Chonnam National University Hospital

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies. The Cancer Genome Atlas reports on molecular evaluation of 295 primary gastric adenocarcinomas and proposes a new classification of gastric cancers into 4 subtypes, which should help with clinical assessment and trials of targeted therapies. This contribution from The Cancer Genome Atlas (TCGA) project describes the molecular evaluation of 295 primary gastric adenocarcinomas. Based on the results, the authors propose a novel classification separating gastric cancers into four subtypes according to: Epstein–Barr virus positive status, microsatellite instability, chromosomal instability or genomic stability. Given the histologic and etiologic heterogeneity of gastric cancer identification of these subtypes, using a schema that can readily be applied to patient samples should help with patient stratification and trials of targeted therapies.

Flexible solid-state supercapacitors (FSSCs) are frontrunners in energy storage device technology and have attracted extensive attention owing to recent significant breakthroughs in modern wearable electronics. In this study, we review the state-of-the-art advancements in FSSCs to provide new insights on mechanisms, emerging electrode materials, flexible gel electrolytes and novel cell designs. The review begins with a brief introduction on the fundamental understanding of charge storage mechanisms based on the structural properties of electrode materials. The next sections briefly summarise the latest progress in flexible electrodes (i.e., freestanding and substrate-supported, including textile, paper, metal foil/wire and polymer-based substrates) and flexible gel electrolytes (i.e., aqueous, organic, ionic liquids and redox-active gels). Subsequently, a comprehensive summary of FSSC cell designs introduces some emerging electrode materials, including MXenes, metal nitrides, metal-organic frameworks (MOFs), polyoxometalates (POMs) and black phosphorus. Some potential practical applications, such as the development of piezoelectric, photo-, shape-memory, self-healing, electrochromic and integrated sensor-supercapacitors are also discussed. The final section highlights current challenges and future perspectives on research in this thriving field.

When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

Signaling by receptor activator of NF-kappaB (nuclear factor-kappaB) ligand (RANKL) is essential for differentiation of bone marrow monocyte-macrophage lineage (BMM) cells into osteoclasts. Here, we show RANKL stimulation of BMM cells transiently increased the intracellular level of reactive oxygen species (ROS) through a signaling cascade involving TNF (tumor necrosis factor) receptor-associated factor (TRAF) 6, Rac1, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) 1. A deficiency in TRAF6 or expression of a dominant-interfering mutant of TRAF6 blocks RANKL-mediated ROS production. Application of N-acetylcysteine (NAC) or blocking the activity of Nox, a protein leading to the formation of ROS, with diphenylene iodonium (DPI) inhibits the responses of BMM cells to RANKL, including ROS production, activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK), and osteoclast differentiation. Moreover, both RANKL-mediated ROS production and osteoclast differentiation were completely blocked in precursors depleted of Nox1 activity by RNA interference or by expressing a dominant-negative mutant of Rac1. Together, these results indicate that ROSs act as an intracellular signal mediator for osteoclast differentiation.

BACKGROUND: Percutaneous coronary intervention (PCI) is increasingly used to treat unprotected left main coronary artery stenosis, although coronary-artery bypass grafting (CABG) has been considered to be the treatment of choice. METHODS: We randomly assigned patients with unprotected left main coronary artery stenosis to undergo CABG (300 patients) or PCI with sirolimus-eluting stents (300 patients). Using a wide margin for noninferiority, we compared the groups with respect to the primary composite end point of major adverse cardiac or cerebrovascular events (death from any cause, myocardial infarction, stroke, or ischemia-driven target-vessel revascularization) at 1 year. Event rates at 2 years were also compared between the two groups. RESULTS: The primary end point occurred in 26 patients assigned to PCI as compared with 20 patients assigned to CABG (cumulative event rate, 8.7% vs. 6.7%; absolute risk difference, 2.0 percentage points; 95% confidence interval [CI], -1.6 to 5.6; P=0.01 for noninferiority). By 2 years, the primary end point had occurred in 36 patients in the PCI group as compared with 24 in the CABG group (cumulative event rate, 12.2% vs. 8.1%; hazard ratio with PCI, 1.50; 95% CI, 0.90 to 2.52; P=0.12). The composite rate of death, myocardial infarction, or stroke at 2 years occurred in 13 and 14 patients in the two groups, respectively (cumulative event rate, 4.4% and 4.7%, respectively; hazard ratio, 0.92; 95% CI, 0.43 to 1.96; P=0.83). Ischemia-driven target-vessel revascularization occurred in 26 patients in the PCI group as compared with 12 patients in the CABG group (cumulative event rate, 9.0% vs. 4.2%; hazard ratio, 2.18; 95% CI, 1.10 to 4.32; P=0.02). CONCLUSIONS: In this randomized trial involving patients with unprotected left main coronary artery stenosis, PCI with sirolimus-eluting stents was shown to be noninferior to CABG with respect to major adverse cardiac or cerebrovascular events. However, the noninferiority margin was wide, and the results cannot be considered clinically directive. (Funded by the Cardiovascular Research Foundation, Seoul, Korea, and others; PRECOMBAT ClinicalTrials.gov number, NCT00422968.).

OBJECTIVE: This study presents a preliminary report on the chest radiographic and computed tomography (CT) findings of the 2019 novel coronavirus disease (COVID-19) pneumonia in Korea. MATERIALS AND METHODS: As part of a multi-institutional collaboration coordinated by the Korean Society of Thoracic Radiology, we collected nine patients with COVID-19 infections who had undergone chest radiography and CT scans. We analyzed the radiographic and CT findings of COVID-19 pneumonia at baseline. Fisher's exact test was used to compare CT findings depending on the shape of pulmonary lesions. RESULTS: = 0.006). CONCLUSION: COVID-19 pneumonia in Korea primarily manifested as pure to mixed ground-glass opacities with a patchy to confluent or nodular shape in the bilateral peripheral posterior lungs. A considerable proportion of patients with COVID-19 pneumonia had normal chest radiographs.

Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicemia and necrotizing wound infections in humans. Virulent V. vulnificus isolates produce a catechol siderophore called vulnibactin, made up of one residue of 2, 3-dihydroxybenzoic acid (2, 3-DHBA) and two residues of salicylic acid (SA). Vulnibactin biosynthetic genes (VV2_0828 to VV2_0844) are clustered at one locus of chromosome 2, expression of which is significantly up-regulated in vivo. In the present study, we decipher the biosynthetic network of vulnibactin, focusing specifically on genes around SA and 2, 3-DHBA biosynthetic steps. Deletion mutant of isochorismate pyruvate lyase (VV2_0839) or 2, 3-dihydroxybenzoate-2, 3-dehydrogenase (VV2_0834) showed retarded growth under iron-limited conditions though the latter showed more significant growth defect than the former, suggesting a dominant role of 2, 3-DHBA in the vulnibactin biosynthesis. A double deletion mutant of VV2_0839 and VV2_0834 manifested additional growth defect under iron limitation. Though the growth defect of respective single deletion mutants could be restored by exogenous SA or 2, 3-DHBA, only 2, 3-DHBA could rescue the double mutant when supplied alone. However, double mutant could be rescued with SA only when hydrogen peroxide was supplied exogenously, suggesting a chemical conversion of SA to 2, 3-DHBA. Assembly of two SA and one 2, 3-DHBA into vulnibactin was mediated by two AMP ligase genes (VV2_0836 and VV2_0840). VV2_0836 deletion mutant showed more significant growth defect under iron limitation, suggesting its dominant function. In conclusion, using molecular genetic analytical tools, we confirm that vulnibactin is assembled of both 2, 3-DHBA and SA. However, conversion of SA to 2, 3-DHBA in presence of hydrogen peroxide and growth profile of AMP ligase mutants suggest a plausible existence of yet unidentified alternative siderophore that may be composed solely of 2, 3-DHBA.

OBJECTIVE: To investigate the efficacy and safety of prostate-specific antigen (PSA) testing to screen for prostate cancer. DESIGN: Systematic review and meta-analysis. DATA SOURCES: Electronic search of Cochrane Central Register of Controlled Trials, Web of Science, Embase, Scopus, OpenGrey, LILACS, and Medline, and search of scientific meeting abstracts and trial registers to April 2018. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Randomised controlled trials comparing PSA screening with usual care in men without a diagnosis of prostate cancer. DATA EXTRACTION: Rapid Recommendation) provided input on the design and interpretation of the systematic review, including selection of outcomes important to patients. We used a random effects model to obtain pooled incidence rate ratios (IRR) and, when feasible, conducted subgroup analyses (defined a priori) based on age, frequency of screening, family history, ethnicity, and socioeconomic level, as well as a sensitivity analysis based on the risk of bias. The quality of the evidence was assessed with the GRADE approach. RESULTS: Five randomised controlled trials, enrolling 721 718 men, were included. Studies varied with respect to screening frequency and intervals, PSA thresholds for biopsy, and risk of bias. When considering the whole body of evidence, screening probably has no effect on all-cause mortality (IRR 0.99, 95% CI 0.98 to 1.01; moderate certainty) and may have no effect on prostate-specific mortality (IRR 0.96, 0.85 to 1.08; low certainty). Sensitivity analysis of studies at lower risk of bias (n=1) also demonstrates that screening seems to have no effect on all-cause mortality (IRR 1.0, 0.98 to 1.02; moderate certainty) but may have a small effect on prostate-specific mortality (IRR 0.79, 0.69 to 0.91; moderate certainty). This corresponds to one less death from prostate cancer per 1000 men screened over 10 years. Direct comparative data on biopsy and treatment related complications from the included trials were limited. Using modelling, we estimated that for every 1000 men screened, approximately 1, 3, and 25 more men would be hospitalised for sepsis, require pads for urinary incontinence, and report erectile dysfunction, respectively. CONCLUSIONS: At best, screening for prostate cancer leads to a small reduction in disease-specific mortality over 10 years but has does not affect overall mortality. Clinicians and patients considering PSA based screening need to weigh these benefits against the potential short and long term harms of screening, including complications from biopsies and subsequent treatment, as well as the risk of overdiagnosis and overtreatment. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number CRD42016042347.

For the last 20 years, a great amount of evidence has accumulated through epidemiological studies that most of the dry eye disease encountered in daily life, especially in video display terminal (VDT) workers, involves short tear film breakup time (TFBUT) type dry eye, a category characterized by severe symptoms but minimal clinical signs other than short TFBUT. An unstable tear film also affects the visual function, possibly due to the increase of higher order aberrations. Based on the change in the understanding of the types, symptoms, and signs of dry eye disease, the Asia Dry Eye Society agreed to the following definition of dry eye: "Dry eye is a multifactorial disease characterized by unstable tear film causing a variety of symptoms and/or visual impairment, potentially accompanied by ocular surface damage." The definition stresses instability of the tear film as well as the importance of visual impairment, highlighting an essential role for TFBUT assessment. This paper discusses the concept of Tear Film Oriented Therapy (TFOT), which evolved from the definition of dry eye, emphasizing the importance of a stable tear film.

resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Together, these findings provide evidence that nonvirulent tumor-targeting bacteria releasing multiple TLR ligands can be used as cancer immunotherapeutics.

Massive hemoptysis is one of the most dreaded of all respiratory emergencies and can have a variety of underlying causes. In 90% of cases, the source of massive hemoptysis is the bronchial circulation. Diagnostic studies for massive hemoptysis include radiography, bronchoscopy, and computed tomography (CT) of the chest. Bronchoscopy and chest radiography have been considered the primary methods for the diagnosis and localization of hemoptysis. Many researchers currently suggest that CT should be performed prior to bronchoscopy in all cases of massive hemoptysis. Bronchial artery embolization (BAE) is a safe and effective nonsurgical treatment for patients with massive hemoptysis. However, nonbronchial systemic arteries can be a significant source of massive hemoptysis and a cause of recurrence after successful BAE. Knowledge of the bronchial artery anatomy, together with an understanding of the pathophysiologic features of massive hemoptysis, are essential for planning and performing BAE in affected patients. In addition, interventional radiologists should be familiar with the techniques, results, and possible complications of BAE and with the characteristics of the various embolic agents used in the procedure.

Mesenchymal stem cells (MSCs) have been widely studied for their applications in stem cell-based regeneration. During myocardial infarction (MI), infiltrated macrophages have pivotal roles in inflammation, angiogenesis and cardiac remodeling. We hypothesized that MSCs may modulate the immunologic environment to accelerate regeneration. This study was designed to assess the functional relationship between the macrophage phenotype and MSCs. MSCs isolated from bone marrow and bone marrow-derived macrophages (BMDMs) underwent differentiation induced by macrophage colony-stimulating factor. To determine the macrophage phenotype, classical M1 markers and alternative M2 markers were analyzed with or without co-culturing with MSCs in a transwell system. For animal studies, MI was induced by the ligation of the rat coronary artery. MSCs were injected within the infarct myocardium, and we analyzed the phenotype of the infiltrated macrophages by immunostaining. In the MSC-injected myocardium, the macrophages adjacent to the MSCs showed strong expression of arginase-1 (Arg1), an M2 marker. In BMDMs co-cultured with MSCs, the M1 markers such as interleukin-6 (IL-6), IL-1β, monocyte chemoattractant protein-1 and inducible nitric oxide synthase (iNOS) were significantly reduced. In contrast, the M2 markers such as IL-10, IL-4, CD206 and Arg1 were markedly increased by co-culturing with MSCs. Specifically, the ratio of iNOS to Arg1 in BMDMs was notably downregulated by co-culturing with MSCs. These results suggest that the preferential shift of the macrophage phenotype from M1 to M2 may be related to the immune-modulating characteristics of MSCs that contribute to cardiac repair. Bone marrow–derived mesenchymal stem cells (MSCs) transplanted into the heart appear to alter the immunologic environment and may help with recovery following a heart attack. A team led by Youngkeun Ahn and Yong Sook Kim from Chonnam National University Hospital, South Korea, induced heart attacks in rats and then injected MSCs into the damaged heart muscle tissue. The researchers analyzed the surrounding macrophages, a type of white blood cell that rushes to the site of injury, and found a strong expression of the enzyme arginase-1 — a sign that the macrophages were helping with tissue repair. The researchers also characterized macrophages before and after being cultured together with MSCs. They showed that the presence of MSCs shifted the macrophages from the M1 subtype, involved in pro-inflammatory responses, toward the ‘M2’ subtype, involved in dampening inflammation.

Candida auris is a newly described species whose clinical significance is not clear. Here, we describe the first three cases of nosocomial fungemia caused by C. auris, which confirms that it is a causative agent of bloodstream infections. All three patients presented persistent fungemia for 10 to 31 days. The isolates obtained from the three patients were misidentified as Candida haemulonii and Rhodotorula glutinis by the Vitek 2 and the API 20C systems, respectively. C. auris was confirmed by sequence analysis of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA of the rRNA gene. The MIC ranges of amphotericin B (AMB), fluconazole (FLU), itraconazole, and voriconazole were 0.5 to 1, 2 to 128, 0.125 to 2, and 0.06 to 1 μg/ml, respectively. All isolates were susceptible to caspofungin (MIC = 0.06 μg/ml) and micafungin (MIC = 0.03 μg/ml). One patient developed breakthrough fungemia while receiving FLU therapy, and two patients who received FLU therapy followed by AMB showed therapeutic failure and fatal outcomes. Our cases show that C. auris fungemia can be persistent, despite FLU or AMB therapy, which emphasizes the importance of accurately identifying this species.

BACKGROUND: Most trials comparing percutaneous coronary intervention (PCI) with coronary-artery bypass grafting (CABG) have not made use of second-generation drug-eluting stents. METHODS: We conducted a randomized noninferiority trial at 27 centers in East Asia. We planned to randomly assign 1776 patients with multivessel coronary artery disease to PCI with everolimus-eluting stents or to CABG. The primary end point was a composite of death, myocardial infarction, or target-vessel revascularization at 2 years after randomization. Event rates during longer-term follow-up were also compared between groups. RESULTS: After the enrollment of 880 patients (438 patients randomly assigned to the PCI group and 442 randomly assigned to the CABG group), the study was terminated early owing to slow enrollment. At 2 years, the primary end point had occurred in 11.0% of the patients in the PCI group and in 7.9% of those in the CABG group (absolute risk difference, 3.1 percentage points; 95% confidence interval [CI], -0.8 to 6.9; P=0.32 for noninferiority). At longer-term follow-up (median, 4.6 years), the primary end point had occurred in 15.3% of the patients in the PCI group and in 10.6% of those in the CABG group (hazard ratio, 1.47; 95% CI, 1.01 to 2.13; P=0.04). No significant differences were seen between the two groups in the occurrence of a composite safety end point of death, myocardial infarction, or stroke. However, the rates of any repeat revascularization and spontaneous myocardial infarction were significantly higher after PCI than after CABG. CONCLUSIONS: Among patients with multivessel coronary artery disease, the rate of major adverse cardiovascular events was higher among those who had undergone PCI with the use of everolimus-eluting stents than among those who had undergone CABG. (Funded by CardioVascular Research Foundation and others; BEST ClinicalTrials.gov number, NCT00997828.).

BACKGROUND: The potential benefits and risks of the use of dual antiplatelet therapy beyond a 12-month period in patients receiving drug-eluting stents have not been clearly established. METHODS: In two trials, we randomly assigned a total of 2701 patients who had received drug-eluting stents and had been free of major adverse cardiac or cerebrovascular events and major bleeding for a period of at least 12 months to receive clopidogrel plus aspirin or aspirin alone. The primary end point was a composite of myocardial infarction or death from cardiac causes. Data from the two trials were merged for analysis. RESULTS: The median duration of follow-up was 19.2 months. The cumulative risk of the primary outcome at 2 years was 1.8% with dual antiplatelet therapy, as compared with 1.2% with aspirin monotherapy (hazard ratio, 1.65; 95% confidence interval [CI], 0.80 to 3.36; P=0.17). The individual risks of myocardial infarction, stroke, stent thrombosis, need for repeat revascularization, major bleeding, and death from any cause did not differ significantly between the two groups. However, in the dual-therapy group as compared with the aspirin-alone group, there was a nonsignificant increase in the composite risk of myocardial infarction, stroke, or death from any cause (hazard ratio, 1.73; 95% CI, 0.99 to 3.00; P=0.051) and in the composite risk of myocardial infarction, stroke, or death from cardiac causes (hazard ratio, 1.84; 95% CI, 0.99 to 3.45; P=0.06). CONCLUSIONS: The use of dual antiplatelet therapy for a period longer than 12 months in patients who had received drug-eluting stents was not significantly more effective than aspirin monotherapy in reducing the rate of myocardial infarction or death from cardiac causes. These findings should be confirmed or refuted through larger, randomized clinical trials with longer-term follow-up. (ClinicalTrials.gov numbers, NCT00484926 and NCT00590174.)

BACKGROUND: Several studies have compared the treatment effects of coronary stenting and coronary-artery bypass grafting (CABG). However, there are limited data regarding the long-term outcomes of these two interventions for patients with unprotected left main coronary artery disease. METHODS: We evaluated 1102 patients with unprotected left main coronary artery disease who underwent stent implantation and 1138 patients who underwent CABG in Korea between January 2000 and June 2006. We compared adverse outcomes (death; a composite outcome of death, Q-wave myocardial infarction, or stroke; and target-vessel revascularization) with the use of propensity-score matching in the overall cohort and in separate subgroups according to type of stent. RESULTS: In the overall matched cohort, there was no significant difference between the stenting and CABG groups in the risk of death (hazard ratio for the stenting group, 1.18; 95% confidence interval [CI], 0.77 to 1.80) or the risk of the composite outcome (hazard ratio for the stenting group, 1.10; 95% CI, 0.75 to 1.62). The rates of target-vessel revascularization were significantly higher in the group that received stents than in the group that underwent CABG (hazard ratio, 4.76; 95% CI, 2.80 to 8.11). Comparisons of the group that received bare-metal stents with the group that underwent CABG and of the group that received drug-eluting stents with the group that underwent CABG produced similar results, although there was a trend toward higher rates of death and the composite end point in the group that received drug-eluting stents. CONCLUSIONS: In a cohort of patients with unprotected left main coronary artery disease, we found no significant difference in rates of death or of the composite end point of death, Q-wave myocardial infarction, or stroke between patients receiving stents and those undergoing CABG. However, stenting, even with drug-eluting stents, was associated with higher rates of target-vessel revascularization than was CABG.

Abstract Purpose: The Cancer Genome Atlas (TCGA) project recently uncovered four molecular subtypes of gastric cancer: Epstein–Barr virus (EBV), microsatellite instability (MSI), genomically stable (GS), and chromosomal instability (CIN). However, their clinical significances are currently unknown. We aimed to investigate the relationship between subtypes and prognosis of patients with gastric cancer. Experimental Design: Gene expression data from a TCGA cohort (n = 262) were used to develop a subtype prediction model, and the association of each subtype with survival and benefit from adjuvant chemotherapy was tested in 2 other cohorts (n = 267 and 432). An integrated risk assessment model (TCGA risk score) was also developed. Results: EBV subtype was associated with the best prognosis, and GS subtype was associated with the worst prognosis. Patients with MSI and CIN subtypes had poorer overall survival than those with EBV subtype but better overall survival than those with GS subtype (P = 0.004 and 0.03 in two cohorts, respectively). In multivariate Cox regression analyses, TCGA risk score was an independent prognostic factor [HR, 1.5; 95% confidence interval (CI), 1.2–1.9; P = 0.001]. Patients with the CIN subtype experienced the greatest benefit from adjuvant chemotherapy (HR, 0.39; 95% CI, 0.16–0.94; P = 0.03) and those with the GS subtype had the least benefit from adjuvant chemotherapy (HR, 0.83; 95% CI, 0.36–1.89; P = 0.65). Conclusions: Our prediction model successfully stratified patients by survival and adjuvant chemotherapy outcomes. Further development of the prediction model is warranted. Clin Cancer Res; 23(15); 4441–9. ©2017 AACR.

Baseline correction methods based on penalized least squares are successfully applied to various spectral analyses. The methods change the weights iteratively by estimating a baseline. If a signal is below a previously fitted baseline, large weight is given. On the other hand, no weight or small weight is given when a signal is above a fitted baseline as it could be assumed to be a part of the peak. As noise is distributed above the baseline as well as below the baseline, however, it is desirable to give the same or similar weights in either case. For the purpose, we propose a new weighting scheme based on the generalized logistic function. The proposed method estimates the noise level iteratively and adjusts the weights correspondingly. According to the experimental results with simulated spectra and measured Raman spectra, the proposed method outperforms the existing methods for baseline correction and peak height estimation.

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. IntroductionAMPK 3The abbreviations used are: AMPK5′-adenosine monophosphate-activated protein kinaseROSreactive oxygen speciesATZ3-amino-1,2,4-triazoleAIDautoinhibitory domainGOglucose oxidaseDCF2′,7′-dichlorodihydrofluoresceinDCF-DA2′,7′-dichlorodihydrofluorescein diacetateGSSS-glutathionylation ofBIAMbiotinylated iodoacetamideACCacetyl-CoA carboxylase. is a serine/threonine kinase that consists of three subunits, of which the α subunit has inducible kinase activity and the β and γ subunits have regulatory function. Formation of the αβγ complex is required for optimal allosteric activation of AMPK, which is induced by binding of AMP to the γ subunit (1Baron S.J. Li J. Russell 3rd, R.R. Neumann D. Miller E.J. Tuerk R. Wallimann T. Hurley R.L. Witters L.A. Young L.H. Circ. 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Nature. 2009; 459: 1146-1149Crossref PubMed Scopus (161) Google Scholar), whereas AMP-induced conformational changes within the αβγ complex diminish function of the AID and lead to kinase activation.The regulation of AMPK activity is primarily thought to result from alterations in the intracellular AMP/ATP ratio, arising from diminished ATP generation due to hypoxia, glucose deprivation, heat shock, or reduction in mitochondrial oxidative phosphorylation or from increased ATP consumption, such as occurs during strenuous exercise (2Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. 2006; 574: 7-15Crossref PubMed Scopus (644) Google Scholar, 10Evans A.M. Mustard K.J. Wyatt C.N. Peers C. Dipp M. Kumar P. Kinnear N.P. Hardie D.G. J. Biol. Chem. 2005; 280: 41504-41511Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 11Yun H. Lee M. Kim S.S. Ha J. J. Biol. Chem. 2005; 280: 9963-9972Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 12Winder W.W. Holmes B.F. Rubink D.S. Jensen E.B. Chen M. Holloszy J.O. J. Appl. Physiol. 2000; 88: 2219-2226Crossref PubMed Scopus (591) Google Scholar). Once activated, AMPK can phosphorylate and modulate the function of essential metabolic pathways participating in the regulation of glucose and lipid homeostasis (13Dolinsky V.W. Dyck J.R. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H2557-H2569Crossref PubMed Scopus (115) Google Scholar, 14Long Y.C. Zierath J.R. J. Clin. Invest. 2006; 116: 1776-1783Crossref PubMed Scopus (745) Google Scholar, 15Hardie D.G. Sakamoto K. Physiology. 2006; 21: 48-60Crossref PubMed Scopus (427) Google Scholar). A major effect of AMPK activation is in preserving energy for use under conditions where ATP is limiting (4Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1039) Google Scholar, 16Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (705) Google Scholar). AMPK activation appears to prevent or diminish inflammation-associated organ injury, including the development of atherosclerotic cardiovascular disease in diabetes (17Knowler W.C. Barrett-Connor E. Fowler S.E. Hamman R.F. Lachin J.M. Walker E.A. Nathan D.M. N. Engl. J. Med. 2002; 346: 393-403Crossref PubMed Scopus (14330) Google Scholar), ischemia-induced cardiac dysfunction (18Shibata R. Sato K. Pimentel D.R. Takemura Y. Kihara S. Ohashi K. Funahashi T. Ouchi N. Walsh K. Nat. Med. 2005; 11: 1096-1103Crossref PubMed Scopus (847) Google Scholar, 19Shin E.J. Schram K. Zheng X.L. Sweeney G. J. Cell. Physiol. 2009; 221: 490-497Crossref PubMed Scopus (54) Google Scholar, 20Gundewar S. Calvert J.W. Jha S. Toedt-Pingel I. Ji S.Y. Nunez D. Ramachandran A. Anaya-Cisneros M. Tian R. Lefer D.J. Circ. Res. 2009; 104: 403-411Crossref PubMed Scopus (315) Google Scholar), and hepatic dysfunction in animal models of nonalcoholic steatohepatitis as well as in humans with this condition (21Lin H.Z. Yang S.Q. Chuckaree C. Kuhajda F. Ronnet G. Diehl A.M. Nat. Med. 2000; 6: 998-1003Crossref PubMed Scopus (608) Google Scholar, 22Marchesini G. Brizi M. Bianchi G. Tomassetti S. Zoli M. Melchionda N. Lancet. 2001; 358: 893-894Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Our studies have also suggested that therapeutic approaches to increase AMPK activity diminish the severity of LPS-induced acute lung injury in mice (23Zhao X. Zmijewski J.W. Lorne E. Liu G. Park Y.J. Tsuruta Y. Abraham E. Am. J. Physiol. Lung Cell Mol. Physiol. 2008; 295: L497-L504Crossref PubMed Scopus (249) Google Scholar, 24Zmijewski J.W. Lorne E. Zhao X. Tsuruta Y. Sha Y. Liu G. Siegal G.P. Abraham E. Am. J. Respir. Crit. Care Med. 2008; 178: 168-179Crossref PubMed Scopus (126) Google Scholar).Although increased formation of reactive oxygen species (ROS) is generally thought to be associated with pathophysiological situations leading to cellular injury and organ dysfunction, recent studies have shown beneficial effects of ROS in modulating inflammation, including TLR4-induced neutrophil activation and LPS-associated acute lung injury (24Zmijewski J.W. Lorne E. Zhao X. Tsuruta Y. Sha Y. Liu G. Siegal G.P. Abraham E. Am. J. Respir. Crit. Care Med. 2008; 178: 168-179Crossref PubMed Scopus (126) Google Scholar, 25Zmijewski J.W. Lorne E. Zhao X. Tsuruta Y. Sha Y. Liu G. Abraham E. Am. J. Respir. Crit. Care Med. 2009; 179: 694-704Crossref PubMed Scopus (79) Google Scholar, 26Zmijewski J.W. Zhao X. Xu Z. Abraham E. Am. J. Physiol. Cell Physiol. 2007; 293: C255-C266Crossref PubMed Scopus (59) Google Scholar, 27Strassheim D. Asehnoune K. Park J.S. Kim J.Y. He Q. Richter D. Mitra S. Arcaroli J. Kuhn K. Abraham E. Am. J. Physiol. Cell Physiol. 2004; 286: C683-C692Crossref PubMed Scopus (52) Google Scholar). Several studies have demonstrated that increased intracellular concentrations of H2O2 result in activation of AMPK and enhancement of AMPK-mediated cellular adaptation (28Horie T. Ono K. Nagao K. Nishi H. Kinoshita M. Kawamura T. Wada H. Shimatsu A. Kita T. Hasegawa K. J. Cell. Physiol. 2008; 215: 733-742Crossref PubMed Scopus (103) Google Scholar, 29Irrcher I. Ljubicic V. Hood D.A. Am. J. Physiol. Cell Physiol. 2009; PubMed Scopus Google Scholar, Zhang S.J. J. H. A. J. Physiol. 2006; PubMed Scopus Google Scholar), including of redox homeostasis E. J. S. Chen K. A. P. T. 2008; PubMed Google Scholar, S. Biochem. J. 2009; PubMed Scopus Google Scholar). In cardiac diminished activation of AMPK and resulted in increased severity of cardiac injury R.R. G. N. J. S.A. 2009; PubMed Scopus Google the ability of increased intracellular concentrations of H2O2 to induce AMPK activation in the for this effect has well that activation of AMPK resulted from ATP and increased AMP/ATP Kim S.J. Lee Kim J. J. Kim S. Ha J. Biochem. Res. 2001; PubMed Scopus (249) Google Scholar), studies demonstrated that increased intracellular concentrations of H2O2 were associated with activation of AMPK or without in the ratio F. C. Z. B. Biol. Med. 2009; PubMed Scopus Google Scholar, T. T. L. S. M. S. K. H. K. G. A. Sato K. T. K. Am. J. Physiol. 2004; PubMed Scopus Google Scholar, M. Y. Xu J. Xie Z. Wu Y. P. M. Wu J. Circ. Res. 2008; PubMed Scopus Google can pathways as a result of oxidative modification of cysteine in Physiol. 2002; PubMed Scopus Google Scholar, D. R. A. R. I. J. Cell Mol. Med. 2004; PubMed Scopus Google Scholar, I. R. D. R. A. Biol. Med. 2007; PubMed Scopus Google Scholar). We that a by which increased intracellular concentrations of H2O2 can activate AMPK is through of in or AMPK subunits. Our present experiments demonstrate that exposure to H2O2 is associated with cysteine in the AMPKα subunit and is to directly activate this we demonstrated that H2O2 can directly activate AMPK in and in through a associated with oxidative including S-glutathionylation of cysteine of the AMPKα subunit. shown that exposure of to H2O2 or to that increased intracellular concentrations of such as by mitochondrial result in increased AMPK activity I. Ljubicic V. Hood D.A. Am. J. Physiol. Cell Physiol. 2009; PubMed Scopus Google Scholar, F. C. Z. B. Biol. Med. 2009; PubMed Scopus Google Scholar, M. Y. Xu J. Xie Z. Wu Y. P. M. Wu J. Circ. Res. 2008; PubMed Scopus Google Scholar, K. A. N. M. M. Cell 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). The ability of H2O2 to activate AMPK has to be and to through intracellular levels of ATP and the ratio of AMP to enhancing binding of AMP to the subunit with allosteric activation of the AMPKα kinase domain Kim S.J. Lee Kim J. J. Kim S. Ha J. Biochem. Res. 2001; PubMed Scopus (249) Google Scholar). A recent that an of the subunit suggested that the effects of H2O2 on AMPK activation are by a diminished ratio S.A. C. Green K.A. A. S. M.C. A.M. Hardie D.G. Cell 11: Full Text Full Text PDF PubMed Scopus Google Scholar). in the present we found that exposure of cells to increased levels of H2O2 and AMPK decrease in ATP levels or in the ratio we that H2O2 directly activate AMPK by that of the AMPKαβγ complex with H2O2 increased kinase activity. exposure of HEK 293 and to H2O2 also results in diminished intracellular ATP levels and increased AMP/ATP in addition to intracellular concentrations of the ability of H2O2 to directly activate AMPK imply that this is the major by which increased generation of H2O2 activation of AMPK in experiments be to the of of the AMPKα subunit as with alterations in AMP/ATP in AMPK during such as injury, that are associated with increased of exposure to H2O2 resulted in oxidative modification and S-glutathionylation of the α and β subunits of AMPK, modification of the AMPKα subunit was to increase kinase activity. exposure of AMPKα (amino acids which the binding domain for with the AMPKβ and subunits, to H2O2 increased AMPK activity, that with the β and γ subunits was for AMPK activation by Recent studies have shown that between of the AID and of the AMPKα subunit is responsible for of the of the AMPKα subunit within the AMPKαβγ complex (7Crute B.E. Seefeld K. Gamble J. Kemp B.E. Witters L.A. J. Biol. Chem. 1998; 273: 35347-35354Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 8Pang T. Xiong B. Li J.Y. Qiu B.Y. Jin G.Z. Shen J.K. Li J. J. Biol. Chem. 2007; 282: 495-506Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 9Chen L. Jiao Z.H. Zheng L.S. Zhang Y.Y. Xie S.T. Wang Z.X. Wu J.W. Nature. 2009; 459: 1146-1149Crossref PubMed Scopus (161) Google Scholar). exposure of AMPKα or AMPKα truncation mutants the AID to H2O2 resulted in AMPK kinase activity, that the AID is to an in this of cysteine 299 and mutation of cysteine 304 totally the activation of AMPKα by that oxidative modification of an in the ability of H2O2 to induce activation of results that exposure to H2O2 enhances the kinase activity of AMPK, phosphorylation of AMPK was also present under such In of the AMPKαβγ complex with H2O2 increased phosphorylation of the AMPKα and AMPKβ subunits. findings are with studies that that AMPK during activation D. Dyck J. Gamble J. C. Witters L.A. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar).Although H2O2 is a H2O2 is of cellular to of intracellular and to modulate activity in pathways Physiol. 2002; PubMed Scopus Google Scholar). The results of the present and of that exposure of the AMPKαβγ complex or of the AMPKα subunit to H2O2 increased kinase activity and diminished that the by which H2O2 such effects is through oxidative modification of cysteine The ability of H2O2 to S-glutathionylation of the AMPKα and AMPKβ subunits is with this Although the present findings that exposure to H2O2 is to and activate AMPK, is that such as from H2O2 to we found that generation of in diminished the activity of AMPK results that H2O2 is responsible for activation of AMPK under in conditions associated with increased generation of to the effects of H2O2 in we found that increased intracellular concentrations of H2O2 in the lungs under in conditions also were associated with AMPK activation L.H. 2008; PubMed Scopus Google Scholar). A for H2O2 in modulating AMPK activity in was cardiac increased levels of H2O2 in the were by activation of AMPK and from a (1Baron S.J. Li J. Russell 3rd, R.R. Neumann D. Miller E.J. Tuerk R. Wallimann T. Hurley R.L. Witters L.A. Young L.H. Circ. Res. 2005; 96: 337-345Crossref PubMed Scopus (86) Google Scholar, N. R.L. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. C. Physiol. 2008; 88: PubMed Scopus Google Scholar, 3rd, R.R. Li J. M. C. M. J. Young L.H. J. Clin. Invest. 2004; PubMed Scopus Google Scholar, A. S. Q. G. Shen M. Sakamoto K. Tian R. A. J. Cell. Physiol. 2007; PubMed Scopus (115) Google Scholar). with experiments with AMPK and with HEK 293 cells, we found activation of AMPK and oxidative modification of the AMPKα subunit in the lungs of acatalasemic mice and in mice treated with the catalase inhibitor conditions in which intracellular concentrations of H2O2 are J.W. Lorne E. Zhao X. Tsuruta Y. Sha Y. Liu G. Abraham E. Am. J. Respir. Crit. Care Med. 2009; 179: 694-704Crossref PubMed Scopus (79) Google experiments demonstrate a for AMPK activation that oxidative modification of the AMPKα subunit as a result of exposure to H2O2 studies have shown that activation of AMPK as well as increased intracellular concentrations of H2O2 have including diminished severity of LPS-induced acute lung injury J.W. Lorne E. Zhao X. Tsuruta Y. Sha Y. Liu G. Abraham E. Am. J. Respir. Crit. Care Med. 2009; 179: 694-704Crossref PubMed Scopus (79) Google Scholar). The present experiments that by which H2O2 effects in is through directly studies be to the primary for the effects of increased intracellular concentrations of H2O2 is through activation of IntroductionAMPK 3The abbreviations used are: AMPK5′-adenosine monophosphate-activated protein kinaseROSreactive oxygen speciesATZ3-amino-1,2,4-triazoleAIDautoinhibitory domainGOglucose oxidaseDCF2′,7′-dichlorodihydrofluoresceinDCF-DA2′,7′-dichlorodihydrofluorescein diacetateGSSS-glutathionylation ofBIAMbiotinylated iodoacetamideACCacetyl-CoA carboxylase. is a serine/threonine kinase that consists of three subunits, of which the α subunit has inducible kinase activity and the β and γ subunits have regulatory function. Formation of the αβγ complex is required for optimal allosteric activation of AMPK, which is induced by binding of AMP to the γ subunit (1Baron S.J. Li J. Russell 3rd, R.R. Neumann D. Miller E.J. Tuerk R. Wallimann T. Hurley R.L. Witters L.A. Young L.H. Circ. Res. 2005; 96: 337-345Crossref PubMed Scopus (86) Google Scholar, 2Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. 2006; 574: 7-15Crossref PubMed Scopus (644) Google Scholar, 3Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Invest. 2004; 113: 274-284Crossref PubMed Scopus (599) Google Scholar, 4Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1039) Google Scholar). In addition to activation by AMP, phosphorylation of the Thr172 residue of the α subunit enhances kinase activity (5Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (484) Google Scholar, 6Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (995) Google Scholar). Recent studies have shown that the autoinhibitory domain (AID), located between amino acids 312 and 335 of the AMPKα subunit, is responsible for the lack of kinase activity under basal conditions (7Crute B.E. Seefeld K. Gamble J. Kemp B.E. Witters L.A. J. Biol. Chem. 1998; 273: 35347-35354Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 8Pang T. Xiong B. Li J.Y. Qiu B.Y. Jin G.Z. Shen J.K. Li J. J. Biol. Chem. 2007; 282: 495-506Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 9Chen L. Jiao Z.H. Zheng L.S. Zhang Y.Y. Xie S.T. Wang Z.X. Wu J.W. Nature. 2009; 459: 1146-1149Crossref PubMed Scopus (161) Google Scholar), whereas AMP-induced conformational changes within the αβγ complex diminish function of the AID and lead to kinase activation.The regulation of AMPK activity is primarily thought to result from alterations in the intracellular AMP/ATP ratio, arising from diminished ATP generation due to hypoxia, glucose deprivation, heat shock, or reduction in mitochondrial oxidative phosphorylation or from increased ATP consumption, such as occurs during strenuous exercise (2Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. 2006; 574: 7-15Crossref PubMed Scopus (644) Google Scholar, 10Evans A.M. Mustard K.J. Wyatt C.N. Peers C. Dipp M. Kumar P. Kinnear N.P. Hardie D.G. J. Biol. Chem. 2005; 280: 41504-41511Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 11Yun H. Lee M. Kim S.S. Ha J. J. Biol. Chem. 2005; 280: 9963-9972Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 12Winder W.W. Holmes B.F. Rubink D.S. Jensen E.B. Chen M. Holloszy J.O. J. Appl. Physiol. 2000; 88: 2219-2226Crossref PubMed Scopus (591) Google Scholar). Once activated, AMPK can phosphorylate and modulate the function of essential metabolic pathways participating in the regulation of glucose and lipid homeostasis (13Dolinsky V.W. Dyck J.R. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H2557-H2569Crossref PubMed Scopus (115) Google Scholar, 14Long Y.C. Zierath J.R. J. Clin. Invest. 2006; 116: 1776-1783Crossref PubMed Scopus (745) Google Scholar, 15Hardie D.G. Sakamoto K. Physiology. 2006; 21: 48-60Crossref PubMed Scopus (427) Google Scholar). A major effect of AMPK activation is in preserving energy for use under conditions where ATP is limiting (4Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1039) Google Scholar, 16Hardie D.G. Scott J.W. Pan D.A. Hudson E.R. FEBS Lett. 2003; 546: 113-120Crossref PubMed Scopus (705) Google Scholar). AMPK activation appears to prevent or diminish inflammation-associated organ injury, including the development of atherosclerotic cardiovascular disease in diabetes (17Knowler W.C. Barrett-Connor E. Fowler S.E. Hamman R.F. Lachin J.M. Walker E.A. Nathan D.M. N. Engl. J. Med. 2002; 346: 393-403Crossref PubMed Scopus (14330) Google Scholar), ischemia-induced cardiac dysfunction (18Shibata R. Sato K. Pimentel D.R. Takemura Y. Kihara S. Ohashi K. Funahashi T. Ouchi N. Walsh K. Nat. Med. 2005; 11: 1096-1103Crossref PubMed Scopus (847) Google Scholar, 19Shin E.J. Schram K. Zheng X.L. Sweeney G. J. Cell. Physiol. 2009; 221: 490-497Crossref PubMed Scopus (54) Google Scholar, 20Gundewar S. Calvert J.W. Jha S. Toedt-Pingel I. Ji S.Y. Nunez D. Ramachandran A. Anaya-Cisneros M. Tian R. Lefer D.J. Circ. Res. 2009; 104: 403-411Crossref PubMed Scopus (315) Google Scholar), and hepatic dysfunction in animal models of nonalcoholic steatohepatitis as well as in humans with this condition (21Lin H.Z. Yang S.Q. Chuckaree C. Kuhajda F. Ronnet G. Diehl A.M. Nat. Med. 2000; 6: 998-1003Crossref PubMed Scopus (608) Google Scholar, 22Marchesini G. Brizi M. Bianchi G. Tomassetti S. Zoli M. Melchionda N. Lancet. 2001; 358: 893-894Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Our studies have also suggested that therapeutic approaches to increase AMPK activity diminish the severity of LPS-induced acute lung injury in mice (23Zhao X. Zmijewski J.W. Lorne E. Liu G. Park Y.J. Tsuruta Y. Abraham E. Am. J. Physiol. Lung Cell Mol. Physiol. 2008; 295: L497-L504Crossref PubMed Scopus (249) Google Scholar, 24Zmijewski J.W. Lorne E. Zhao X. Tsuruta Y. Sha Y. Liu G. Siegal G.P. Abraham E. Am. J. Respir. Crit. Care Med. 2008; 178: 168-179Crossref PubMed Scopus (126) Google Scholar).Although increased formation of reactive oxygen species (ROS) is generally thought to be associated with pathophysiological situations leading to cellular injury and organ dysfunction, recent studies have shown beneficial effects of ROS in modulating inflammation, including TLR4-induced neutrophil activation and LPS-associated acute lung injury (24Zmijewski J.W. Lorne E. Zhao X. Tsuruta Y. Sha Y. Liu G. Siegal G.P. Abraham E. Am. J. Respir. Crit. Care Med. 2008; 178: 168-179Crossref PubMed Scopus (126) Google Scholar, 25Zmijewski J.W. Lorne E. Zhao X. Tsuruta Y. Sha Y. Liu G. Abraham E. Am. J. Respir. Crit. Care Med. 2009; 179: 694-704Crossref PubMed Scopus (79) Google Scholar, 26Zmijewski J.W. Zhao X. Xu Z. Abraham E. Am. J. Physiol. Cell Physiol. 2007; 293: C255-C266Crossref PubMed Scopus (59) Google Scholar, 27Strassheim D. Asehnoune K. Park J.S. Kim J.Y. He Q. Richter D. Mitra S. Arcaroli J. Kuhn K. Abraham E. Am. J. Physiol. Cell Physiol. 2004; 286: C683-C692Crossref PubMed Scopus (52) Google Scholar). Several studies have demonstrated that increased intracellular concentrations of H2O2 result in activation of AMPK and enhancement of AMPK-mediated cellular adaptation (28Horie T. Ono K. Nagao K. Nishi H. Kinoshita M. Kawamura T. Wada H. Shimatsu A. Kita T. Hasegawa K. J. Cell. Physiol. 2008; 215: 733-742Crossref PubMed Scopus (103) Google Scholar, 29Irrcher I. Ljubicic V. Hood D.A. Am. J. Physiol. Cell Physiol. 2009; PubMed Scopus Google Scholar, Zhang S.J. J. H. A. J. Physiol. 2006; PubMed Scopus Google Scholar), including of redox homeostasis E. J. S. Chen K. A. P. T. 2008; PubMed Google Scholar, S. Biochem. J. 2009; PubMed Scopus Google Scholar). In cardiac diminished activation of AMPK and resulted in increased severity of cardiac injury R.R. G. N. J. S.A. 2009; PubMed Scopus Google the ability of increased intracellular concentrations of H2O2 to induce AMPK activation in the for this effect has well that activation of AMPK resulted from ATP and increased AMP/ATP Kim S.J. Lee Kim J. J. Kim S. Ha J. Biochem. Res. 2001; PubMed Scopus (249) Google Scholar), studies demonstrated that increased intracellular concentrations of H2O2 were associated with activation of AMPK or without in the ratio F. C. Z. B. Biol. Med. 2009; PubMed Scopus Google Scholar, T. T. L. S. M. S. K. H. K. G. A. Sato K. T. K. Am. J. Physiol. 2004; PubMed Scopus Google Scholar, M. Y. Xu J. Xie Z. Wu Y. P. M. Wu J. Circ. Res. 2008; PubMed Scopus Google can pathways as a result of oxidative modification of cysteine in Physiol. 2002; PubMed Scopus Google Scholar, D. R. A. R. I. J. Cell Mol. Med. 2004; PubMed Scopus Google Scholar, I. R. D. R. A. Biol. Med. 2007; PubMed Scopus Google Scholar). We that a by which increased intracellular concentrations of H2O2 can activate AMPK is through of in or AMPK subunits. Our present experiments demonstrate that exposure to H2O2 is associated with cysteine in the AMPKα subunit and is to directly activate

Importance: After percutaneous coronary intervention (PCI), patients with CYP2C19*2 or *3 loss-of-function (LOF) variants treated with clopidogrel have increased risk of ischemic events. Whether genotype-guided selection of oral P2Y12 inhibitor therapy improves ischemic outcomes is unknown. Objective: To determine the effect of a genotype-guided oral P2Y12 inhibitor strategy on ischemic outcomes in CYP2C19 LOF carriers after PCI. Design, Setting, and Participants: Open-label randomized clinical trial of 5302 patients undergoing PCI for acute coronary syndromes (ACS) or stable coronary artery disease (CAD). Patients were enrolled at 40 centers in the US, Canada, South Korea, and Mexico from May 2013 through October 2018; final date of follow-up was October 2019. Interventions: Patients randomized to the genotype-guided group (n = 2652) underwent point-of-care genotyping. CYP2C19 LOF carriers were prescribed ticagrelor and noncarriers clopidogrel. Patients randomized to the conventional group (n = 2650) were prescribed clopidogrel and underwent genotyping after 12 months. Main Outcomes and Measures: The primary end point was a composite of cardiovascular death, myocardial infarction, stroke, stent thrombosis, and severe recurrent ischemia at 12 months. A secondary end point was major or minor bleeding at 12 months. The primary analysis was in patients with CYP2C19 LOF variants, and secondary analysis included all randomized patients. The trial had 85% power to detect a minimum hazard ratio of 0.50. Results: Among 5302 patients randomized (median age, 62 years; 25% women), 82% had ACS and 18% had stable CAD; 94% completed the trial. Of 1849 with CYP2C19 LOF variants, 764 of 903 (85%) assigned to genotype-guided therapy received ticagrelor, and 932 of 946 (99%) assigned to conventional therapy received clopidogrel. The primary end point occurred in 35 of 903 CYP2C19 LOF carriers (4.0%) in the genotype-guided therapy group and 54 of 946 (5.9%) in the conventional therapy group at 12 months (hazard ratio [HR], 0.66 [95% CI, 0.43-1.02]; P = .06). None of the 11 prespecified secondary end points showed significant differences, including major or minor bleeding in CYP2C19 LOF carriers in the genotype-guided group (1.9%) vs the conventional therapy group (1.6%) at 12 months (HR, 1.22 [95% CI, 0.60-2.51]; P = .58). Among all randomized patients, the primary end point occurred in 113 of 2641 (4.4%) in the genotype-guided group and 135 of 2635 (5.3%) in the conventional group (HR, 0.84 [95% CI, 0.65-1.07]; P = .16). Conclusions and Relevance: Among CYP2C19 LOF carriers with ACS and stable CAD undergoing PCI, genotype-guided selection of an oral P2Y12 inhibitor, compared with conventional clopidogrel therapy without point-of-care genotyping, resulted in no statistically significant difference in a composite end point of cardiovascular death, myocardial infarction, stroke, stent thrombosis, and severe recurrent ischemia based on the prespecified analysis plan and the treatment effect that the study was powered to detect at 12 months. Trial Registration: ClinicalTrials.gov Identifier: NCT01742117.