Diagnostic kit based on halloysite nanoclay-ionic liquid nanocomposite modified electrode for electrochemical determination of cancer biomarker.

Nucleic acid hybridization is occurred between the selective single-stranded nucleic acid sequence and its target sequence, which is one of the essential procedure for electrochemical detection of nucleic acid. microRNA-21 (miRNA-21) is known as a biomarker in various cancers. The determination of miRNA-21 was attained through by hybridization of inosine substituted miRNA-21 specific DNA probe (Pinosine) with its target miRNA-21. In this study, the surface of pencil graphite electrode (PGE) was firstly modified with halloysite nanoclay-ionic liquid (HNT/IL) nanocomposite. The characterization of surface was performed by Scanning Electron Microscope (SEM) images and Energy Dispersive X-Ray Analysis (EDX) analysis, and the differences at surface modifications were also shown by electrochemical methods with electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). For sensitive and selective determination of miRNA-21, Pinosine and target miRNA concentration, immobilization and hybridization time were optimized by using HNT/IL modified PGE in combination with differential pulse voltammetry (DPV). The detection limit was achieved as 0.17 µg/mL (equals to 23.69 nM) in the linear range of 0.25-2 µg/mL miRNA-21. The selectivity of voltammetric method based on HNT/IL-PGE developed for miRNA-21 was examined in the presence of mismatch (MM) and non-complementary (NC) sequences. Because miRNA-21 is over-expressed in cancer cells, it has been tested in total RNA samples isolated from cancer cell line (breast cancer cell line, MCF-7). In the total RNA samples obtained from MCF-7, the detection limit was calculated as 0.28 µg/mL in the linear range of 1-4 µg/mL. Besides, the healthy cell line (human embryonic kidney cell line, HEK-293) was used as a control group and the results obtained by MCF-7 total RNA samples were compared to the results using HEK-293 total RNA samples in terms of miRNA-21 level.

Implications of Possible HBV-Driven Regulation of Gene Expression in Stem Cell-like Subpopulation of Huh-7 Hepatocellular Carcinoma Cell Line.

Elevated levels of STIM1, an endoplasmic reticulum Ca2+ sensor/buffering protein, appear to be correlated with poor cancer prognosis in which microRNAs are also known to play critical roles. The purpose of this study is to investigate possible HBV origins of specific microRNAs we identified in a stem cell-like subpopulation of Huh-7 hepatocellular carcinoma (HCC) cell lines with enhanced STIM1 and/or Orai1 expression that mimicked poor cancer prognosis. Computational strategies including phylogenetic analyses were performed on miRNome data we obtained from an EpCAM- and CD133-expressing Huh-7 HCC stem cell-like subpopulation with enhanced STIM1 and/or Orai1 expression originally cultured in the present work. Results revealed two putative regions in the HBV genome based on the apparent clustering pattern of stem loop sequences of microRNAs, including miR3653. Reciprocal analysis of these regions identified critical human genes, of which their transcripts are among the predicted targets of miR3653, which was increased significantly by STIM1 or Orai1 enhancement. Briefly, this study provides phylogenetic evidence for a possible HBV-driven epigenetic remodeling that alters the expression pattern of Ca2+ homeostasis-associated genes in STIM1- or Orai1 overexpressing liver cancer stem-like cells for a possible mutual survival outcome. A novel region on HBV-X protein may affect liver carcinogenesis in a genotype-dependent manner. Therefore, detection of the viral genotype would have a clinical impact on prognosis of HBV-induced liver cancers.

Silencing acpP gene via antisense oligonucleotide-niosome complex in clinical Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is one of the major causes of nosocomial infections. In addition to its physiological adaptation capacity, it can develop resistance to disinfectants and antibiotics through various mechanisms. Recently, new eradication methods are gaining attention. Therefore, in this study, an LNA-2'-O-methyl hybrid antisense oligonucleotide targeting the acyl carrier protein P (acpP) gene was introduced into P. aeruginosa isolates. The design was determined through sequence analysis and prediction of the secondary structure of mRNA by software. Niosomes were used for enhancing cellular uptake. The control of the binding and transfection ability of the sequence was determined fluorometrically by labeling with 6-Fam. The effects were determined with broth microdilution method and qPCR studies. Eight different formulations were prepared. Among these, one formulation has shown to have ASO complexation ability whose composition was 312 µl Span 80 + 69.5 mg Cholesterol+ 36.4 mg CTAB+1 ml Chloroform and 5 ml dH2O. Thus this formulation was determined as the delivery system for the next stages. Significant gene inhibition was detected at the six isolates. Results of this study suggested that niosomes can be used as a delivery system for cellular uptake of ASO and could eliminate bacterial growth.

Mechanism of adrenomedullin 2/intermedin mediated vasorelaxation in rat main pulmonary artery.

Adrenomedullin 2/intermedin (AM2/IMD) is a member of calcitonin related gene peptide family and an important nitric oxide mediated vasorelaxant in various vascular beds. However, the mechanism of post receptor-interaction is not clear and may differ depending on tissue type and species. In this study, we aimed to investigate the exact mechanism and the role of BKCa and calcium channels on the vasorelaxant effect of AM2/IMD in rat PA. Changes in the AM2/IMD-mediated vasorelaxation were evaluated in the presence of various inhibitors. CGRP(8-37) (10-6 M), L-NAME (10-4 M), ODQ (10-5 M), SQ22536 (10-4 M), H89 (10-6 M), TEA (10-2 M), iberiotoxin (3 × 10-7 M), and verapamil (10-5 M), all partly or completely inhibited the vasorelaxation. The relaxation was also abolished by removal of the endothelium, or in KCl precontracted PAs. AM2/IMD did not elicit vasorelaxation in the Ca2+-free conditions. However, the vasorelaxation was not inhibited with AM(22-52) (10-6 M), 4-AP (3 × 10-3 M), glibenclamide (10-5 M), apamin (3 × 10-7 M), TRAM-34 (10-5 M), and La+3 (10-4 M). AM2/IMD -induced changes in intracellular calcium levels and isometric force were monitored simultaneously in fura-2-loaded, endothelium-intact PAs. The AM2/IMD-induced increase in intracellular Ca2+ concentration was inhibited in the presence of iberiotoxin and verapamil, whereas no change was observed with La3+ incubation. Our data suggest that the cAMP/PKA pathway is one of the important pathways AM2/IMD-induced vasorelaxation. AM2/IMD acts through activation of endothelial BKCa and subsequently causes hyperpolarization of the endothelial cell membrane. The hyperpolarization induces Ca2+ influx, which leads to NO production and subsequent vasorelaxation.

Electrochemical detection of interaction between capsaicin and nucleic acids in comparison to agarose gel electrophoresis.

In this study, the biomolecular interaction occurred between nucleic acids and Capsaicin (CPS), the active compound in chilli peppers, which has been reported to have anti-carcinogenic properties, was investigated for the first time herein using disposable electrochemical biosensor. It is aimed to perform the surface-confined interaction between CPS and different types of nucleic acids and under this aim, the experimental conditions were optimized; such as, the concentration of CPS and DNA, DNA immobilization time and interaction time etc. The detection limit of DNA was estimated based on guanine oxidation signal in the linear concentration range of DNA from 1 to 5 µg/mL, and it was found to be 0.62 µg/mL. The effect of time-dependent manner from 1 min to 30 min on the interaction of CPS with nucleic acids was explored upon to the changes at guanine signal coming from double stranded DNA and cDNA as well as PCR samples. The interaction of CPS with double stranded DNA was also determined by agarose gel electrophoresis.

Effects of cell seeding density on real-time monitoring of anti-proliferative effects of transient gene silencing.

BACKGROUND: Real-time cellular analysis systems enable impedance-based label-free and dynamic monitoring of various cellular events such as proliferation. In this study, we describe the effects of initial cell seeding density on the anti-proliferative effects of transient gene silencing monitored via real-time cellular analysis. We monitored the real-time changes in proliferation of Huh7 hepatocellular carcinoma and A7r5 vascular smooth muscle cells with different initial seeding densities following transient receptor potential canonical 1 (TRPC1) silencing using xCELLigence system. Huh7 and A7r5 cells were seeded on E-plate 96 at 10,000, 5000, 1250 and 5000, 2500 cells well-1, respectively, following silencing vector transfection. The inhibitory effects of transient silencing on cell proliferation monitored every 30 min for 72 h. RESULTS: TRPC1 silencing did not inhibit the proliferation rates of Huh7 cells at 10,000 cells well-1 seeding density. However, a significant anti-proliferative effect was observed at 1250 cells well-1 density at each time point throughout 72 h. Furthermore, significant inhibitory effects on A7r5 proliferation were observed at both 5000 and 2500 cells well-1 for 72 h. CONCLUSIONS: Data suggest that the effects of transient silencing on cell proliferation differ depending on the initial cell seeding density. While high seeding densities mask the significant changes in proliferation, the inhibitory effects of silencing become apparent at lower seeding densities as the entry into log phase is delayed. Using the optimal initial seeding density is crucial when studying the effects of transient gene silencing. In addition, the results suggest that TRPC1 may contribute to proliferation and phenotypic switching of vascular smooth muscle cells.

Resveratrol Stimulates Hydrogen Sulfide (H2 S) Formation to Relax Murine Corpus Cavernosum.

INTRODUCTION: Resveratrol (RVT) found in red wine protects against erectile dysfunction and relaxes penile tissue (corpus cavernosum) via a nitric oxide (NO) independent pathway. However, the mechanism remains to be elucidated. Hydrogen sulfide (H2 S) is a potent vasodilator and neuromodulator generated in corpus cavernosum. AIMS: We investigated whether RVT caused the relaxation of mice corpus cavernosum (MCC) through H2 S. METHODS: H2 S formation is measured by methylene blue assay and vascular reactivity experiments have been performed by DMT strip myograph in CD1 MCC strips. MAIN OUTCOME MEASURES: Endothelial NO synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 0.1 mM) or H2 S inhibitor aminooxyacetic acid (AOAA, 2 mM) which inhibits both cystathionine-ß-synthase (CBS) and cystathionine-gamma-lyase (CSE) enzyme or combination of AOAA with PAG (CSE inhibitor) has been used in the presence/absence of RVT (0.1 mM, 30 min) to elucidate the role of NO or H2 S pathways on the effects of RVT in MCC. Concentration-dependent relaxations to RVT, L-cysteine, sodium hydrogen sulfide (NaHS) and acetylcholine (ACh) were studied. RESULTS: Exposure of murine corpus cavernosum to RVT increased both basal and L-cysteine-stimulated H2 S formation. Both of these effects were reversed by AOAA but not by L-NNA. RVT caused concentration-dependent relaxation of MCC and that RVT-induced relaxation was significantly inhibited by AOAA or AOAA + PAG but not by L-NNA. L-cysteine caused concentration-dependent relaxations, which are inhibited by AOAA or AOAA + PAG significantly. Incubation of MCC with RVT significantly increased L-cysteine-induced relaxation, and this effect was inhibited by AOAA + PAG. However, RVT did not alter the effect of exogenous H2 S (NaHS) or ACh-induced relaxations. CONCLUSIONS: These results demonstrate that RVT-induced relaxation is at least partly dependent on H2 S formation and acts independent of eNOS pathway. In phosphodiesterase 5 inhibitor (PDE-5i) nonresponder population, combination therapy with RVT may reverse erectile dysfunction via stimulating endogenous H2 S formation.

Silencing of TRPC1 regulates store-operated calcium entry and proliferation in Huh7 hepatocellular carcinoma cells.

PURPOSE: Previously, we observed reciprocal changes in TRPC1 and TRPC6 expression levels in aging rat aorta and A7r5, rat embryonic vascular smooth muscle cells. Furthermore, downregulation of TRPC1 significantly elevated store-operated Ca(2+) entry suggesting the regulatory role of TRPC1 in A7r5 cells. Since TRPC6 upregulation shown to be associated with cell proliferation, the purpose of our study was to investigate the functional consequences of TRPC1 ion channel downregulation by RNA interference in Huh7 human hepatocellular carcinoma cell line. METHODS: Huh7 cells used in quantitative gene and protein expression as well as in functional analyses. To determine mRNA and protein levels, quantitative real-time RT-PCR and western blot analyses were performed, respectively. In functional analyses, real-time changes in proliferation, migration and intracellular Ca(2+) levels were monitored. RESULTS: In shTRPC1-transfected Huh7 cells, TRPC1 mRNA and protein levels significantly decreased whereas store-operated Ca(2+) entry significantly elevated. TRPC1-silencing suppressed cell proliferation without affecting cell migration in real-time cellular analyses. CONCLUSION: These results suggest that TRPC1 may take part both in regulation of store-operated Ca(2+) entry and proliferation of hepatocellular carcinoma cells.

Simultaneous measurement of cytosolic and mitochondrial calcium levels: observations in TRPC1-silenced hepatocellular carcinoma cells.

INTRODUCTION: The measurement of intracellular Ca(2+), cytosolic or stored in organelles, i.e., mitochondria, gave valuable data for numerous areas of research. In case of tumor cells, mitochondrial Ca(2+) levels play essential roles in apoptosis along with endoplasmic reticulum (ER) Ca(2+). In this study, we describe a Ca(2+) monitoring system that allows studying both adherent cells and tissues and discuss data obtained from hepatocellular carcinoma cells and rat thoracic aorta by using this system. METHODS: For this purpose, two apparatus, one for adherent cells and the other for intact rat aorta, were designed and produced. With this system, changes in cytosolic Ca(2+) levels following store-operated calcium (SOC) entry induced by sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) blockers were recorded in different hepatocellular carcinoma cells. Furthermore, cytosolic and mitochondrial Ca(2+) levels were simultaneously measured in TRPC1-silenced Huh7 hepatocellular carcinoma cells. In addition, the effects of trifluoromethylphenylimidazole (TRIM) on cyclopiazonic acid (CPA)-, serotonin (5-HT)-, and phenylephrine (PE)-induced changes in isometric force and cytosolic Ca(2+) levels were determined simultaneously in rat thoracic aorta. The effects of aging on PE-induced responses were also investigated. RESULTS: After SOC entry activation, cytosolic Ca(2+) levels were increased, as expected in all hepatocellular carcinoma cells. Mitochondrial Ca(2+) levels following CPA-induced ER depletion were significantly (p<.05) diminished in TRPC1-silenced Huh7 cells. In addition, TRIM partially inhibited both 5-HT-induced contractions and cytosolic Ca(2+) levels without affecting CPA and PE responses. PE-induced contractions and cytosolic Ca(2+) levels were similar in aorta from young and old (3 and 22 months, respectively) rats. DISCUSSION: We confirmed that the system provides valuable data about intracellular Ca(2+) dynamics by allowing simultaneous measurements and sequential addition of compounds in adherent cells. The decrease in mitochondrial Ca(2+) loading following CPA-induced ER depletion in TRPC1-silenced Huh7 cells suggests a possible role of TRPC1 in hepatocellular carcinoma cell apoptosis. The system also enables the simultaneous measurement of isometric force and cytosolic Ca(2+) levels and promotes understanding vascular physiology and disease.

Electrochemical based detection of microRNA, mir21 in breast cancer cells.

In this work, a novel electrochemical microRNA (miRNA) detection method based on enzyme amplified biosensing of mir21 from cell lysate of total RNA was demonstrated. The proposed enzymatic detection method was detailed and compared with the conventional guanine oxidation based assay in terms of detection limit and specificity. For the detection of mir21, capture probes and/or cell lysates were covalently attached onto the pencil graphite electrode (PGE) by coupling agents of N-(dimethylamino)propyl-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). Having immobilized the capture probe onto the surface of PGE, hybridization was achieved with a biotinylated (from its 3' end) complementary target. Extravidin labeled alkaline phosphatase (Ex-Ap) binds to the biotinylated target due to the interaction between biotin-avidin and the enzyme converts electro-inactive alpha naphtyl phosphate (the substrate) to electro-active alpha naphtol (α-NAP, the product). α-NAP was oxidized at +0.23 V vs Ag/AgCl and this signal was measured by Differential Pulse Voltammetry (DPV). The signals obtained from α-NAP oxidation were compared for the probe and hybrid DNA. The specificity of the designed biosensor was proved by using non-complementary sequences instead of complementary sequences and the detection limit of the assay was calculated to be 6 pmol for cell lysates.

Post-transcriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca2+ entry in A7r5 vascular smooth muscle cells.

This study investigates functional consequences of TRPC1 ion channel downregulation observed in aging rat aorta by employing RNA interference in cultured vascular smooth muscle cells. For this purpose, A7r5 aortic smooth muscle cells were used in quantitative gene and protein expression as well as in functional analyses. According to quantitative RT-PCR results, TRPC3, TRPC4 and TRPC5 mRNAs were not at detectable levels. In siTRPC1-transfected cells, TRPC1 mRNA and protein levels were decreased by 40% and 64%; however, those of TRPC6 were drastically increased by 100% and 200%, respectively. In fura-2-loaded TRPC1 knockdown cells, despite the decreased TRPC1 levels, cyclopiazonic acid-induced Ca2+ entry and store-operated Ca2+ entry following Ca2+ addition were elevated by 77% and 135%, respectively. Results suggest that decrease in TRPC1 may be compensated by upregulated TRPC6 that possibly takes part in store-operated Ca2+ entry in vascular smooth muscle cells.