Proteomics and secretomics of Penicillium chrysogenum: Molecular characterization of proteins relevant for penicillin biosynthesis = Proteómica y secretómica de "Penicillium chrysogenum": caracterización molecular de proteínas importantes para la biosíntesis de penicilina

366 p.Eighty years after the discovery of penicillin, the interest of this ß-lactam antibiotic at industrial level is still remarkable. Despite the good background knowledge, little is known on how Penicillin chrysogenum became a good penicillin overproducer, and much of the molecular basis for improved productivity remains obscure. Some light has been shed on this issue after the recent publication of the P. chrysogenum genome (van den Berg et al., 2008). These authors reported that transcription of genes involved in biosynthesis of the amino acid precursors for penicillin biosynthesis, as well as of genes encoding microbody proteins was higher in the high-producing strain DS17690. However, full exploitation of P. chrysogenum requires the integration of knowledge from other -omics such as proteomics. Proteomics studies are one of the most powerful methods to evaluate the final result of gene expression and 2-DE has been the technique of choice to obtain a reference global picture of the proteomic and secretomic map. This technique has been successfully applied to other ascomycete fungi, such as Schizosaccharomyces pombe (Hwang et al., 2006), some Aspergilli (Melin et al., 2002; Kniemeyer et al., 2006; Vödisch et al., 2009) or Saccharomyces cerevisiae (Shevchenko et al., 1996; Bruckmann et al., 2009). But only Kiel and coworkers (2009) have performed a proteomics-based inventory of the proteins present in the microbody matrix of P. chrysogenum. Therefore, it was necessary to study in depth the cytosolic proteome and secretome of this important fungus. In this work, we have optimized the methods to extract the P. chrysogenum cytosolic proteins (proteome) as well as secreted protein to the culture (secretome). As a result, the cytosolic proteome reference map and secretome of this filamentous fungus has been developed. We have also characterized the relevant modifications produced during the industrial strain improvement program through the proteome analysis and of three P. chrysogenum strains: i) the wild-type strain NRRL 1951 isolated in Peoria, IL, ii) the genome reference strain Wisconsin 54-1255 (an improved but still low-producer) and iii) the high-producing strain AS-P-78 developed by Antibioticos S.A. (León, Spain)

Association of the length of CA Dinucleotide repeat in the epidermal growth factor receptor with risk and age of breast cancer onset in Isfahan

Background: The epidermal growth factor receptor (EGFR) also known as HER1 or ErbB1 is a prominent member of the erbB proto-oncogene family, which encodes a receptor tyrosine kinase with a pivotal role in the regulation of cell growth and differentiation. The aim of this research was to study the CA repeat polymorphism in intron I of EGFR among the patients with breast cancer and healthy controls. We have also evaluated risks for breast cancer, age of onset and grade of tumor associated with the CA repeat polymorphism. Methods: In the

Genetics and epidemiology of Middle East Respiratory Syndrome-Coronavirus (MERS-CoV)

Background: Middle East respiratory syndrome (MERS) is a viral respiratory illness caused by a coronavirus. After the primary onset of MERS in Saudi Arabia, in September 2015 cases began to increase. The number of laboratory-affirmed cases by MERS-CoV in the Middle East has been being increased recently. Method: In this current review article, by using the terms “MERS” and “coronavirus” we first searched for English language articles in the PubMed database, published in last five years. Then by a detailed review of related articles, we provided a comprehensive information about epidemiology, genetic, host and coronavirus treatment. Result: More importantly, evidences of human-to-human transmission in Europe and America indicate that the viral adaptations in humans may precede a large-scale epidemic. The genome of Coronaviruses is a linear positive-sense single stranded large RNA and they are enveloped viruses that have a helical symmetric nucleocapsid. Some new insights have been provided in previous few months in to the animal Coronavirus hosts, transmissibility, contagion of MERS Co-V and ideal laboratory diagnostic methods. Conclusion: It seems crucial to control this new human infection “MERS-CoV” by collaborating global and local health authorities and their continual support for further research on it

The effects of pBudCE4.1-azurin-MAM-A recombinant vector on IL-2, IL-6, IL-7, and IL-10 expressions in laboratory mice

Background and aims: Breast cancer is one of the most common types of malignancy in women with morbidity and mortality (15.0%) in the world. The antitumor activity of azurin protein produced by Pseudomonas aeruginosa has been described before. Mammaglobin-A (MAM-A) protein is especially expressed in 40%-80% of breast cancer types and this protein is a very specific molecular marker for stimulating the immune system. Accordingly, this study investigated the effects of pBudCE4.1-azurin-MAM-A recombinant vector on the induction of the immune system in laboratory mice by the real-time polymerase chain reaction (PCR) method. Methods: The pBudCE4.1-azurin-MAM-A recombinant and empty vectors were purchased and then separately transformed into Escherichia coli for multiplying. Next, each plasmid was extracted and the accuracy of transformation was confirmed by the PCR. These recombinant and empty (control) vectors were separately infused into the thigh muscle of the animals and the healthy group was infused with phosphatebuffered saline. The infusion sites, blood specimens, as well as the serum of the animals were collected and examined by serological and molecular tests. Results: Molecular and serological studies showed that the serum and expression levels of IL-2, IL-6, IL-7, and IL-10 in infused mice with pBudCE4.1-azurin-MAM-A recombinant vector significantly increased compared to healthy animals and injected mice with an empty vector (P<0.05). Conclusion: In general, the findings revealed that the pBudCE4.1-azurin-MAM-A recombinant vector can stimulate the immune system of the mouse by an increase in the expression levels of IL-2, IL-6, IL-7, and IL-10. Thus, it would be better to examine the effects of this recombinant vector as a DNA vaccine on the prevention and treatment of breast cancer. Keywords: Azurin, MAM-A, Recombinant vector, Breast cance

Detection of Toxoplasma gondii from Native Cattle in Southwest of Iran

Abstract: Infections by protozoan parasite Toxoplasma gondii are prevalent worldwide in animals and human. T. gondii is the causative agent of toxoplasmosis, one of the most prevalent parasitic infections to humans and domestic animals. If first be during pregnancy, T. gondii may be transferred vertically by tachyzoites that are passed to the embryo via the placenta. T. gondii may be transmitted horizontally in three phases of the life cycle, ingesting infectious oocysts from the environment or tissue cysts or tachyzoites which are contained in gastrointestinal of many different animals. Transmission may also occur via tachyzoites contained in blood products, texture transplants or non-pasteurized milk. Like rest of the world toxoplasmosis is prevalent in Iran. The present study aimed to determine T. gondii isolates from native cattle in Chaharmahal Va Bakhtiari province located in south west of Iran by molecular methods. In this study, 155 blood samples were collected from native cattle. Genomic DNA was extracted using DNA extraction Kit (Cinna Gen, Iran) according to the manufacturer protocol and PCR was performed using specific primers (ITS-F and ITS-R). Sixteen (6.95%) cattle were positive to T. gondii infection. The positive control samples showed the excepted amplification product specific for T. gondii (171 bp). Although the present results showed relatively low prevalence of T.gondii infection in Chaharmahal Va Bakhtiari native cattle, control and eradication programs seem to be still necessary to prevent the prevalence of this infection factor and economic losses

Charcot–Marie–Tooth disease: Genetics, epidemiology and complications

Background and aims: Charcot Marie Tooth disease (CMT) is the most prevalent hereditary neuropathy and its frequency is 1 in 2500. CMT is a heterogeneous disease and has different clinical symptoms. The prevalence of CMT and involved genes differ in different countries. CMT patients experience considerable sleep problems and a higher risk of decreased quality of life. In this work it was aimed to provide a review on the genetic and epidemiologic aspects of this disease. Methods: In the current review article, we performed a literature search on the epidemiology of Charcot–Marie–Tooth disease” and provided a brief review on epidemiology, genetic, and complications of CMT. Databases Web of Science and PubMed were searched using the Endnote software for the publications on CMT during 2000 to 2016. Results: Charcot Marie Tooth disease has different prevalence around the world and is the most common neuropathy. Epidemiological studies have estimated the prevalence of CMT in Japan 1/9200, in Iceland 1/8300, in Spain 1/3500 and in Italy 1/5700.The patients have different phenotype and the age of onset. There is a variety of inherited patterns of disease and many genes have been identified responsible whose mutations are main cause of the disease. Conclusion: Due to the impact of this kind of disabilities on the national health, further studies seem to be necessary to gain better knowledge of the disease particularly in the regions with higher prevalence. Moreover molecular biology services offered by genetic laboratories can reduce the incidence of disorder

Prevalence of aadA1, aadA2, aadB, strA and strB genes and their associations with multidrug resistance phenotype in Salmonella Typhimurium isolated from poultry carcasses

This study aimed to assess the prevalence of aminoglycoside resistance genes in S. Typhimurium isolated from poultry carcasses in Iran, and to reveal the most prevalent patterns of antimicrobial resistance. A total number of 300 samples of poultry carcasses were analyzed. Salmonella was isolated from 245 samples (81.66%). Multiplex PCR showed that 56.3% of the samples belonged to serovar S. Typhimurium and the remainder (43.6%) contained the rest of serovars. The highest rate of drug resistance was observed for tetracycline (97.0%), nalidixic acid (87.0%) and amoxicillinclavulanic acid (67.4%). These serovars, however, were sensitive to cefotaxime (84.8%), sulfamethoxazole trimethoprim (77.6%) and gentamicin (71.0%). aadA1 gene was detected in 63 isolates (45.6%), aadA2 in 48 isolates (34.7%), aadB in 43 isolates (31.1%), strA in 52 isolates (37.6%) and strB in 31 isolates (22.4%). High prevalence of aminoglycoside resistance genes in S. Typhimurium was shown. Furthermore, there was a significant association (P < 0.02) between the presence of aadA1, aadA2, strA and strB genes and resistance to streptomycin. Also, there was a significant association (P < 0.001) between the presence of aadB gene and resistance to kanamycin and gentamicin

CRISPR/Cas9-mediated LINC00511 knockout strategies, increased apoptosis of breast cancer cells via suppressing antiapoptotic genes

Background The growing detection of long noncoding RNAs (lncRNAs) required the application of functional approaches in order to provide absolutely precise, conducive, and reliable processed information along with effective consequences. We utilized genetic knockout (KO) techniques to ablate the Long Intergenic Noncoding RNA 00,511 gene in several humans who suffered from breast cancer cells and at the end we analyzed and examined the results. Results The predictive relevance of LINC00511 expression pattern was measured by using a pooled hazard ratio (HR) with a 95% confidence interval (CI). The link among LINC00511 expression profiles and cancer metastasis was measured by using a pooled odds ratio (OR) with a 95% confidence interval. This meta- analysis was composed of fifteen studies which contained a total of 1040 tumor patients. We used three distinct CRISPR/Cas9-mediated knockdown techniques to prevent the LINC00511 lncRNA from being transcribed. RT-PCR was used to measure lncRNA and RNA expression. We used CCK-8, colony formation tests, and the invasion transwell test to measure cell proliferation and invasion. The stemness was measured by using a sphere-formation test. To validate molecular attachment, luciferase reporter assays were performed. The functional impacts of LINC00511 gene deletion in knockdown breast cancer cell lines were confirmed by using RT-qPCR, MTT, and a colony formation test. This meta-analysis was composed of 15 trials which contained a total of 1040 malignant tumors. Greater LINC00511 expression was ascribed to a lower overall survival (HR = 1.93, 95% CI 1.49-2.49, < P 0.001) and to an increased proportion of lymph node metastasis (OR = 3.07, 95% CI 2.23-4.23, P < 0.001) in the meta-analysis. It was found that the role of LINC00511 was overexpressed in breast cancer samples, and this overexpression was ascribed to a poor prognosis. The gain and loss-of-function tests demonstrated findings such as LINC00511 increased breast cancer cell proliferation, sphere-forming ability, and tumor growth. Additionally, the transcription factor E2F1 binds to the Nanog gene's promoter site to induce transcription. P57, P21, Prkca, MDM4, Map2k6, and FADD gene expression in the treatment group (LINC00511 deletion) was significantly higher than in the control group (P < 0.01). In addition, knockout cells had lower expression of BCL2 and surviving genes than control cells P < 0.001). In each of the two target alleles, the du-HITI approach introduced a reporter and a transcription termination signal. This strategy's donor vector preparation was significantly easier than "CRISPR HDR," and cell selection was likewise much easier than "CRISPR excision." Furthermore, when this approach was used in the initial transfection attempt, single-cell knockouts for both alleles were generated. Conclusions The methods employed and described in this work could be extended to the production of LINC00511 knockout cell lines and, in theory, to the deletion of other lncRNAs to study their function

Molecular analysis of the clavulanic acid regulatory gene isolated from an Iranian strain of Streptomyces clavuligerus, PTCC 1709

Objective: The clavulanic acid regulatory gene (claR) is in the clavulanic acid biosynthetic gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus). Materials and Methods: In this experimental study, two different strains of S. clavuligerus were used (PTCC 1705 and DSM 738), of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were subjected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction (PCR) using DNA isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (PCR-RFLP), and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript (pBs) vector and transformed into E. coli. Results: The entire sequence of the isolated claR (Iranian strain) was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes. Conclusion: The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The overproduced protein could also be used for other related investigations, such as a mobility shift assa

The functions of azurin of Pseudomonas aeruginosa and human mammaglobin-A on proapoptotic and cell cycle regulatory genes expression in the MCF-7 breast cancer cell line

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated