Horizon College and Seminary

Cystathionine γ-lyase (CSE) is a key enzyme in the trans-sulfuration pathway. CSE uses L-cysteine as a substrate to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we used CSE stably overexpressed HEK-293 cells to explore the effect of the CSE/H2S system on cell growth and proliferation. The overexpression of CSE resulted in increases in CSE mRNA levels, CSE proteins, and intracellular H2S production rates, as well as the inhibition of cell proliferation and DNA synthesis. These effects were accompanied by a sustained ERK activation and up-regulation of the cyclin-dependent kinase inhibitor p21Cip/WAK-1. Blocking the action of ERK with U0126 inhibited the induction of p21Cip/WAK-1, suggesting that ERK activation functions upstream of p21Cip/WAK-1 activation to initiate the CSE overexpression-induced cell growth inhibition. The antiproliferative effect of CSE is likely mediated by endogenously produced H2S because the H2S scavenger methemoglobin (10 μm) significantly decreased the H2S production rate and reversed the antiproliferative effect afforded by CSE. Exogenous H2S (100 μm) also inhibited cell proliferation. However, the other CSE-catalyzed products, ammonium and pyruvate, failed to inhibit cell proliferation. Methemoglobin also abolished the inhibitory effect of exogenous H2S on cell proliferation. Moreover, exogenous H2S induced a sustained ERK and p21Cip/WAK-1 activation. These findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival. Cystathionine γ-lyase (CSE) is a key enzyme in the trans-sulfuration pathway. CSE uses L-cysteine as a substrate to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we used CSE stably overexpressed HEK-293 cells to explore the effect of the CSE/H2S system on cell growth and proliferation. The overexpression of CSE resulted in increases in CSE mRNA levels, CSE proteins, and intracellular H2S production rates, as well as the inhibition of cell proliferation and DNA synthesis. These effects were accompanied by a sustained ERK activation and up-regulation of the cyclin-dependent kinase inhibitor p21Cip/WAK-1. Blocking the action of ERK with U0126 inhibited the induction of p21Cip/WAK-1, suggesting that ERK activation functions upstream of p21Cip/WAK-1 activation to initiate the CSE overexpression-induced cell growth inhibition. The antiproliferative effect of CSE is likely mediated by endogenously produced H2S because the H2S scavenger methemoglobin (10 μm) significantly decreased the H2S production rate and reversed the antiproliferative effect afforded by CSE. Exogenous H2S (100 μm) also inhibited cell proliferation. However, the other CSE-catalyzed products, ammonium and pyruvate, failed to inhibit cell proliferation. Methemoglobin also abolished the inhibitory effect of exogenous H2S on cell proliferation. Moreover, exogenous H2S induced a sustained ERK and p21Cip/WAK-1 activation. These findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival. The endogenous production of hydrogen sulfide (H2S) and its physiological functions, including membrane hyperpolarization and smooth muscle cell relaxation, place this gas in the family of gas transmitters, together with nitric oxide and carbon monoxide (1Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1549) Google Scholar, 2Wang R. Antioxid. Redox Signal. 2003; 5: 493-501Crossref PubMed Scopus (429) Google Scholar). Two pyridoxal-5′-phosphate-dependent enzymes, cystathionine β-synthase (EC 4.2.1.22) and cystathionine γ-lyase (CSE) 1The abbreviations used are: CSE, cystathionine γ-lyase; cdk, cyclin-dependent kinase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; EGFP, enhanced green fluorescent protein; HEK, human embryonic kidney; MAPK, mitogen-activated protein kinase; PCR, polymerase chain reaction. (EC 4.4.1.1), are responsible for the endogenous production of H2S in mammalian tissues, which use L-cysteine as the main substrate (3Kery V. Bukovska G. Kraus J.P. J. Biol. Chem. 1994; 269: 25283-25288Abstract Full Text PDF PubMed Google Scholar, 4Erickson P.F. Maxwell I.H. Su L.J. Baumann M. Glode L.M. Biochem. J. 1990; 269: 335-340Crossref PubMed Scopus (112) Google Scholar, 5Stipanuk M.H. Beck P.W. Biochem. J. 1982; 206: 267-277Crossref PubMed Scopus (616) Google Scholar). Cystathionine β-synthase is a predominant H2S-generating enzyme in the brain and nervous system (6Eto K. Kimura H. J. Neurochem. 2002; 83: 80-86Crossref PubMed Scopus (71) Google Scholar), and CSE is mainly expressed in liver, kidney, and vascular smooth muscles (7Barber T. Triguero A. Martinez-Lopez I. Torres L. Garcia C. Miralles V.J. Vina J.R. J. Nutr. 1999; 129: 928-933Crossref PubMed Scopus (29) Google Scholar, 8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar). Cystathionine β-synthase and CSE are important for the metabolism of sulfur-containing amino acids (e.g. cystathionine), as well as the production of H2S, ammonium, and pyruvate from L-cysteine. H2S inhibits cell proliferation (9Valitutti S. Castellino F. Musiani P. Ann. Allergy. 1990; 65: 463-468PubMed Google Scholar) and induces cell death predominantly by an apoptotic mechanism in polymorphonuclear cells (10Mariggio M.A. Minunno V. Riccardi S. Santacroce R. De Rinaldis P. Fumarulo R. Immunopharmacol. Immunotoxicol. 1998; 20: 399-408Crossref PubMed Scopus (57) Google Scholar). H2S treatment has also been shown to lead to nasal lesions and olfactory epithelial necrosis (11Brenneman K.A. Meleason D.F. Sar M. Marshall M.W. James R.A. Gross E.A. Martin J.T. Dorman D.C. Toxicol. Pathol. 2002; 30: 200-208Crossref PubMed Scopus (37) Google Scholar). On the other hand, H2S induces serum-independent cell cycle entry in rat intestinal epithelial cells and increases the fraction of colonic mucosa cells in the S phase (12Eplancke B. Gaskins H.R. FASEB J. 2003; 17: 1310-1312Crossref PubMed Scopus (149) Google Scholar, 13Hristl S.U. Eisner H.D. Dusel G. Kasper H. Scheppach W. Dig. Dis. Sci. 1996; 41: 2477-2481Crossref PubMed Google Scholar). However, little is known about the cellular consequences of an elevated CSE expression or about the associated increase in endogenously produced H2S. In the present study, we overexpressed the CSE gene using a highly effective expression system. The successful overexpression of CSE was confirmed by measuring the CSE protein contents, CSE mRNA expression level, and endogenous H2S production rate. Proliferation of the transfected HEK-293 cells was monitored. We determined whether the CSE overexpression-induced cellular changes were because of overproduced H2S. Finally, the effect of both the cyclin-dependent kinase (cdk) inhibitor p21Cip/WAK-1 and the ERK/mitogen-activated protein kinase (MAPK) pathway on the CSE/H2S system was examined. Our findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival. Cell Culture and Measurement of H2S Production—HEK-293 cells (American Type Culture Collection, Manassas, VA) were cultured at 37 °C in a humidified incubator with 95% air and 5% CO2 in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 100 units of penicillin, and 100 μg of streptomycin/ml. The cultured cells were subjected to gene transfection when they had grown to 70–80% confluence. The H2S production rate in CSE stably transfected HEK-293 cells was measured as described previously (8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar). Cloning of CSE cDNA and Stable Transfection—PCR was used to amplify the open reading frame of CSE (GenBank™ accession number AY032875) from rat vascular tissues using the primers 5′-CGTCCCAGCATGCAGAAGAA-3′ and 5′-CAGTTATTCAGAAGGTCTGGCCC-3′. The amplified open reading frame of CSE was subcloned into TA cloning vector (PCR4-TOPO). Positive clone containing CSE open reading frame insert was sequenced to confirm the accuracy of the inserted CSE sequence. The constructs containing CSE cDNA were cleaved and subcloned into the mammalian expression vector pIRES2-EGFP (Clontech), which contained the human cytomegalovirus immediate early promoter/enhancers and the SV40 poly(A) signal. For stable transfection, the constructs were linearized with KpnI (MBI Fermentas) and then subjected to phenol-chloroform extraction and ethanol precipitation. Linearized constructs were mixed with a FuGENE 6 transfection reagent (Roche Applied Science) in a ratio of 1 μg to 3 μl in 100 μl of FBS-free minimal essential medium (14Cao K. Tang G. Hu D. Wang R. Biochem. Biophys. Res. Commun. 2002; 296: 463-469Crossref PubMed Scopus (43) Google Scholar). After incubating for 45 min at room temperature (20–22 °C), the mixture was added to HEK-293 cells in 2 ml of FBS-free minimal essential medium. After 48 h of transfection, the cells were trypsinized, counted, and replated at 1 × 105 cells/plate in 35-mm plates, which contained 500 μg/ml G418 for antibiotic selection. Mock (empty vector) transfection was also performed. In the pIRES2-EGFP vector, the EGFP gene (which encodes the enhanced green fluorescent protein) was expressed separately from the gene of interest and was used as a transfection marker. Non-transfected HEK-293 cells were included as the negative control for antibiotic selection. After 5 weeks of the antibiotic selective culturing, survival-transfected cells were harvested and grown to establish the sublines, which were subsequently examined for the presence of the CSE gene. Stable transfectants were used at passage numbers <15. Wild-type HEK-293 cells were maintained in identical conditions but without selection antibiotic G418 treatment. Western Immunoblotting—Cultured cells (3 × 106) were harvested and lysed in a lysis buffer (0.5 m EDTA, 1 m Tris-Cl, pH 7.4, 0.3 m sucrose, 1 μg/ml antipain hydrochloride, 1 mm benzamidine hydrochloride hydrate, 1 μg/ml leupeptin hemisulfate, 1 mm 1,10-phenanthroline monohydrate, 1 μm pepstatin A, 0.1 mm phenylmethylsulfonyl fluoride, and 1 mm iodoacetamide). The extracts were clarified by centrifugation at 14,000 × g for 15 min at 4 °C. SDS-PAGE and Western blot analysis were performed as described previously (15Sun X. Cao K. Yang G. Huang Y. Hanna S.T. Wang R. Biochem. Pharmacol. 2004; 67: 147-156Crossref PubMed Scopus (33) Google Scholar). The primary antibody dilutions were 1:1000 for phosphorylated or total ERK, p38 MAPK, and c-Jun NH -terminal kinase, 1:500 for p21Cip/WAF-1 and cyclin D1, and 1:5000 for β-actin. Horseradish peroxidase-conjugated secondary antibody was used at 1:5000. The immunoreactions were visualized by ECL and exposed to x-ray film (Kodak Scientific Imaging film, X-Omat Blue XB-1). Membranes were stripped by incubation in a buffer containing 100 mm β-mercaptoethanol, 2% SDS, and 62.5 mm Tris-HCl, pH 6.8. mRNA Collection and Reverse Transcription—Total cellular RNA from wild-type, mock, or CSE-transfected cells was harvested after the cells were plated for 48 h. Monolayers were rinsed twice with phosphate-buffered saline (pH 7.6), and RNA was collected using TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH). Contaminated DNA was removed using the DNA-free kit (Ambion, Austin, TX), and total RNA (2 μg) was reverse transcribed into cDNA with avian myeloblastosis virus reverse transcriptase using random hexamer primers according to the manufacturer's protocol (Roche Applied Science). Controls without reverse transcriptase were used to check for genomic DNA contamination in each sample. Real Time Quantitative PCR—Real time PCR was performed in an iCycler iQ apparatus (Bio-Rad) associated with the iCycler optical system software (version 3.1) using SYBR Green PCR Master Mix. All PCRs were performed in a 20-μl volume using 96-well optical grade PCR plates and optical sealing tape. Negative controls for this experiment were samples without a template. The cycling conditions were 95 °C for 90 s followed by 38 cycles of 95 °C for 10 s and 60 °C for 20 s. For quantification, the target gene was normalized to the internal standard gene β-actin. The primers of CSE (GenBank™ accession number AY032875) were 5′-AGCGATCACACCACAGACCAAG-3′ (sense, position and position These primers produced a of The primers of produced a of standard was with a of dilutions of total RNA transcribed to cDNA using the protocol to confirm the in the standard analysis was performed using a cycling that at 95 °C for 10 to °C for 15 and then to 95 °C in to confirm the of was determined by PCR on a mRNA was by using the P. V. G. A. D. Chem. 2004; PubMed Scopus Google Scholar, L.J. Huang J. 2003; 41: PubMed Scopus Google Scholar), is the the cycle of a target cDNA and an endogenous this the of the target normalized to an endogenous Measurement of was performed to the cell growth in CSE-transfected HEK-293 an number of cells (2 × were in 35-mm and then cells were using a The medium was 3 DNA was by the of number of cells in the plates (2 × were cultured in growth medium for 48 h. were with medium and then in the medium for h for After the cells were with 10% in minimal essential medium for 6 0.1 was added to each well in growth medium for 6 h. The medium was and the cells were with ml of phosphate-buffered saline containing 1 mm and 1 mm The cells were with ml of 5% for 10 and then the were lysed with ml of SDS, 0.1 of cell were into and mixed with 5 ml of was determined using a and was by with H2S gas to the H2S m at (8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar). H2S was to different in cell and the pH of the medium was to The CSE antibody was by The protein signal-regulated kinase inhibitor and p21Cip/WAK-1 antibody were from antibody was from was from ammonium and pyruvate were from Horseradish peroxidase-conjugated antibody was from are expressed as and at were using the or analysis of followed by a analysis The of was at of CSE cDNA in HEK-293 cells were transfected with a CSE or an identical vector a cDNA insert as a control The transfection of HEK-293 cells with CSE cDNA or the control vector the of the In HEK-293 cells transfected with CSE cDNA or the control vector, green by green fluorescent protein was In green was in HEK-293 cells The expression of CSE was by Western blot time PCR and the H2S production rate. Western blot and time PCR analysis that transfection of CSE but of the resulted in increases in both CSE protein expression and CSE mRNA with HEK-293 cells and of HEK-293 cells with CSE cDNA also resulted in a increase in the H2S production rate with cells Proliferation and DNA in CSE-transfected HEK-293 whether overexpression of the CSE gene proliferation and DNA of HEK-293 cell growth were with or HEK-293 cells transfected with CSE cDNA a significantly cell growth rate on the the the number of cells was of the cells antiproliferative effect of CSE overexpression was also by the of shown in HEK-293 cells transfected with CSE cDNA had cells transfected with or cells of and of p21Cip/WAK-1 by CSE including ERK, p38 MAPK, and c-Jun kinase, an important role in the proliferation and of of HEK-293 cells with CSE but the resulted in a of phosphorylated ERK and p38 the of c-Jun kinase total of were cells with 10 μm U0126 or 20 μm p38 for h significantly decreased the expression of phosphorylated ERK and p38 p21Cip/WAF-1 is a inhibitory and cell cycle by with cyclin and or to a cell S. D. B. 1994; PubMed Scopus Google Scholar, J. M.W. A. PubMed Scopus Google Scholar). p21Cip/WAF-1 expression is an important for cell growth A. B. H. Biol. 17: PubMed Scopus Google Scholar, D. D. H. M. Biol. 17: PubMed Scopus Google Scholar, X. Huang D. Y. C. Wang J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in the expression of p21Cip/WAF-1 significantly in CSE-transfected HEK-293 cells that in or the of activation with CSE p21Cip/WAF-1 we the effects of U0126 and on the expression of After incubating cells with 10 μm the expression of p21Cip/WAF-1 in CSE-transfected HEK-293 cells decreased to the level, μm) had little that p21Cip/WAF-1 induction by CSE overexpression may from activation of the ERK pathway. is an of and which the rate of cell cycle V. P. M. J. C. Res. 2003; PubMed Scopus (29) Google Scholar). p21Cip/WAF-1 inhibit cyclin by to cyclin D1, cyclin T. S. Y. M. Biol. 2004; PubMed Scopus Google Scholar). Our that cyclin expression was in of and U0126 and had little effect on cyclin expression of CSE on Cell Proliferation to the mechanism by which CSE overexpression inhibited cell we the effects of H2S, ammonium, and pyruvate, of CSE-catalyzed on cell HEK-293 cells were with 10% in the presence of H2S (100 DNA decreased However, 100 μm ammonium and 100 μm pyruvate failed to inhibit DNA we the effect of the H2S scavenger methemoglobin (1Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1549) Google Scholar, T. Res. 2002; PubMed Scopus Google Scholar) on CSE cell growth inhibition. shown in methemoglobin at 10 μm but significantly reversed the antiproliferative effect of CSE HEK-293 cells with 10 μm methemoglobin for 1 h to 100 μm H2S significantly abolished the antiproliferative effect of H2S H2S production in cells by methemoglobin also that methemoglobin the endogenous H2S the role of exogenous H2S on and cell cycle protein we the of ERK and p21Cip/WAF-1 in HEK-293 cells after to 100 μm H2S. The expression of ERK and p21Cip/WAF-1 after incubating HEK-293 cells with H2S for 2 h that H2S likely the antiproliferative effect of H2S increases the ERK and p21Cip/WAK-1 The HEK-293 cells were cultured in the presence or of H2S (100 μm) for the and Western blot analysis was used to the activation of ERK and p21Cip/WAK-1 The the ratio of phosphorylated ERK optical to total ERK optical to from Our present that the CSE expression is an important for cell the endogenous production of H2S, the overexpression of CSE significantly inhibited cell proliferation. In this of ERK and p21Cip/WAF-1 were by H2S, to cell growth inhibition. CSE is a key enzyme of the trans-sulfuration which and L-cysteine. also uses L-cysteine as an substrate to H2S P.F. Maxwell I.H. Su L.J. Baumann M. Glode L.M. Biochem. J. 1990; 269: 335-340Crossref PubMed Scopus (112) Google Scholar). a CSE is expressed in a of mammalian cells and tissues, and to the main enzyme in the liver, kidney, and system (7Barber T. Triguero A. Martinez-Lopez I. Torres L. Garcia C. Miralles V.J. Vina J.R. J. Nutr. 1999; 129: 928-933Crossref PubMed Scopus (29) Google Scholar, 8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar). of in as which are by of and early of (9Valitutti S. Castellino F. Musiani P. Ann. Allergy. 1990; 65: 463-468PubMed Google Scholar, T. Res. 2002; PubMed Scopus Google Scholar, H. X. Yang F. L. Yang X. W. 2003; PubMed Scopus Google Scholar). on H2S on the the physiological of the the functions of H2S, including hyperpolarization of cell of smooth muscle and decreased (1Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1549) Google Scholar, 2Wang R. Antioxid. Redox Signal. 2003; 5: 493-501Crossref PubMed Scopus (429) Google Scholar, K. Kimura H. J. Neurochem. 2002; 83: 80-86Crossref PubMed Scopus (71) Google Scholar, 8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar, R.A. J. 2004; PubMed Scopus Google Scholar, W. Wang R. J. Pharmacol. 2003; PubMed Scopus Google Scholar). is known about the effect of endogenous CSE/H2S on cell growth and proliferation. In the present study, we used CSE stably transfected HEK-293 cells to explore the effects of CSE overexpression on cell growth and proliferation. Our that the CSE overexpression intracellular H2S inhibition of cell and DNA 1 and CSE induced the expression of inhibitor p21Cip/WAF-1 sustained ERK suggesting a cell cycle The antiproliferative effect of CSE is likely mediated the of H2S because the H2S scavenger methemoglobin (1Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1549) Google Scholar, T. Res. 2002; PubMed Scopus Google Scholar) and significantly reversed the antiproliferative effect of CSE Exogenous H2S also inhibited cell by decreased The other CSE products, ammonium and pyruvate, failed to inhibit cell proliferation. of HEK-293 cells with methemoglobin for 1 h to the of H2S abolished the antiproliferative effect of H2S Moreover, exogenous H2S induced sustained ERK and p21Cip/WAF-1 activation These findings support the hypothesis that endogenously produced H2S an important role in cell proliferation and survival. The mitogen-activated protein kinase a role in extracellular into growth and The ERK pathway is in the of cellular proliferation J. Sci. S. A. PubMed Scopus Google Scholar, C. J. A. M. V. D. 2004; PubMed Scopus Google Scholar, F. T. Y. S. T. J. 2003; PubMed Scopus Google Scholar), growth inhibition has also been T. Sci. S. A. PubMed Scopus Google Scholar, S. L. R. C. R. Biochem. Pharmacol. 2003; 65: PubMed Scopus Google Scholar). we that a sustained increase in ERK induced by CSE overexpression to inhibited cell growth The by the that the pathway in both and inhibition of cell proliferation D. D. H. M. Biol. 17: PubMed Scopus Google Scholar, Sci. S. A. PubMed Scopus Google Scholar). The cellular cell cycle or cellular to activation of the pathway on the and of the signal. or activation may to cell cycle sustained of ERK may lead to cell growth inhibition. in the has been that the and of activation changes in gene which in whether a cell the cell cell cycle or p38 was also in CSE-transfected may in CSE overexpression-induced p21Cip/WAF-1 up-regulation and because the inhibition of p38 by the expression of p21Cip/WAF-1 is of the inhibitory proteins, and an important role in growth cellular DNA cell and Biophys. Google Scholar, 1999; PubMed Scopus Google Scholar). p21Cip/WAF-1 expression is also an important for cell growth A. B. H. Biol. 17: PubMed Scopus Google Scholar, D. D. H. M. Biol. 17: PubMed Scopus Google Scholar, H. Zhang L. S. F. D. B. 2004; PubMed Scopus Google Scholar, M.W. Lu K. J. PubMed Scopus Google Scholar). Our that the inhibition of growth by CSE overexpression in HEK-293 cells was accompanied by an induction of the inhibitor which was on the ERK activation p21Cip/WAF-1 ERK and the inhibition of p21Cip/WAF-1 by U0126 confirmed that p21Cip/WAF-1 induction after ERK activation. In the of the cell activation of has been V. P. M. J. C. Res. 2003; PubMed Scopus (29) Google Scholar). The kinase of is by as p21Cip/WAF-1 has been shown to with and DNA polymerase a cell S. D. B. 1994; PubMed Scopus Google Scholar). Blocking the activation of ERK the induction of p21Cip/WAF-1 suggesting that ERK activation is with CSE overexpression-induced up-regulation of p21Cip/WAF-1 and cell growth inhibition. that sustained ERK activation for the induction of cyclin A. B. H. Biol. 17: PubMed Scopus Google Scholar, D. D. H. M. Biol. 17: PubMed Scopus Google Scholar). p21Cip/WAF-1 inhibit cyclin activation and cyclin T. S. Y. M. Biol. 2004; PubMed Scopus Google Scholar). However, that cyclin was in CSE overexpression-induced cell growth inhibition. ERK and p21Cip/WAF-1 likely play in CSE cell growth is also that other that were are the sustained ERK activation and expression of p21Cip/WAF-1 to In this for the time that CSE cell proliferation a of ERK and p21Cip/WAK-1. the of the CSE/H2S system to inhibit cell growth that to CSE expression and H2S production may a in the treatment of CSE/H2S

Abstract Recognizing ECG cardiac arrhythmia automatically is an essential task for diagnosing the abnormalities of cardiac muscle. The proposal of few algorithms has been made for classifying the ECG cardiac arrhythmias, however the system of classification efficiency is determined on the basis of its prediction and diagnosis accuracy. Hence, in this study the proposal of an efficient system has been made for classifying the ECG cardiac arrhythmia as an expertise. Discrete Wavelet Transform (DWT) is being utilized for the preprocessing mechanism of ECG signal, Independent Component Analysis (ICA) is being utilized for dimensionality reduction and Feature Extraction process of ECG signal and Multi-Layer Perceptron (MLP) neural network is being utilized for performing the task of classification. As an outcome of classification, the results have been acquired on categorizing Normal Beats under the class of Non-Ectopic beat, Atrial Premature Beat under the class of Supra-Ventricular ectopic beat and Ventricular Escape beat under the class of Ventricular ectopic beat on the basis of standardization given by ANSI/AAMI EC57: 1998. For the acquisition of ECG signal, MIT-BIH physionet arrhythmia database is being utilized in this study added to that its being utilized for training process and testing process of the classifier on the basis of MLP-NN. The results obtained from the simulation has been inferred that the accuracy of classification of the proposed algorithm is 96.50% on utilizing 10 files inclusive of normal beats, Atrial Premature Beat and Ventricular Escape beat.

The development of new 1,3,5-triazine-based pyrazole derivatives as effective anticancer agents.

The present study utilized the catalytic pyrolysis method for extracting oil from waste high-density polyethylene (WHDPE) plastics. Experiments were carried out with D70H30 (diesel-70%, WHDPE oil-30%) blend under the influence of three start of pilot injection (SoPI) timings [45° before top dead center (bTDC), 50°bTDC, and 55°bTDC] and three pilot fuel injection quantity (PFIQ) (10%, 20%, and 30%) at the engine’s rated power output. Later, the impact of exhaust gas recirculation (EGR) (0%, 10%, and 20%) was studied with the optimum SoPI and PFIQ. Experimental results indicated that at SoPI timing of 55°bTDC and at PFIQ of 30%, the brake thermal efficiency (BTE) increased by 8.5%, nitrogen oxides (NOx) increased by 19.25%, while smoke, hydrocarbons (HC), and CO emission got lowered with baseline operation of the blend and found to be best among other modifications. In the next phase of the work, exhaust gas is recirculated at 10% and 20%. The results portray that inclusion of 10% EGR in the intake at SoPI 55°bTDC and at PFIQ 30% has shown 1.7% higher BTE, with 3.7% and 28.9% lower NOx and smoke emission compared to the single main injection of the blend. It is concluded that pilot injection strategy with 30% PFIQ and SoPI timing of 55°bTDC at 10% EGR rate can be adopted to utilize WHDPE oil/diesel in diesel engines effectively.

Plant disease is defined as an abnormal physiological process that distorts the plant's normal structure, growth and function. Disease reduces quality as well as quantity of the crops which in turn affects the economy of country like Bangladesh where agriculture is the main occupation. Since Rice is the major crop, classification of disease in paddy is very important as it prevents the losses in the yields and quantity. Classification of rice disease includes visually observable patterns and color of the affected portion. Manual observation of patterns and colors to classify the diseases require excessive work and appears to be less useful while dealing with non-native diseases. This paper presents a new technique to detect and classify the diseases based on percentage of RGB value of the affected portion using image processing. Once the percentage of RGB from the affected region is extracted and grouped into various classes, they are fed to a simple classifier called Naive Bayes which classifies the disease into various categories. This technique has successfully detected and identified three rice diseases namely rice brown spot, rice bacterial blight, and rice blast. This technique is efficient and faster because it uses only one feature i.e. RGB values of the affected portion which requires minimum computation time to identify and classify the diseases. Rather than processing the whole leaf, this technique even successfully detects the diseases using only a small sample of leaf containing the affected portion for rice disease.

BACKGROUND: Earlier detection of oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMD) is essential for dental professionals to improve patient survival rates. The aim of this systematic review is to to evaluate the effectiveness of devices that utilise the principles of chemiluminescence and tissue autofluorescence as adjuncts in the detection of OSCC and OPMD. MATERIAL AND METHODS: The electronic retrieval systems and databases searched for relevant articles were PubMed [MEDLINE] and Science direct. The search was for limited articles published in English or with an English abstract and articles published during the period from January 2005 to April 2014. Clinical trials utilized ViziLite, Microlux TM/DL and Visual Enhanced Light scope (VELscope) for early detection of OPMD and OSCC. RESULTS: Twenty primary studies published satisfied our criteria for selection - 10 utilised chemiluminescence and 10 tissue autofluorescence. Senstivity of Vizilite for detecting OSCC nad OPMD ranged from 77.1 % to 100% and specificity was low that ranged from 0% to 27.8%.Most have shown that chemiluminescence increases the brightness and margins of oral mucosal white lesions and thus assist in identification of mucosal lesions not considered under Conventional visual examination. However, it preferentially detects leukoplakia and may fail to spot red patches. Clinical trials demonstrated that sensitivity of VELscope in detecting malignancy and OPMD ranged from 22 % to 100 % and specificity ranged from 16 % to 100%. Most studies concluded that VELscope can help the experienced clinician to find oral precursor malignant lesions. But it couldnot differentiate between dysplasia and benign inflammatory conditions. CONCLUSIONS: Both devices are simple, non-invasive test of the oral mucosa but are suited for clinicians with sufficient experience and training. More clinical trials in future should be conducted to establish optical imaging as an efficacious adjunct tool in early diagnosis of OSCC and OPMD.

BACKGROUND: Plasma fractionators have implemented many improvements over the past decade directed toward reducing the likelihood of pathogen transmission by purified blood products, yet little has been published attempting to assess the overall impact of these improvements on the probability of safety of the final product. STUDY DESIGN AND METHODS: Safety margins for human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), hepatitis A virus (HAV), parvovirus B19, and variant form of Creutzfeldt-Jakob disease (vCJD) were calculated for the two fibrin sealants licensed in the United States and for thrombin. These products were selected because their use in a clinical setting is, in most cases, optional, and both were relatively recently approved for marketing by the US Food and Drug Administration (FDA). Moreover, thrombin and fibrinogen both undergo two dedicated virus inactivation steps and/or removal steps in accord with the recommendations of regulatory agencies worldwide. Safety margins were determined by comparing the potential maximum viral loads in contaminated units to viral clearance factors, ultimately leading to the calculation of the residual risk per vial. RESULTS: The residual risk of pathogen transmission per vial was calculated to be less than 1 in 10(-15) for HIV, HCV, HBV, and HAV for both fibrinogen and thrombin. Owing to the greater quantities that can be present and its greater thermal stability, the calculated risk for parvovirus transmission was 1 in 500,000 vials for fibrinogen and less than 1 in 10(7) per vial for thrombin. Assuming that vCJD is found to be present in plasma donations, its risk of transmission by these purified and processed plasma derivatives would appear to be very low. CONCLUSIONS: The pathogen safety initiatives implemented by plasma fractionators over the past 10 to 20 years have resulted in products with excellent pathogen safety profiles. Of the agents examined, parvovirus continues to have the lowest calculated margin of safety. Despite this, parvovirus transmissions should be rare. Manufacturers are encouraged to continue exploring processes to further enlarge parvovirus safety margins and to continue exploring ways of eliminating prions.

An artificial intelligence (AI) program called ChatGPT that generates text in response to typed commands has proven to be highly popular, as evidenced by the fact that OpenAI makes it available online. The goal of the present investigation was to investigate ChatGPT's potential applications as an outstanding instance of large language models (LLMs) in the fields of public dental health schooling, writing for academic use, research in public dental health, and clinical practice in public dental health based on the available data. Importantly, the goals of the current review included locating any drawbacks and issues that might be connected to using ChatGPT in the previously mentioned contexts in healthcare settings. Using search phrases including chatGPT, implications, artificial intelligence (AI), public health dentistry, public health, practice in public health dentistry, education in public health dentistry, academic writing in public health dentistry, etc., a thorough search was carried out on the Pubmed database, the Embase database, the Ovid database, the Global Health database, PsycINFO, and the Web of Science. The dates of publication were not restricted. Systematic searches were carried out for all publications according to inclusion and exclusion criteria between March 31, 2018, and March 31, 2023. Eighty-four papers were obtained through a literature search using search terms. Sixteen similar and duplicate papers were excluded and 68 distinct articles were initially selected. Thirty-three articles were excluded after reviewing abstracts and titles. Thirty-five papers were selected, for which full text was managed. Four extra papers were found manually from references. Thirty-nine articles with full texts were eligible for the study. Eighteen inadequate articles are excluded from the final 21 studies that were finally selected for systemic review. According to previously published studies, ChatGPT has demonstrated its effectiveness in helping scholars with the authoring of scientific research and dental studies. If the right structures are created, ChatGPT can offer suitable responses and more time to concentrate on the phase of experimentation for scientists. Risks include prejudice in the training data, undervaluing human skills, the possibility of fraud in science, as well as legal and reproducibility concerns. It was concluded that practice considering ChatGPT's potential significance, the research's uniqueness, and the premise-the activity of the human brain-remains. While there is no question about the superiority of incorporating ChatGPT into the practice of public health dentistry, it does not, in any way, take the place of a dentist since clinical practice involves more than just making diagnoses; it also involves relating to clinical findings and providing individualized patient care. Even though AI can be useful in a number of ways, a dentist must ultimately make the decision because dentistry is a field that involves several disciplines.

INTRODUCTION: Aloe vera is well known for its medicinal properties which lead to its application in treating various diseases. Its use in treating oral lesions has not been much documented in literature. AIM: Although, systematic reviews on aloe vera and its extracts have been done earlier, but in relation to oral diseases this is the first systematic review. The aim of the present systematic review was to compile evidence based studies on the effectiveness of Aloe vera in treatment of various oral diseases. MATERIALS AND METHODS: Computerized literature searches were performed to identify all published articles in the subject. The following databases were used: PUBMED [MEDLINE], SCOPUS, COCHRANE DATABASE, EMBASE and SCIENCE DIRECT using specific keywords. The search was limited to articles published in English or with an English Abstract. All articles (or abstracts if available as abstracts) were read in full. Data were extracted in a predefined fashion. Assessment was done using Jadad score. RESULTS: Fifteen studies satisfied the inclusion criteria. Population of sample study ranged from 20 patients to 110 patients with clinically diagnosed oral mucosal lesions. Out of 15 studies, five were on patients with oral lichen planus, two on patients with oral submucous fibrosis, other studies were carried on patients with burning mouth syndrome, radiation induced mucositis, candida associated denture stomatitis, xerostomic patients and four were on minor recurrent apthous stomatitis. Most studies showed statistically significant result demonstrating the effectiveness of Aloe vera in treatment of oral diseases. CONCLUSION: Although there are promising results but in future, more controlled clinical trials are required to prove the effectiveness of Aloe vera for management of oral diseases.

Background: With introduction of many herbal medicines, dentistry has recently evidenced shift of approach for treating many inflammatory oral diseases by using such modalities. Aloe vera is one such product exhibiting multiple benefits and has gained considerable importance in clinical research recently. Aims: To compare the efficacy of Aloevera and Chlorhexidine mouthwash on Periodontal Health. Material and Methods: Thirty days randomized controlled trial was conducted among 390 dental students. The students were randomized into two intervention groups namely Aloe Vera (AV) chlorhexidine group (CHX) and one control (placebo) group. Plaque index and gingival index was recorded for each participant at baseline, 15 days and 30 days. The findings were than statistically analyzed, ANOVA and Post Hoc test were used. Results: There was significant reduction (p<0.05) in the mean scores of all the parameters with Aloe Vera (AV) and chlorhexidine group. Post hoc test showed significant difference (p<0.000) in mean plaque and gingival index scores of aloe Vera and placebo and chlorhexidine and placebo group. No significant difference (p<0.05) was observed between AloeVera and chlorhexidine group. Conclusions: Being an herbal product AloeVera has shown equal effectiveness as Chlorhexidine. Hence can be used as an alternative product for curing and preventing gingivitis.

Abstract Education and Health care sectors are two predominant areas where societal growth is expected through innovation and technology development. Machine Learning and Deep learning classification models have been entertained in predicting, detecting, and diagnosing major diseases in the early stage. In this research paper, we have analyzed the impact of hidden dense layers in the Convolution neural network to improve the performance of the classification model. Three different classification deep network models have been constructed, analyzed and the result was tested with a diabetes dataset. Results concluded that the more layers with deeper the network better was the classification performance. The classification model with six hidden dense layers outperforms all other less number of hidden dense layers.

Abstract Over the past ten years, autonomous driving has garnered a great deal of interest from both the scientific community and business. Strong technological advancements have made automated driving more practical because human driving abilities seem limited in terms of driving experience, reaction time, and the effectiveness of real‐time decisions. The development of highly autonomous driving algorithms is inextricably tied to planning and changing a vehicle path that must be user‐acceptable, efficient, and collision‐free. Path planning for road vehicles is a difficult problem due to the high speed involved and the requirement to assure passenger safety. Here, a new path‐planning method is developed for both connected and disconnected automatic road vehicles on multilane highways. This paradigm states that the right phrases to describe the objectives of vehicle improvement, passenger comfort, prevention of vehicle‐to‐vehicle collisions and road deviations are included in the objective function. Hunger Games improved Archimedes optimization (HGE‐ARCO) is used to optimize the paths for achieving better‐planned outcomes. At the 100th penetration rate, the HGE‐ARCO scheme reached a top speed of about 99 km/h. The results shows unmistakably that the proposed HGE‐ARCO produces a time of 12.3021 s, which is less than other conventional methods.

Docking studies, structural data of DNA binding and molecular dynamics simulations of substituted quinazolin-4(1<italic>H</italic>)-ones.

Photo physical properties of fluorescent organic compounds give an immense improved knowledge on characteristics of excited state that is beneficial to devise innovate molecules and understand their performance in particular applications. Coumarin derivatives have been extensively investigated in this regard. This article narrates steady state fluorescence quenching measurements of a coumarin derivative namely 3-hydroxy-3-[2-oxo-2-(3-oxo-3H-benzo[f]chromen-2-yl)-ethyl]-1,3-dihydro-indol-2-one (3HBCD) in a binary mixture of acetonitrile and 1,4-dioxane. Aniline is used as quencher. Fluorescence intensity is large in acetonitrile and decreases as the percentage of 1,4-dioxane in the solvent mixture increases. With modest quencher concentration a deviation towards the x axis is noticed in the Stern-Volmer (S-V) plots. This downward curvature is interpreted as due to the presence of 3HBCD in different conformers in the lowest energy level. Ground state intramolecular hydrogen bonding formation is observed due to the conformational changes in the solute. Figured estimations of various quenching parameters recommend that, while dynamic quenching prompts linearity in S-V plot at lower quencher concentration, increasing quenching efficiency with increasing medium viscosity suggests that reaction is not entirely controlled by material diffusion. Stern-Volmer constant increases with decreasing medium dielectric constant.

Summary Non‐orthogonal multiple access (NOMA) is a great contender for future cellular modulation due to its desirable properties like massive connectivity, high data rate transmission, and high spectral efficiency. However, its peak‐to‐average power ratio (PAPR) is significant, which becomes a significant disadvantage for the efficient operability of the NOMA waveform compared to current techniques. Several PAPR reduction algorithms like selective mapping (SLM), partial transmission sequence (PTS), and companding techniques have been proposed to lower the PAPR of multicarrier waveforms (MCWs). PTS reduces the PAPR but has high complexity. On the other hand, SLM has a less complex framework, but its PAPR performance is not as efficient as PTS. Companding methods reduce the PAPR by compressing the signals at the transmitter, which unfortunately reduces the dynamic range of the signal. In this work, we propose a hybrid algorithm (SLM + PTS) with a companding method for the first time for the NOMA waveform, which efficiently reduces the PAPR with low computational complexity. Furthermore, we compare the performances of a host of candidate algorithms like SLM, PTS, hybrid (SLM + PTS), hybrid + A law (SLM–PTS–A law), and hybrid + Mu law (SLM–PTS–Mu law). The results of the experiments show that the hybrid + Mu law did a better job than the existing PAPR reduction algorithms.

Since mid-December of 2019, coronavirus disease 2019 (COVID-19) has been spreading from Wuhan, China. As of February 21, total 75,773 confirmed cases worldwide have spread to more than two dozen countries. Transmission of COVID-19 can occur early in the course of infection since SARS-CoV-2 viral loads in asymptomatic patients are similar to that in the symptomatic patients. Therefore, more sensitive diagnostic methods are needed to detect early phase of the infection to prevent secondary or tertiary spreads. Here, we compare the RT-PCR confirmatory test results using two different SARS-CoV-2 viral RNAs from two Korean COVID-19 confirmed cases.RT-PCR method targeting the RdRP gene, which was recommended by WHO guideline, was less sensitive than targeting N genes (as per CDC guideline). Because many countries follow the WHO guideline, our findings may contribute to the early diagnosis of COVID-19.

IoT - a new proliferation in the technological advancement, changed the way object is perceived and used. It enables connecting smart objects to the internet and aims to develop new promising future to Intelligent Transportation System (ITS). ITS uses techniques such as wireless communication, computational technologies, GPS, and sensor technologies to provide smart and quick services to users and to be better informed and make safer, more coordinated, and 'smarter' use of transportation medium. As number of objects connected to ITS application increases, the amount of data generated also increases and they are send to cloud for data analysis and knowledge discovery. However, sending and retrieving of data across cloud is less useful due to delay latency and others. An alternative to cloud is fog (edge) model that overcomes the weakness of cloud by analyzing and discovering knowledge at the edge. However, the fog computing model has limited computational capability. For an IoT enabled Intelligent Transportation System with enormous number of objects connected, neither cloud nor fog computing model addresses the issues alone. This paper focuses on presenting an IoT hybrid model for Intelligent Transportation System (ITS). We also address the effectiveness of the model by discussing use case scenarios.

Three clay minerals, a kaolinite, an illite and a montmorillonite, were saturated with different cations (Li + , Na + , K + , Mg ++ , Ca ++ , Sr ++ , and Ba ++ ) before equilibration with dilute phosphorus solutions. The phosphorus adsorption data conformed to the Langmuir adsorption isotherm. The relative magnitude of the phosphorus adsorption constants (b and k) could be qualitatively predicted from the type of exchangeable cations for any one clay mineral.The patterns of the adsorption isotherms and adsorption constants, b and k, can be explained on the basis of P adsorption at the edges of the clay minerals; the possibility of precipitation of the phosphorus with divalent cations or aluminum in solution was considered but rejected.

Solar energy is the base for both photovoltaic (PV) power generation and plant growth. Inspired by this biological phenomenon, a novel crowded plant height optimisation (CPHO) algorithm was developed for solar PV maximum power point tracking (MPPT). This CPHO‐tuned MPPT algorithm was developed with the aim of obtaining the optimal duty cycle ( d ) for DC‐DC boost converter for maximum solar power extraction from PV panels with the help of a proportional‐integral controller. Crowded plants regulate the growth of their stem height in relation to neighbouring plants, also known as height convergence. Using this CPHO‐algorithm, the stable height of the plant found in a numerical value is taken as the optimal height of the plant. This optimal numerical value was converted into ( d ) for the converter. Under dynamic weather conditions, the ( d ) was optimally adjusted by the proposed algorithm to regulate the DC output of the converter. On the utility side, d–q vector control‐based voltage source inverter was used for PV power integration into the grid. The performance of the converter control strategy of the proposed CPHO algorithm was compared with perturb and observe algorithm‐based MPPT control, which was analysed on MATLAB/Simulink platform.

The agricultural sector is facing significant challenges in meeting the increasing demands for food production while ensuring sustainability and resource efficiency. To address these challenges, the integration of Internet of Things (IoT) technology into farming practices has gained attention as a promising solution. This research focuses on the development and implementation of an IoT-enabled smart farming system aimed at enhancing crop growth efficiency. The proposed system leverages IoT sensors and devices to monitor and collect real-time data on various parameters such as environmental conditions, soil moisture levels, and crop health. The collected data is then analyzed using advanced analytics techniques to gain valuable insights and make informed decisions regarding irrigation, fertilization, and pest control. By utilizing IoT technology, farmers can optimize their resource utilization, reduce waste, and maximize crop productivity. This research aims to investigate the potential benefits and challenges associated with implementing the IoT-enabled smart farming system. In this paper, a cutting-edge Internet of Things (IoT) technology is explored for monitoring weather and soil conditions for efficient crop development. The system was built to monitor temperature, humidity, and soil moisture using Node MCU and several linked sensors. Additionally, a Wi-Fi connection is used to send a notification through SMS to the farmer's phone about the field's environmental state. The results will help in developing strategies and guidelines for the widespread adoption of IoT-enabled smart farming practices, ultimately leading to sustainable and efficient crop production to meet the demands of a growing population.