. Using a gel-free proteomic technique, 154 and 94 proteins in Hyola308 and 100 and 144 proteins in Sarigol were uniquely identified in non-inoculated and bacterial-inoculated cultivars, respectively. By PEG fractionation, a total of 132 and 207 proteins were identified in non-inoculated and inoculated Hyola308, respectively. Notably, the abundance of copper/zinc superoxide dismutase 1 was significantly increased in inoculated Hyola308 under severe salt stress and decreased under moderate salt stress. In addition, the enzyme activity of delta-1-pyrroline-5-carboxylate synthase was significantly increased non-inoculated Hyola308 and the activity of succinate dehydrogenase was increased in inoculated Hyola308 leaves exposed to salt stress. Taken together, these results suggest that the bacterial inoculation of canola increases salt tolerance by inducing an increase in the abundance of proteins related to glycolysis, tricarboxylic acid cycle, and amino acid metabolism.

Proteomic analysis of canola root inoculated with bacteria under salt stress.

Plant-growth promoting bacteria can ameliorate the negative effects of salt stress on canola. To better understand the role of bacteria in canola under salt stress, salt-sensitive (Sarigol) and salt-tolerant (Hyola308) cultivars were inoculated with Pseudomonas fluorescens and protein profiles of roots were compared. Bacterial inoculation increased the dry weight and length of canola roots under salt stress. Using a gel-free proteomic technique, 55 commonly changed proteins were identified in Sarigol and Hyola308 roots inoculated with bacteria under salt stress. In both canola cultivars, proteins related to amino acid metabolism and tricarboxylic acid cycle were affected. Hierarchical cluster analysis divided the identified proteins into three clusters. Proteins related to Clusters II and III, which were secretion-associated RAS super family 1, dynamin-like protein, and histone, were increased in roots of both Sarigol and Hyola308 inoculated with bacteria under salt stress. Based on pathway mapping, proteins related to amino acid metabolism and the tricarboxylic acid cycle significantly changed in canola cultivars inoculated with or without bacteria under salt stress. These results suggest that bacterial inoculation of canola roots increases tolerance to salt stress by proteins related to energy metabolism and cell division. BIOLOGICAL SIGNIFICANCE: Plant-growth promoting bacteria as an emerging aid can ameliorate the negative effect of salt stress on canola. To understand the role of bacteria in canola under salt stress, salt sensitive Sarigol and tolerant Hyola308 cultivars were used. Dry weight and length of canola root were improved by inoculation of bacteria under salt stress. Using gel-free proteomic technique, 55 commonly changed proteins identified in Sarigol and Hyola308 inoculated with bacteria under salt stress. In both canola cultivars, the number of proteins related to amino acid metabolism and tricarboxylic acid cycle was more than other categories with higher change in protein abundance. Hierarchical cluster analysis divided into 3 clusters. Cluster II including secretion-associated RAS super family 1 and dynamin-like protein and Cluster III including histones H2A were increased by bacterial inoculation in both cultivars. Furthermore, pathway mapping highlighted the importance of S-denosylmethionine synthetase and malate dehydrogenase that decreased in canola inoculated with bacteria under salt stress. These results suggest that bacterial inoculation helps the canola to endure salt stress by modulating the proteins related to energy metabolism and cell division.

The TP53 intron 6 G13964C polymorphism and risk of thyroid and breast cancer development in the Iranian Azeri population.

BACKGROUND: TP53 mutations are the most common genetic alterations in human cancers. There are also several polymorphisms in both exons and introns of TP53 that may influence its anti-tumor functions and increase the risk of cancer development. Associations of the TP53 intron 6 G13964C polymorphism with increased risk of development of several cancers have been investigated in numerous studies, but the results were controversial and conflicting. In this study, we aimed to investigate the probable association of this polymorphism with risk of both thyroid and breast cancers among the Iranian-Azeri population. MATERIALS AND METHODS: We performed two separate case control studies on associations of the intron 6 polymorphism with two different kinds of cancer. In one case-control study, a total of 75 patients with thyroid carcinoma and 180 controls were analyzed and the other study included 170 patients with breast cancer and 135 healthy women. The intron 6 genotype was determined by RFLP-PCR and the SPSS 16 program was applied for data analysis. RESULTS: For thyroid cancer, the frequencies of GG genotype were 96.0% in patients and 93.3% in controls. The GC genotype had a frequency of 4.0% in patients and 6.7% in controls. In the study on breast cancer, the frequency of GG and GC genotypes in patients were 95.3% and 4.7%, respectively. In breast related control group, the frequency of GG genotype was 93.3% and the frequency of GC genotype was 6.7%. None of the cases and controls had the CC genotype. CONCLUSIONS: There was no significant association between the TP53 intron 6 G13964C polymorphism and risk of development of both thyroid and breast cancer in Iranian-Azeri patients.

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli.

PURPOSE: The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. METHODS: To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. RESULTS: The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. CONCLUSION: It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein.

Antibacterial activity of Pseudonocardia sp. JB05, a rare salty soil actinomycete against Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium that causes many harmful and life-threatening diseases. Some strains of this bacterium are resistant to available antibiotics. This study was designed to evaluate the ability of indigenous actinomycetes to produce antibacterial compounds against S. aureus and characterize the structure of the resultant antibacterial compounds. Therefore, a slightly modified agar well diffusion method was used to determine the antibacterial activity of actinomycete isolates against the test microorganisms. The bacterial extracts with antibacterial activity were fractionated by silica gel and G-25 sephadex column chromatography. Also, the active fractions were analyzed by thin layer chromatography. Finally, the partial structure of the resultant antibacterial compound was characterized by Fourier transform infrared spectroscopy. One of the isolates, which had a broad spectrum and high antibacterial activity, was designated as Pseudonocardia sp. JB05, based on the results of biochemical and 16S rDNA gene sequence analysis. Minimum inhibitory concentration for this bacterium was 40 AU mL(-1) against S. aureus. The antibacterial activity of this bacterium was stable after autoclaving, 10% SDS, boiling, and proteinase K. Thin layer chromatography, using anthrone reagent, showed the presence of carbohydrates in the purified antibacterial compound. Finally, FT-IR spectrum of the active compound illustrated hydroxyl groups, hydrocarbon skeleton, and double bond of polygenic compounds in its structure. To the best of our knowledge, this is the first report describing the efficient antibacterial activity by a local strain of Pseudonocardia. The results presented in this work, although at the initial stage in bioactive product characterization, will possibly contribute toward the Pseudonocardia scale-up for the production and identification of the antibacterial compounds.

Prevalence of thrombophilic gene polymorphisms in an azari population of iran.

There is several evidence suggests that thrombophilic gene polymorphisms may influence susceptibility to thromboembolic events. The prevalence of these polymorphisms is different in various races and ethnics. Accordingly, we studied the prevalence of Factor V (G1691A and A4070G), prothrombin G20210A and PAI-1 4G/5G in healthy northwest population of Iran. In this prospective study, 500 healthy individuals, who had no history of both personal and family history of thromboembolic disorders, were selected as a sample of healthy population in northwestern Iran. Genotyping of these polymorphisms was performed using the amplification refractory mutation system-polymerase chain reaction method. No significant differences were detected between the expected and observed frequencies of FV G1691A and A4070G, prothrombin G20210A polymorphisms (P>0.05), while the expected frequency of 4G allele was significantly more than observed frequency in the studied population (P<0.01). These findings were compared with other reports from various populations. In conclusion, the allele frequency for FV G1691A and PAI-1 4G/5G polymorphisms showed relative consistency compared to those of previous studies, while the incidence pattern of FV A4070G polymorphism in Northwestern population of Iran showed conflicting results regarding other studied population. The prothrombin G20210A polymorphism was observed at a higher frequency than other studied populations.

Apolipoprotein E genotyping in women with recurrent pregnancy loss: an in silico and experimental hybrid study.

The role of apolipoprotein E gene polymorphisms in the pathogenesis of recurrent pregnancy loss remains controversial. Therefore, our objective was to investigate the association between recurrent pregnancy loss and apolipoprotein E gene polymorphisms among northwest Iranian women, and also to predict the impact of these nonsynonymous single nucleotide polymorphisms on structure and function of apolipoprotein E protein. The subjects of our current study consisted of 100 women that have had two or more consecutive idiopathic first trimester miscarriages, and one hundred healthy women from the same geographical areas were used as a control group. After DNA extraction, we used a polymerase chain reaction-restriction fragment length polymorphism to genotype of the apolipoprotein E gene. In addition, we predicted the possible effects of amino acid substitutions at codons 112 and/or 158 on the structure and function of apolipoprotein E protein using Polymorphism Phenotyping online software v2. Our results showed that the rate of apolipoprotein E ε4 carriers and the frequency of the ε4 allele in the case group were statistically and significantly higher than those in the control group (P<0.05). Therefore, our data support the association of the Apo ε4 allele with RPL; however, in silico analysis predicted that the amino acid substitution at residue 112 (Apo ε4 allele) is a benign mutation. Accordingly, further studies are required to elucidate the mechanism(s) underlying the link between RPL pathogenesis and the Apo ε4 allele.

Lack of association of intron 3 16 bp polymorphism of TP53 with breast cancer among Iranian-Azeri patients.

BACKGROUND: p53 gene is a well-known tumor suppressor gene that has several polymorphisms in both its exons and introns. It has been suggested that intron 3 16 bp duplication polymorphism may affect the gene function resulting in reduction or suppression of p53 anti tumor activity. In most case control studies a duplicated allele has been noticeably more frequent in cases rather than controls but there are also conflicting results. The aim of this study was to assess the association of intron 3 16 bp duplication polymorphism of p53 with breast cancer risk among Iranian-Azeri population. We also analyzed the clinicopathological information of patients as an epidemiological description of breast cancer in the north-west of Iran. MATERIALS AND METHODS: This case-control study was performed on 221 breast cancer patients and 170 controls. Genomic DNA was extracted from peripheral blood samples and tumor tissues. p53 PIN3 genotype was determined using electrophoresis of PCR products on 8% non-denaturing polyacrylamide gels and silver staining. RESULTS: In the control and case groups, respectively, 62.9% and 61.1% had no 16 bp insertion (A1A1 genotype), 7.1% and 7.7% had insertion in both p53 alleles (A2A2) and 30% and 31.2% were heterozygous (A1A2). There was no significant difference between genotype frequencies as well as allelic frequencies in two case and control groups. CONCLUSIONS: According to the result of the present study, the intron 3 16 bp duplication polymorphism of p53 could not be assessed as a marker of risk factor for predisposition to breast cancer in Azeri population. However, a high frequency of A2 allele (22.1%) in our population suggested that intron 3 16 bp duplication polymorphism may be a valuable marker for study in other cancers with well designed large groups.