Effect of angiotensin converting enzyme gene polymorphism on patients with in-stent restenosis after percutaneous coronary intervention

Purpose: To evaluate the association between common single nucleotide polymorphisms (SNPs) in angiotensin converting enzyme (ACE) gene and the risk of in-stent restenosis (ISR) and/or the response to angiotensin converting enzyme inhibitor ACEI in individuals with stable coronary artery disease (CAD) after stent implantation. Methods: The total population of this study consisted of 200 Egyptian individuals divided into 2 groups - in-stent restenosis (ISR) and non ISR group). Genomic DNA was withdrawn from EDTA whole blood applying a spin column approach and ACE gene insertion/deletion (I/D) polymorphisms were determined by polymerase chain reaction (PCR). Results: Carriers of allele D of ACE gene were significantly more liable to ISR occurrence. However, carriers of allele I were significantly more liable to ISR occurrence after administration of ACEI. There is a negative interaction between DD genotype of ACE gene and ACEI administration on ISR after percutaneous coronary intervention (PCI). However, there is a positive interaction between II and ID genotype of ACE gene and ACEI administration on ISR after PCI with bare metal stents (BMS). Conclusion: It is beneficial to implement ACEI in therapeutic regimen in individuals with ID or II genotypes of ACE gene, especially with BMS implementation.

Bottom-up/cross-linking mass spectrometry via simplified sample processing on anion-exchange solid-phase extraction spin column

Capturing proteins on anion-exchange discs facilitates concentration of diluted samples and removal of contaminants, allowing more efficient sample pretreatment for bottom-up/cross-linking mass spectrometry than in-solution and in-gel.

Modified Spin Column-Based RNA Extraction Methods of Staphylococcus aureus using PureLink® RNA Mini Kit and Basic Laboratory Instrument

RNA extraction is an important process before gene expression assessment at the transcriptomic level. RNA is a sensitive material to environmental factors such as temperature and contaminants, so the RNA extraction process generally requires sophisticated and expensive laboratory instruments. In this study, we extract RNA from Staphylococcus aureus bacteria using the PureLink® RNA Mini Kit. The instruments used in this study are basic instruments such as a hand homogenizer and non-thermal centrifuge. The results of RNA extraction were visualized using agarose gel electrophoresis. These results indicate that bacterial RNA extraction can be performed using the PureLink® RNA Mini Kit even with inexpensive basic laboratory instruments.

Wildlife pathogen detection: evaluation of alternative DNA extraction protocols

Accurate detection of wildlife pathogens is critical in wildlife disease research. False negatives or positives can have catastrophic consequences for conservation and disease-mitigation decisions. Quantitative polymerase chain reaction is commonly used for molecular detection of wildlife pathogens. The reliability of this method depends on the effective extraction of the pathogen’s DNA from host samples. A wildlife disease that has been in the centre of conservationist’s attention is the amphibian disease Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Here, we compare the efficiency of a spin column extraction kit (QIAGEN), commonly used in Bd DNA extraction, to an alternative spin column kit (BIOKÈ) used in extractions from other types of samples, which is considerably cheaper but not typically used for Bd DNA extraction. Additionally, we explore the effect of an enzymatic pre-treatment on detection efficiency. Both methods showed similar efficiency when extracting Bd DNA from zoospores from laboratory-created cell-cultures, as well as higher efficiency when combined with the enzymatic pre-treatment. Our results indicate that selecting the optimal method for DNA extraction is essential to ensure minimal false negatives and reduce project costs.

Simple, inexpensive and RNase-free purification of plasmid DNA by fractional precipitation with isopropanol

We present a modified alkaline lysis method for purification of plasmid DNA (pDNA) from bacterial extract using fractional precipitation with isopropanol (FPI). This method includes two successive precipitations with 0.33 and 0.36 volumes of isopropanol and separates pDNA from total RNA and most of the lipopolysaccharides. Using different quality control tests, we demonstrate that plasmids purified with FPI show superior quality compared to plasmids prepared with commercial kits based on spin-column chromatography.

Exosome microRNAs in Amyotrophic Lateral Sclerosis: A Pilot Study

The pathogenesis of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease, remains undisclosed. Mutations in ALS related genes have been identified, albeit the majority of cases are unmutated. Clinical pathology of ALS suggests a prion-like cell-to-cell diffusion of the disease possibly mediated by exosomes, small endocytic vesicles involved in the propagation of RNA molecules and proteins. In this pilot study, we focused on exosomal microRNAs (miRNAs), key regulators of many signaling pathways. We analyzed serum-derived exosomes from ALS patients in comparison with healthy donors. Exosomes were obtained by a commercial kit. Purification of miRNAs was performed using spin column chromatography and RNA was reverse transcribed into cDNA. All samples were run on the miRCURY LNATM Universal RT miRNA PCR Serum/Plasma Focus panel. An average of 29 miRNAs were detectable per sample. The supervised analysis did not identify any statistically significant difference among the groups indicating that none of the miRNA of our panel has a strong pathological role in ALS. However, selecting samples with the highest miRNA content, six biological processes shared across miRNAs through the intersection of the GO categories were identified. Our results, combined to those reported in the literature, indicated that further investigation is needed to elucidate the role of exosome-derived miRNA in ALS.

HEPG2 EXPRESSION OF MIR-202 & TRIB-1 UNDER METABOLIC AND INFLAMMATORY STRESS.

Background: The burden of cardiovascular disease (CVD) affects both developed and developing countries with high rates of mortality and morbidity. Cardiovascular diseases are highly polymorphic across their various risk factors. Human polymorphisms of the trib-1 gene have been implicated to be associated with risk factors for CVD. Trib-1 gene is a known target for microRNA-202, which consequently could affect its stability. The objective of this study was to evaluate the expression of miR-202 in an hepatic cell line under in vitro conditions of metabolic and inflammatory stress and the effect on the trib-1 level. Materials and Methods: HepG2 cells cultured under in vitro conditions of high glucose and cytokine stimulation of concentrations of varying time intervals were harvested and mRNA/microRNA extracted using the spin column-based centrifugation, reversed transcribed and analysed for endogenous expressions of trib-1 and miR-202 using qPCR. One-ANOVA followed by Dunnetts multiple comparison test was used to test for significance (P<0.05) across samples. Results: It was observed that there was a significant decrease in trib-1 levels under these conditions of high glucose and cytokine stimulation and also with the combination of both whilst there was a consistent pattern of upregulation of MIR-202 under these conditions. Conclusion: This study reveals that miR-202 is expressed in HepG2 cells, and there is a possible interaction between trib-1 and MIR-202 which could affect trib-1 stability, and also the potentials for MIR-202 to be involved in some cellular activities in HepG2 cells relating to these conditions.