Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing

We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes.     

A method for genetic modification of human embryonic stem cells using electroporation

The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.     

Prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, Northeast Iran

doi: 10.1111/j.1741‐2358.2011.00588.x
Prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, Northeast Iran Objective: The aim of this study was to determine the prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, northeast of Iran. Background: This study was conducted to assess the prevalence of oral lesions in the institutionalized elderly. Methods: In this survey, we studied all of the nursing homes in Mashhad; a total of five encompassing 237 residents. The subjects were examined and lesions recorded in a designed checklist. T‐test, chi‐square and Fisher’s exact tests were used for the analysis. Results: The mean age of the patients was 79.59 ± 8.88. Ninety‐eight per cent of cases had at least one oral mucosal lesion. The most common lesions were fissured tongue (66.5%), atrophic glossitis (48.8%), sublingual varicosity (42%) and xerostomia (38%). There was no significant difference in the prevalence of denture‐related lesions (DRLs) between men and women (p > 0.05). Xerostomia was more prevalent in 70–79‐year‐old than in 60–69‐year‐old subjects. No case of oral malignant lesion was detected. Discussion: These findings revealed a higher prevalence of oral mucosal lesions in Mashhad city compared with other studies conducted in other major cities in Iran and abroad. The results emphasise the necessity of national programmes towards oral health promotion.     

MicroRNA-590-3P suppresses cell survival and triggers breast cancer cell apoptosis via targeting sirtuin-1 and deacetylation of p53

Downregulation of microRNA-590-3p (miR-590-3p) is a frequently occurring, nonphysiological event which is observed in several human cancers, especially breast cancer. However, the significance of miR-590-3p still remain unclear in the progression of this disease. This study explored the role of miR-590-3p in apoptosis of breast cancer cells. Gene expression of miR-590-3p, Sirtuin-1 (SIRT1), Bcl-2 associated X protein (BAX), and p21 was evaluated with real-time polymerase chain reaction (PCR) and SIRT1 protein expression was assessed by Western blot analysis in breast cancer cell lines. Bioinformatics analysis and luciferase reporter assay were used to evaluate targeting of SIRT1 messenger RNA (mRNA) by miR-590-3p. Cells were transfected with miR-590-3p mimic and inhibitor and their effects on the expression and activity of SIRT1 were evaluated. The effects of miR-590-3p upregulation on the acetylation of p53 as well as cell viability and apoptosis were assessed by Western blot analysis, WST-1 assay, and flow cytometry, respectively. miR-590-3p expression was considerably downregulated in breast cancer cells which was accompanied by upregulation of SIRT1 expression. SIRT1 was recognized as a direct target for miR-590-3p in breast cancer cells and its protein expression and activity was dramatically inhibited by the miR-590-3p. In addition, there was an increase in p53 and its acetylated form that ultimately led to upregulation of BAX and p21 expression, suppression of cell survival, and considerable induction of apoptosis in breast cancer cells. These findings suggest that miR-590-3p exerts tumor-suppressing effects through targeting SIRT1 in breast cancer cells, which makes it a potential therapeutic target for developing more efficient treatments for breast cancer.     

PIA and rSesC Mixture Arisen Antibodies Could Inhibit the Biofilm-Formation in Staphylococcus aureus

Background:Staphylococcus aureus as a causative agent of hospital-acquired infections has been considered as the primary concern in biomaterial-related infections (BAIs).Methods:Following the purification of polysaccharide intercellular adhesion (PIA) as an efficient macromolecule in biofilm formation in the native condition, recombinant S. epidermidis surface-exposed rSesC protein, with the most homology to clumping factor A (ClfA) in S. aureus was cloned and expressed in a prokaryotic host as well. Fourier transform infrared spectrometry (FTIR) and Western blotting procedure analyzed purified PIA and protein, respectively. Then, the immune response was evaluated by measuring total IgG titers. Moreover, the capacity of Anti-biofilm forming activity of arisen antibodies to a biofilm-forming S. aureus strains was assessed by the semi-quantitative micro-plate procedure.Results:Data showed that the total IgGs were boosted in mice immunized sera. By performing an inhibition assay, the biofilm inhibitory effect of secreted antibodies to test strain was observed. Arisen antibodies against the mixture significantly were more potent than PIA and rSesC, when comparing individual antigens in a biofilm inhibition assay.Conclusion:immunization of mice with mentioned antigens especially a mixture of them, could eliminate the biofilm formation process in S. aureus. Hopefully, this study corresponds to the suggestion that the immunization of mice with PIA and rSesC candidate vaccines could protect against S. aureus infection.Key Words: PIA, Purification, Staphylococcus aureus, rSesC, Vaccine candidates     

The expanding role of CDR1‐AS in the regulation and development of cancer and human diseases

CircRNAs are a superabundant and highly conserved group of noncoding RNAs (ncRNAs) that are characterized by their high stability and integrity compared with linear forms of ncRNAs. Recently, their critical role in gene expression regulation has been shown; thus, it is not far‐fetched to believe that their abnormal expression can be a cause of different kinds of diseases such as cancer, neurodegenerative, and autoimmune diseases. They can have a function in variety of biological processes such as microRNA (miRNA) sponging, interacting with RNA‐binding proteins, or even an ability to translate to proteins. A huge challenge in finding diagnostic biomarkers is finding noninvasive biomarkers that can be detected in human fluids, especially blood samples. CircRNAs are becoming candidate biomarkers for diagnosis and prognosis of these diseases through their ability to transverse from the blood‐brain barrier and their broad presence in circulating exosomes. The circRNA for miRNA‐7 (ciRS‐7) is newly recognized, and acknowledged to being related to human pathology and cancer progression. In this review, we first briefly summarize the latest studies about their characteristics, biogenesis, and their mechanisms of action in the regulation and development of human diseases. Finally, we provide a list of diseases that are linked to one member of this novel class of ncRNAs called ciRS‐7.     

Experimental Analysis of E2BB (LTIIb) Signal Peptide in Secretory Production of Reteplase in Escherichia coli

International Journal of Peptide Research and Therapeutics - Recombinant reteplase is the truncated form of tissue plasminogen activator. Signal peptides play a pivotal role in the secretion of...     

Zebrafish models to study hypoxia-induced pathological angiogenesis in malignant and nonmalignant diseases

Most in vivo preclinical disease models are based on mouse and other mammalian systems. However, these rodent-based model systems have considerable limitations to recapitulate clinical situations in human patients. Zebrafish have been widely used to study embryonic development, behavior, tissue regeneration, and genetic defects. Additionally, zebrafish also provides an opportunity to screen chemical compounds that target a specific cell population for drug development. Owing to the availability of various genetically manipulated strains of zebrafish, immune privilege during early embryonic development, transparency of the embryos, and easy and precise setup of hypoxia equipment, we have developed several disease models in both embryonic and adult zebrafish, focusing on studying the role of angiogenesis in pathological settings. These zebrafish disease models are complementary to the existing mouse models, allowing us to study clinically relevant processes in cancer and nonmalignant diseases, which otherwise would be difficult to study in mice. For example, dissemination and invasion of single human or mouse tumor cells from the primary site in association with tumor angiogenesis can be studied under normoxia or hypoxia in zebrafish embryos. Hypoxia-induced retinopathy in the adult zebrafish recapitulates the clinical situation of retinopathy development in diabetic patients or age-related macular degeneration. These zebrafish disease models offer exciting opportunities to understand the mechanisms of disease development, progression, and development of more effective drugs for therapeutic intervention.