Identification of novel potential drugs and miRNAs biomarkers in lung cancer based on gene co-expression network analysis

Non–small cell lung cancer (NSCLC) is an important cause of cancer-associated deaths worldwide. Therefore, the exact molecular mechanisms of NSCLC are unidentified. The present investigation aims to identify the miRNAs with predictive value in NSCLC. The two datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs (DEmiRNA) and mRNAs (DEmRNA) were selected from the normalized data. Next, miRNA-mRNA interactions were determined. Then, co-expression network analysis was completed using the WGCNA package in R software. The co-expression network between DEmiRNAs and DEmRNAs was calculated to prioritize the miRNAs. Next, the enrichment analysis was performed for DEmiRNA and DEmRNA. Finally, the drug-gene interaction network was constructed by importing the gene list to dgidb database. A total of 3,033 differentially expressed genes and 58 DE miRNA were recognized from two datasets. The co-expression network analysis was utilized to build a gene co-expression network. Next, four modules were selected based on the Zsummary score. In the next step, a bipartite miRNA-gene network was constructed and hub miRNAs (let-7a-2-3p, let-7d-5p, let-7b-5p, let-7a-5p, and let-7b-3p) were selected. Finally, a drug-gene network was constructed while SUNITINIB, MEDROXYPROGESTERONE ACETATE, DOFETILIDE, HALOPERIDOL, and CALCITRIOL drugs were recognized as a beneficial drug in NSCLC. The hub miRNAs and repurposed drugs may act a vital role in NSCLC progression and treatment, respectively; however, these results must validate in further clinical and experimental assessments.

Induction effects of bacteroides fragilis derived outer membrane vesicles on toll like receptor 2, toll like receptor 4 genes expression and cytokines concentration in human intestinal epithelial cells

Objective: Gastrointestinal [GI] tract, like other mucosal surface, is colonized with a microbial population known as gut microbiota. Outer membrane vesicles [OMVs] which are produced by gram negative bacteria could be sensed by Toll like receptors [TLRs]. The interaction between gut microbiota and TLRs affects homeostasis and immune responses. In this study, we evaluated TLR2, TLR4 genes expression and cytokines concentration in Caco-2 cell line treated with Bacteroides fragilis [B. fragilis] and its OMVs

Materials and Methods: In this experimental study, OMVs were extracted using sequential centrifugation and their physicochemical properties were evaluated as part of quality control assessment. Caco-2 cells were treated with B. fragilis and its OMVs [180 and 350 microg/ml]. Quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR] was performed to assess TLR2 and TLR4 mRNA expression levels. Pro-inflammatory [IFN?] and anti-inflammatory [IL- 4 and IL-10] cytokines were evaluated by ELISA

Results: B. fragilis significantly decreased TLR2 and slightly increased TLR4 mRNA levels in Caco-2 cell line. The TLR2 mRNA level was slightly increased at 180 and 350 microg/ml of OMVs. Conversely, the TLR4 mRNA level was decreased at 180 microg/ml of OMVs, while it was significantly increased at 350 microg/ml of OMVs. Furthermore, B. fragilis and its OMVs significantly increased and decreased IFN? concentration, respectively. Anti-inflammatory cytokines were increased by B. fragilis and its OMVs

Conclusion: B. fragilis and its OMVs have pivotal role in the cross talk between gut microbiota and the host especially in the modulation of the immune system. Based on the last studies on immunomodulatory effect of B. fragilis derived OMVs on immune cells and our results, we postulate that B. fragilis derived OMVs could be possible candidates for the reduction of immune responses

Arylamine n-acetyltransferase 2 polymorphisms and the risk of endometriosis

Background: Human arylamine N-acetyltransferase 2 [NAT2] gene has a key role in xenobiotic metabolism through the conjugation of acetyl group to xenobiotic substances. NAT2 has been suggested as a susceptibility factor in endometriosis; however, the results of studies have been controversial. In this study, the association of NAT2 polymorphisms with susceptibility to endometriosis was evaluated in an Iranian population

Methods: This is an association study and totally 141 women with diagnosis of endometriosis and 158 healthy women as control group were analyzed for NAT2 gene polymorphisms [C481T, A803G, G857A and G590A] by PCR-RFLP methods

Results: The 590 GA genotype was significantly lower [p=0.001; OR=0.42, 95% CI: 0.25-0.71] in the patients [38.3%] than the control group [55.1%]. The 590A allele was significantly lower [p=0.033; OR=0.69, 95% CI: 0.49-0.79] in the patients [31.2%] compared with the controls [39.6%]. Analysis of haplotypes showed that NAT2 481C, 803A, 590A, 587A combination was significantly different between the case and control women [p= 0.029; OR=3.11, 95% CI: 1.13-8.52]

Conclusion: The NAT2 G590A SNP may be associated with susceptibility to endometriosis and the 590A allele may have a protective role in development of endometriosis. The NAT2 481C, 803A, 590A, 587A haplotype was associated with a higher risk of endometriosis in Iranian population

Peroxisome proliferator-activated receptor gamma activity is required for appropriate cardiomyocyte differentiation

Objective: Peroxisome proliferator-activated receptor gamma [PPAR gamma] is a member of the PPAR nuclear receptor superfamily. Although PPAR gamma acts as a master transcription factor in adipocyte differentiation, it is also associated with a variety of cell functions including carbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of PPAR gamma in order to address its role in cardiac cell differentiation of mouse embryonic stem cells [mESCs]

Materials and Methods: In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chain reaction [qPCR] was carried out to estimate level of gene expression. Furthermore, the requirement of PPAR gamma in cardiac differentiation of mESCs, during cardiac progenitor cells [CPCs] formation, was examined by applying the respective agonist and antagonist

Results: The obtained data revealed an elevation in the expression level of PPAR gamma during spontaneous formation of CPCs and cardiomyocytes. Our results indicated that during CPC formation, PPAR gamma inactivation via treatment with GW9662 [GW] reduced expression of CPC and cardiac markers

Conclusion: We conclude that PPAR gamma modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis

[Histopathology of epididymis in adult NMRI male mice treated with zinc oxide nanoparticles]

Background: The rapid development of nanotechnology has given rise to broad applications of nanoparticles [NPs] in biomedicine. This material is distributed in all parts of the body, rapidly after injection, by circulation and reach to all organs and tissues. Before their application as medicine, toxic effects of them on human and animals should be assessed. In this study, the effects of zinc oxide nanoparticle on histopathological changes of epididymis in adult male mice was investigated

Materials and methods: In this experimental study, 30 adult male NMRI mice were used. The animals were assigned as control, sham and three experimental groups [n=6]. Sham and experimental groups received 1ml of distilled water and experimental animals received different doses of nano zinc oxide [250, 500 and 700 mg/kg, i.p. injection, respectively]. Treatments was performed for one day. After a week, effects of zinc oxide nanoparticle on histopathological changes of epididym tissue were studied. Data were analyzed by ANOVA and Tukey test

Results: Administration of zinc oxide nanoparticle in 250 mg/kg dose caused significant reduction in the number of sperm cells. Also, zinc oxide nanoparticle in 250 mg/kg dose lead to degenerated epididymis cells in epididymis tubules. There were no significant changes in diameter of epididymis tubules and number of epididymis tubules

Conclusion: According to the results of this study, zinc oxide nanoparticles may cause adverse effects on the reproductive system. So, we recommend to avoid using products containing nanoparticles of zinc oxide

Expression and purification of HCV core and core-E1E2 proteins in different bacterial strains

Background: hepatitis C virus [HCV] is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma [HCC]. Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses

Objectives: in current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems

Materials and Methods: the expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer

Results: the data showed a clear band of [tilde]573 bp for HCV core and [tilde]2238 bp for core-E1E2 genes in agarose gel. Moreover, a [tilde]21 kDa band of core protein and a [tilde]83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method

Conclusions: this study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system

[Study of breast cancer cells inhibition by cold atmospheric plasma]

Breast cancer is the most prevalent cancer and the second leading cause of cancer death in women. Because of side effects of current treatments, researchers are looking for methods with less possible side effects and highest rate of destruction of cancer cells without influencing healthy cells. Based on this, cold atmospheric plasma [CAP], is used on MCF-7 breast cancer cells. In this method, electromagnetic waves are used for destruction and death of cells. The aim of this study was to evaluate the treatment of breast cancer using CAP. In this study, cold atmospheric plasma [CAP] with helium gas and oxygen was used for treatment of breast cancer cells [MCF-7] in different times and with using MTT assay to determine viability percentage of treated cells. Data were analyzed by one way ANOVA using SPSS software. Viability percentage of cancer cells treated with CAP was significantly reduced compared to cancer cells not treated with plasma. Optimal treatment time of CAP for cancer cells was 45s. Cold atmospheric plasma can be considered as useful method for breast cancer cells treatment

Different expression systems for production of HCV structural proteins

Hepatitis C virus [HCV] is an important agent causing chronic liver infection, which often leads to liver cirrhosis and lethal hepatocellular carcinoma [HCC]. At present, there is no effective HCV vaccine for prevention of hepatic disease and the standard treatment is neither economical nor fully effective in all the patients. However, vaccination based on structural and nonstructural proteins of HCV has attracted a special interest. Different heterologous systems have been used to generate the recombinant HCV core, E1, and E2 proteins including Escherichia coli, yeast, insects and mammalian cells. Further studies showed that the amounts of HCV recombinant proteins in E. coli are more suitable and un-expensive compared to other systems. It should be considered that this system is not efficient for generation of the glycosylated proteins. Thus, the structure of proteins is an important agent of selection for expression systems. The selection of expression systems will be critical for the use of recombinant proteins as an immunogen. In this mini-review, we briefly describe different expression systems for generation of the HCV recombinant structural proteins applied in vaccine design

Immunogenicity evaluation of a DNA vaccine expressing the hepatitis C virus non-structural protein 2 gene in C57BL/6 mice

Most of the hepatitis C virus [HCV] infections elicit poor immune responses and 75% to 85% of cases become chronic; therefore, the development of an effective vaccine against HCV is of paramount importance. In this study, we aimed to evaluate co-administration of HCV non-Structural Protein 2 and IL-12 DNA vaccines in C57BL/6 mice A plasmid encoding full-length HCV NS2 protein [non-structural protein 2] was generated and used to vaccinate mice. Negative control [an empty expression vector] was also employed to evaluate the background response. To investigate immune responses against vaccine, C57BL/6 mice received three doses of the vaccine with a two-week interval. Cellular immunity was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay for lymphocyte proliferation, lactate dehydrogenase release for cytotoxic T lymphocyte [CTL] activity and cytokine assay. The findings demonstrated that immunization of mice with plasmid expressing HCV NS2 induced CTL response, interferon gamma production, and lymphocyte proliferation compared to negative control. The results also demonstrated that co-administration of IL-12 with the HCV NS2 plasmid induced significantly better immune response in C57BL/6 mice. DNA vaccine encoding HCV NS2 is an effective candidate that can trigger CTL-based immune response against HCV. In addition, the results suggested that combining the DNA vaccine approach with immune stimulatory cytokines may significantly enhance antigen-specific immune responses

Design, construction and expression of recombinant vector containing the rabies virus nucleoprotein gene

Rabies is an acute encephalitis that causes more than 60,000 deaths worldwide. The only way to save individuals bitten by a rabies-infected animal is the timely use of effective vaccines. Treatment with new generation vaccines is expensive. Therefore, there is a global movement towards the production of less expensive vaccines which retain and improve upon the quality and effectiveness of the vaccine. Production and evaluation of non-classical vaccines is one of the approaches taken in this regard. In this study, we describe a new eukaryotic expression system to express the nucleoprotein N gene of rabies virus which, if suitable, may be evaluated for anti-rabies vaccine production. The complete sequence of the N gene of rabies virus PV subtype was amplified by real-time polymerase chain reaction and cloned into the pCDNA3.1[+] vector. The cloned gene was excised from the vector by restriction enzyme digestion and sequenced. Due to mutations detected in the N gene, the gene coding sequence was purchased as a recombinant pGH/N vector. Vector pGH/N was amplified and following enzymatic digestion, the excised N gene was once again cloned into vector pCDNA3.1[+]. Successful cloning was confirmed using restriction digests and quick check. The recombinant vector pCDNA3.1[+]/N was transformed into cultured BSR cells and protein N expression was analyzed using fluorescent antibody test [FAT]. Electrophoresis confirmed amplification of the nucleoprotein N gene and subsequent restriction enzyme digestion showed that the N gene had been successfully cloned into the recombinant pCDNA3.1[+]/N vector. However, DNA sequencing revealed the presence of mutations within the N gene. Restriction digest of the commercial pGH/N vector showed that the N gene had been excised from the vector. Successful cloning of the N gene into the pCDNA3.1[+] expression vector was confirmed using restriction digests and quick check. Protein expression in BSR cells was assayed by immunostaining with anti-ribonucleocapsid FITC-conjugated antibody and visually confirmed by fluorescence microscopy. This study showed that the protein N of rabies virus subtype PV can be expressed in a eukaryotic expression system using the pCDNA3.1[+] expression vector

[Effect of insulin-like growth factor-I on apoptosis in Chinese hamster ovary cells for biopharmaceutical production]

The ability to reducing death due to apoptosis and maintaining extensive levels on cell viability under serum free media in cell culture are important subjects in production of recombinant proteins. Insulin-like growth factor-I [IGF-I], is the growth factor of choice for mammalian cell proliferation in serum-free culture. In addition to its mitogenic activity, it has antiapoptotic activity protecting cultures from diverse death inducing stimuli. In this study, the effect of different concentrations of IGF-I examined on CHO-K1 [Chinese hamster ovary K1 cells] cell line for 24 and 48 hours. In this experimental study, the cell line was cultivated in DMEM supplemented with 10% fetal bovine serum [FBS]. Apoptosis process was induced in cells by methotrexate, serum was removed and then 10-50 ng/ml concentrations of IGF-I were added. Apoptosis was assessed by caspase 3 detection kit and cell proliferation and viability determined by MTT assay. Caspase 3 activity decreased significantly by increasing concentrations of IGF-I. In the highest concentration of IGF-I [50 ng/ml], 1.7 and 1.4 equal decreases of caspase 3 activities, compared with negative control group, were noted after 24 and 48 hours, respectively. It confirmed antiapoptotic activity of growth factor to maintain viability and protect cultures from apoptotic inducing stimuli [methotrexate]. IGF-I, as antiapoptotic factor, decreased programmed death of CHO-K1 cells in apoptotic induced conditions