PHD2 enzyme is an intracellular manganese sensor that initiates the homeostatic response against elevated manganese.

Intracellular sensors detect changes in levels of essential metals to initiate homeostatic responses. But, a mammalian manganese (Mn) sensor is unknown, representing a major gap in understanding of Mn homeostasis. Using human-relevant models, we recently reported that: 1) the primary homeostatic response to elevated Mn is upregulation of hypoxia-inducible factors (HIFs), which increases expression of the Mn efflux transporter SLC30A10; and 2) elevated Mn blocks the prolyl hydroxylation of HIFs by prolyl hydroxylase domain (PHD) enzymes, which otherwise targets HIFs for degradation. Thus, the mammalian mechanism for sensing elevated Mn likely relates to PHD inhibition. Moreover, 1) Mn substitutes for a catalytic iron (Fe) in PHD structures; and 2) exchangeable cellular levels of Fe and Mn are comparable. Therefore, we hypothesized that elevated Mn directly inhibits PHD by replacing its catalytic Fe. In vitro assays using catalytically active PHD2, the primary PHD isoform, revealed that Mn inhibited, and Fe supplementation rescued, PHD2 activity. However, a mutation in PHD2 (D315E) that selectively reduced Mn binding without substantially impacting Fe binding or enzymatic activity resulted in complete insensitivity of PHD2 to Mn in vitro. Additionally, hepatic cells expressing full-length PHD2D315E were less sensitive to Mn-induced HIF activation and SLC30A10 upregulation than PHD2wild-type. These results: 1) define a fundamental Mn sensing mechanism for controlling Mn homeostasis-elevated Mn inhibits PHD2, which functions as a Mn sensor, by outcompeting its catalytic Fe, and PHD2 inhibition activates HIF signaling to up-regulate SLC30A10; and 2) identify a unique mode of metal sensing that may have wide applicability.

Melatonin and Exercise Counteract Sarcopenic Obesity through Preservation of Satellite Cell Function.

Sarcopenic obesity (SO) is characterized by atrophic skeletal muscle impairment (sarcopenia) and obesity, which is associated with adverse outcomes of morbidity and mortality in elderly people. We investigated the effects of melatonin and exercise training on SO in 32-week-old senescence-accelerated mouse-prone-8 (SAMP8) mice fed a normal diet or a high-fat diet for 16 weeks. Melatonin, exercise, or melatonin and exercise for 8 weeks displayed reductions in the SO-induced impairment of skeletal muscle function and atrophy. Specifically, a decrease in mitochondrial calcium retention capacity in skeletal muscles observed in the HFD-con group was attenuated in melatonin and/or exercise intervention groups. More importantly, HFD-con mice displayed a lower number of Pax7+ satellite cells (SCs) and higher expression of p16ink than P8ND mice, which were attenuated by melatonin and/or exercise interventions. The cellular senescence in SC-derived primary myoblasts from HFD-con mice was significantly attenuated in myoblasts from the melatonin and/or exercise groups, which was reproduced in a senescence model of H2O2-treated C2C12 myoblasts. Our results suggest that melatonin and exercise training attenuate SO-induced skeletal muscle dysfunction, at least in part, through preserving the SC pool by inhibiting cellular senescence and attenuating mitochondrial dysfunction.

Moderate aerobic exercise training ameliorates impairment of mitochondrial function and dynamics in skeletal muscle of high-fat diet-induced obese mice.

The purpose of this study is to determine whether moderate aerobic exercise training improves high-fat diet-induced alterations in mitochondrial function and structure in the skeletal muscle. Male 4-week-old C57BL/6 mice were randomly divided into four groups: control (CON), control plus exercise (CON + EX), high-fat diet (HFD), and high-fat diet plus exercise (HFD + EX). After obesity was induced by 20 weeks of 60% HFD, treadmill exercise training was performed at 13-16 m/min, 40-50 min/day, and 6 days/week for 12 weeks. Mitochondrial structure, function, and dynamics, and mitophagy were analyzed in the skeletal muscle fibers from the red gastrocnemius. Exercise training increased mitochondrial number and area and reduced high-fat diet-induced obesity and hyperglycemia. In addition, exercise training attenuated mitochondrial dysfunction in the permeabilized myofibers, indicating that HFD-induced decrease of mitochondrial O2 respiration and Ca2+ retention capacity and increase of mitochondrial H2 O2 emission were attenuated in the HFD + EX group compared to the HFD group. Exercise also ameliorated HFD-induced imbalance of mitochondrial fusion and fission, demonstrating that HFD-induced decrease in fusion protein levels was elevated, and increase in fission protein levels was reduced in the HFD + EX groups compared with the HFD group. Moreover, dysregulation of mitophagy induced by HFD was mitigated in the HFD + EX group, indicating a decrease in PINK1 protein level. Our findings demonstrated that moderate aerobic exercise training mitigated obesity-induced insulin resistance by improving mitochondrial function, and reversed obesity-induced mitochondrial structural damage by improving mitochondrial dynamics and mitophagy, suggesting that moderate aerobic exercise training may play a therapeutic role in protecting the skeletal muscle against mitochondrial impairments and insulin resistance induced by obesity.

Characterization of early postzygotic somatic mutations through multi-organ analysis.

Mosaicisms caused by postzygotic mutational events are of increasing interest because of their potential association with various human diseases. Postzygotic somatic mutations have not been well characterized however in terms of their developmental lineage in humans. We conducted whole-genome sequencing (WGS) and targeted deep sequencing in 15 organs across three developmental lineages from a single male fetus with polycystic kidney disease (PKD) of 21 weeks gestational age. This fetus had no detectable neurological abnormalities at autopsy but germline mutations in the PKHD1 gene were identified that may have been associated with the PKD. Eight early embryonic mosaic variants with no alteration of protein function were detected. These variants were thought to have occurred at the two or four cell stages after fertilization with a mutational pattern involving frequent C>T and T>C transitions. In our current analyses, no tendency toward organ-specific mutation occurrences was found as the eight variants were detected in all 15 organs. However different allele fractions of these variants were found in different organs, suggesting a tissue-specific asymmetric growth of cells that reflected the developmental germ layer of each organ. This indicated that somatic mutation occurrences, even in early embryogenesis, can affect specific organ development or disease. Our current analyses demonstrate that multi-organ analysis is helpful for understanding genomic mosaicism. Our results also provide insights into the biological role of mosaicism in embryonic development and disease.

A tunable assay for modulators of genome-destabilizing DNA structures.

Regions of genomic instability are not random and often co-localize with DNA sequences that can adopt alternative DNA structures (i.e. non-B DNA, such as H-DNA). Non-B DNA-forming sequences are highly enriched at translocation breakpoints in human cancer genomes, representing an endogenous source of genetic instability. However, a further understanding of the mechanisms involved in non-B DNA-induced genetic instability is needed. Small molecules that can modulate the formation/stability of non-B DNA structures, and therefore the subsequent mutagenic outcome, represent valuable tools to study DNA structure-induced genetic instability. To this end, we have developed a tunable Förster resonance energy transfer (FRET)-based assay to detect triplex/H-DNA-destabilizing and -stabilizing ligands. The assay was designed by incorporating a fluorophore-quencher pair in a naturally-occurring H-DNA-forming sequence from a chromosomal breakpoint hotspot in the human c-MYC oncogene. By tuning triplex stability via buffer composition, the assay functions as a dual-reporter that can identify stabilizers and destabilizers, simultaneously. The assay principle was demonstrated using known triplex stabilizers, BePI and coralyne, and a complementary oligonucleotide to mimic a destabilizer, MCRa2. The potential of the assay was validated in a 384-well plate with 320 custom-assembled compounds. The discovery of novel triplex stabilizers/destabilizers may allow the regulation of genetic instability in human genomes.

Whole transcriptome analysis of gestational trophoblastic neoplasms reveals altered PI3K signaling pathway in epithelioid trophoblastic tumor.

OBJECTIVE: Genomic characteristics of gestational trophoblastic neoplasm (GTN) are mostly unknown. This study reveals the molecular features of malignant GTN, including choriocarcinoma (CC), epithelioid trophoblastic tumor (ETT), and placental site trophoblastic tumor (PSTT), by whole transcriptome sequencing analysis. METHODS: Data obtained from the total RNA sequencing of 2 CC, 4 ETT, and 4 PSTT were evaluated for differential gene expression, pathway alteration, fusion gene, infiltrating immune cell type, PD-L1 and PTEN expression level, and mutation analysis was performed. RESULTS: The transcriptome data were correlated with known biomarkers, including HDS3B1, p63, hCG, and hPL for all tumor types. ETT and PSTT were more closely clustered compared with CC in clustering analysis using gene expression; however, ETT showed various altered signaling pathways, including PI3K-Akt-mTOR, with frequent loss of PTEN protein expression. This finding was both well correlated with PIK3CA c.3140A > G pathogenic mutation, detected in 1 ETT, and further confirmed using the MassARRAY method. PSTT showed an overexpressed gene cluster associated with muscle contraction and G protein-coupled receptor activity. No significant fusion gene was seen in all 10 cases. In tumor-infiltrating immune cell profiles, CD4 memory T cell and macrophage signature were relatively high in ETT and PSTT. PD-L1 mRNA expression level was high in all cases, which was significantly correlated with the PD-L1 level by immunohistochemistry (p = 0.03) with positivity in all 10 cases. CONCLUSIONS: ETT and PSTT were similar at the transcriptome level, with a high level of PD-L1 expression in all tumor types; however, specific pathways, such as PI3K signaling, were altered in ETT.

Effect of post-operative anticoagulation management in patients who have undergone On-X mechanical heart valve replacement surgery on post-discharge warfarin therapy.

WHAT IS KNOWN AND OBJECTIVE: We evaluated the effect of the proportion of time maintained within the target international normalized ratio (INR) postoperatively in hospitalized patients who underwent On-X mechanical heart valve replacement on warfarin therapy after discharge. METHODS: Inclusion was patients who were ≥18 years, received warfarin for a minimum of 10 days without any interruptions during hospitalization and followed by the anticoagulation service (ACS) clinic after discharge between June 2006 and June 2016. Patients were excluded if they had incomplete medical records, INR goal changes, known as warfarin resistance, transferred to another facility or expired during the study. The patients were divided into 3 groups according to the proportion of time maintained within therapeutic INR range (TTR) from day 4 to 10 of warfarin initiation (low: <30%, moderate: ≥30% to <70%, and high: ≥70%). The number of days needed to reach target INR for 2 consecutive measurements after discharge and the number of ACS visits was compared among the groups. RESULTS AND DISCUSSION: Among 539 postoperative patients, 273 were included. The baseline demographics were similar among the 3 groups. The mean time needed to reach target INR for 2 consecutive measurements was 68.6 ± 106.1 days. The low group required time needed to reach target INR for 2 consecutive measurements of 94.0 ± 140.9 days compared with 44.8 ± 57.1 days in the high group (P = .007). Additionally, the low group had more ACS visits than the high group (low, 6.6 ± 5.2 vs high, 4.6 ± 3.9; P = .025). Patient compliance affected the time needed to reach target INR for 2 consecutive measurements (compliant, 42.36 ± 58.5 days vs non-compliant, 132.0 ± 157.1 days, P < .001). WHAT IS NEW AND CONCLUSION: The study implicated that high postoperative TTR would reduce the time to require post-discharge target INR and the number of ACS visits.

Re-Setting the Circadian Clock Using Exercise against Sarcopenia.

Sarcopenia is defined as the involuntary loss of skeletal muscle mass and function with aging and is associated with several adverse health outcomes. Recently, the disruption of regular circadian rhythms, due to shift work or nocturnal lifestyle, is emerging as a novel deleterious factor for the development of sarcopenia. The underlying mechanisms responsible for circadian disruption-induced sarcopenia include molecular circadian clock and mitochondrial function associated with the regulation of circadian rhythms. Exercise is a potent modulator of skeletal muscle metabolism and is considered to be a crucial preventative and therapeutic intervention strategy for sarcopenia. Moreover, emerging evidence shows that exercise, acting as a zeitgeber (time cue) of the skeletal muscle clock, can be an efficacious tool for re-setting the clock in sarcopenia. In this review, we provide the evidence of the impact of circadian disruption on skeletal muscle loss resulting in sarcopenia. Furthermore, we highlight the importance of exercise timing (i.e., scheduled physical activity) as a novel therapeutic strategy to target circadian disruption in skeletal muscle.

Immunogenomic landscape of hepatocellular carcinoma with immune cell stroma and EBV-positive tumor-infiltrating lymphocytes.

BACKGROUND & AIMS: The immunogenomic characteristics of hepatocellular carcinomas (HCCs) with immune cell stroma (HCC-IS), defined histologically, have not been clarified. We investigated the clinical and molecular features of HCC-IS and the prognostic impact of Epstein-Barr virus (EBV) infection. METHODS: We evaluated 219 patients with conventional HCC (C-HCC) and 47 with HCC-IS using in situ hybridization for EBV, immunohistochemistry, multiplex immunofluorescence staining, and whole exome and transcriptome sequencing. Human leukocyte antigen types were also extracted from the sequencing data. Genomic and prognostic parameters were compared between HCC-IS and C-HCC. RESULTS: CD8 T cell infiltration was more frequent in HCC-IS than C-HCC (mean fraction/sample, 22.6% vs. 8.9%, false discovery rate q <0.001), as was EBV positivity in CD20-positive tumor-infiltrating lymphocytes (TILs) (74.5% vs. 4.6%, p <0.001). CTNNB1 mutations were not identified in any HCC-IS, while they were present in 24.1% of C-HCC (p = 0.016). Inhibitory and stimulatory immune modulators were expressed at similar levels in HCC-IS and EBV-positive C-HCC. Global hypermethylation, and expression of PD-1 and PD-L1 in TILs, and PD-L1 in tumors, were also associated with HCC-IS (p <0.001), whereas human leukocyte antigen type did not differ according to HCC type or EBV positivity. HCC-IS was an independent factor for favorable recurrence-free survival (adjusted hazard ratio [aHR] 0.23; p = 0.002). However, a subgroup of tumors with a high density of EBV-positive TILs had poorer recurrence-free (aHR 25.48; p <0.001) and overall (aHR 9.6; p = 0.003) survival, and significant enrichment of CD8 T cell exhaustion signatures (q = 0.0296). CONCLUSIONS: HCC-IS is a distinct HCC subtype associated with a good prognosis and frequent EBV-positive TILs. However, paradoxically, a high density of EBV-positive TILs in tumors is associated with inferior prognostic outcomes. Patients with HCC-IS could be candidates for immunotherapy. LAY SUMMARY: Hepatocellular carcinomas with histologic evidence of abundant immune cell infiltration are characterized by frequent activation of Epstein-Barr virus in tumor-infiltrating lymphocytes and less aggressive clinical behavior. However, a high density of Epstein-Barr virus-positive tumor-infiltrating lymphocytes is associated with inferior prognostic outcomes, possibly as a result of immune escape due to significant CD8 T cell exhaustion.

Layered Bismuth Oxyfluoride Nitrates Revealing Large Second-Harmonic Generation and Photocatalytic Properties.

Two novel bismuth oxyfluoride nitrates, Bi2OF3(NO3) and Bi6O6F5(NO3), have been synthesized via hydrothermal reactions. Whereas Bi2OF3(NO3) crystallizes in the centrosymmetric (CS) hexagonal space group, P63/ m, Bi6O6F5(NO3) crystallizes in the polar noncentrosymmetric (NCS) trigonal space group, R3. The backbones of the title compounds reveal double layered structures composed of asymmetric BiF3(O/F)3 or BiO3F2 polyhedra and NO3 trigonal planar groups. The diffuse reflectance spectra indicate that Bi2OF3(NO3) and Bi6O6F5(NO3) contain wide band gaps of 3.5 and 4.0 eV, respectively. Powder second-harmonic generation (SHG) measurements suggest that NCS Bi6O6F5(NO3) is Type-I phase-matchable and has a large SHG response of ca. 3 times that of KH2PO4 (KDP). Electron localization function (ELF) analysis indicates that the large SHG efficiency of Bi6O6F5(NO3) is attributed to the synergistic effect of the alignment of NO3- trigonal planar groups and strong interactions between highly polarizable lone pair electrons on Bi3+ and π-delocalized electrons in NO3- groups. Bi2OF3(NO3) also exhibits a very good photocatalytic degradation efficiency of Rhodamine B (RhB) under the UV light irradiation.

Effect of gastrectomy on blood pressure in early gastric cancer survivors with hypertension.

PURPOSE: We investigated the effect of gastrectomy on blood pressure (BP) in early gastric cancer survivors with hypertension and whether well-controlled BP was due solely to surgery-induced weight loss. METHODS: The study enrolled 66 early gastric cancer patients with hypertension, undergoing endoscopic submucosal dissection (ESD), or gastrectomy. Blood analyses, 24-h ambulatory BP monitoring, brachial ankle pulse wave velocity (baPWV), and echocardiography were measured prior to, 3 months after, and 1 year after ESD or gastrectomy. The primary endpoint was remission of hypertension at 1 year. RESULTS: The remission rate of hypertension was significantly higher in the gastrectomy group than in the ESD group (p = 0.006). Those with remission of hypertension had a significant weight loss (p < 0.001), decrease in body mass index (p < 0.001), 24-h total systolic BP (p = 0.047), baPWV (p = 0.042), triglycerides (p = 0.049) and apolipoprotein B/apolipoprotein A1 (p = 0.004), and an increase in high-density lipoprotein cholesterol (p < 0.001) at 1 year. Upon multivariate logistic regression analysis, gastrectomy [odds ratio (OR) = 7.77, 95% confidence interval (CI) = 2.05-35.89], diuretic use (OR = 3.76, 95% CI = 1.14-13.98), and lower 24-h total diastolic BP before treatment (OR = 0.90, 95% CI = 0.82-0.96) were predictive of remission of hypertension after adjusting for percent weight. CONCLUSIONS: In early gastric cancer survivors with hypertension, gastrectomy resulted in better BP control than did ESD, which may be due to the gastrectomy itself, beyond weight loss. Therefore, it should be remembered that the adequate reduction of antihypertensives may be necessary in early gastric cancer survivors after gastrectomy.

Factors related to inappropriate edoxaban use.

WHAT IS KNOWN AND OBJECTIVE: The aim of this study was to evaluate the appropriateness and clinical outcomes of edoxaban use, and to determine the role of clinical pharmacists in improving the efficacy and safety of edoxaban use. METHODS: A retrospective study was performed by using an electronic medical record and anticoagulation clinical data from 600 patients who received edoxaban from 1 March 2016 to 16 July 2017 at a tertiary teaching university hospital. The appropriateness of edoxaban use was assessed using eight criteria based on drug use evaluation criteria developed by the American Society of Health-System Pharmacists drug use evaluation guidelines, details in Korea Food and Drug Administration approval of edoxaban. Clinical outcomes were evaluated between the appropriately prescribed and inappropriately prescribed groups regarding the incidence of thrombosis and bleeding episodes. RESULTS AND DISCUSSION: After excluding 86 patients due to the inability to assess renal function, 514 were eligible. Appropriate use was found in 294 patients (57.2%). The most frequent inappropriate use of edoxaban was dose adjustment (60.8%) in accordance with the dosing recommendation in patients with renal insufficiency (creatinine clearance [CrCl] of 15-50 mL/min) and a low body weight of <60 kg. Moreover, there were three cases of edoxaban use in patients with prosthetic heart valves and moderate-to-severe mitral stenosis, and 15 cases of non-valvular atrial fibrillation in patients with CrCl >95 mL/min in whom edoxaban use is not recommended. Furthermore, we found that the factors related to the appropriateness of edoxaban use were <60 kg body weight (adjusted odds ratio [OR]: 0.310; confidence interval [CI]: 0.197-0.488) and CrCl <50 mL/min (adjusted OR: 0.629; CI: 0.404-0.980). There were 45 events (8.75%) of any bleeding, 9 (1.8%) of stroke/transient ischaemic attack (TIA) and four events (0.8%) of deep vein thrombosis (DVT)/pulmonary embolism (PE). However, there was no difference between the appropriately prescribed group (294 patients) and inappropriately prescribed group (220 patients) in the incidence of bleeding events (27 [9.2%] vs 18 [8.2%]), stroke/TIA (7 [2.4%] vs 2 [0.9%]) and DVT/PE (2 [0.7%] vs 2 [0.9%]), respectively. WHAT IS NEW AND CONCLUSION: Although edoxaban has a broad therapeutic window that does not require routine monitoring, it should be cautiously used in patients with renal insufficiency (CrCl <50 mL/min) and body weight <60 kg.

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay.

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli- or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.

Assessment of reverse remodeling predicted by myocardial deformation on tissue tracking in patients with severe aortic stenosis: a cardiovascular magnetic resonance imaging study.

BACKGROUND: The technique of tissue tracking with balanced steady-state free precession cine sequences was introduced, and allowed myocardial strain to be derived directly, offering advantages over traditional myocardial tagging. The aim of this study was to evaluate the correlation between reverse remodeling as an outcome and left ventricular strain using cardiovascular magnetic resonance imaging (CMR) tissue tracking, and to evaluate prediction of reverse remodeling by myocardial deformation in patients with severe aortic stenosis (AS). METHODS: We enrolled 63 patients with severe AS and normal left ventricular (LV) systolic function (ejection fraction > 60%), who underwent both CMR and transthoracic echocardiography (Echo) before surgical aortic valve replacement (AVR). CMR at 1.5 T, including non and post-contrast T1 mapping for extracellular volume (ECV), was carried out to define the amount of myocardial fibrosis. Cardiac Performance Analysis software was used to derive myocardial deformation as strain parameters from three short-axis cine views (basal, mid and apical levels) and apical 2, 3, and 4 chamber views. The primary outcome was reverse remodeling, as evaluated by regression of left ventricular mass index (LVMI). RESULTS: Median follow-up was 28.8 months (interquartile range 11.3-38.3 months). As evaluated by LVMI between baseline and follow-up, mass regression was significantly improved after AVR (baseline 145.9 ± 37.0 [g/m2] vs. follow-up 97.7 ± 22.2[g/m2], p < 0.001). Statistically significant Pearson's correlations with LVMI regression were observed for longitudinal global strain (r = -0.461, p < 0.001), radial strain (r = 0.391, p = 0.002), and circumferential strain (r = -0.334, p = 0.009). A simple linear regression analysis showed that all strain parameters could predict the amount of LVMI regression (P < 0.05), as well as non-contrast T1 value (beta = -0.314, p < 0.001) and ECV (beta = -2.546, p = 0.038). However, ECV had the lowest predictive power (multiple r2 = 0.071). Multiple regression analysis showed strain could independently predict the amount of LVMI regression and the longitudinal global strain (beta = -3.335, p < 0.001). CONCLUSION: Longitudinal global strain measured by CMR tissue tracking as a technique was correlated with reverse remodeling as LVMI regression and was predictive of this outcome. As a simple and practical method, tissue tracking is promising to assess strain and predict reverse remodeling in severe AS, especially in patients with suboptimal Echo image quality.

Discovery of a potent inhibitor of MELK that inhibits expression of the anti-apoptotic protein Mcl-1 and TNBC cell growth.

Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.

Effects of increased left ventricular wall thickness on the myocardium in severe aortic stenosis with normal left ventricular ejection fraction: Two- and three-dimensional multilayer speckle tracking echocardiography.

BACKGROUND: The aim of this study was to determine the capability of real time three-dimensional echocardiography (RT3DE) and two-dimensional (2D) multilayer speckle tracking echocardiography (MSTE) for evaluation of early myocardial dysfunction triggered by increased left ventricular (LV) wall thickness in severe aortic stenosis (AS) with normal LV ejection fraction (EF≥55%). METHODS: Conventional, RT3D STE and 2D MSTE were performed in 45 patients (mean 68.9±9.0 years) with severe AS (aortic valve area <1 cm2 , aortic velocity Vmax >4 m/s or mean PG >40 mm Hg) and normal left ventricular ejection fraction (LVEF) without overt coronary artery disease and in 18 age-, sex-matched healthy controls. Global longitudinal strain (GLS), global circumferential strain (GCS), global area strain (GAS), and global radial strain (GRS) were calculated using RT3DE and MSTE. RESULTS: The severe AS group had lower 3D GLS, GRS, GAS and 2D epicardium, and mid-wall and endocardium GLS compared to healthy controls. In MSTE analysis, 2D LS and CS values decreased from the endocardial layer toward the epicardial layer. Severe AS patients with increased LV wall thickness had lower 3D GLS and 2D epicardium, and mid-wall and endocardium GLS compared with severe AS patients without LV wall thickening. GLS on RT3D STE was correlated with GLS on 2D MSTE, left ventricular mass index, LVEF, left atrial volume index, and lnNT-proBNP. CONCLUSION: RT3DE and 2D MSTE can be used to identify subtle contractile dysfunction triggered by increased LV wall thickness in severe AS with normal LVEF. Therefore, RT3D STE and 2D MSTE may provide additional information that can facilitate decision-making regarding severe AS patients with increased LV wall thickness and normal LV function.

Identification of Factors that Predict whether the Right Parasternal View Is Required for Accurate Evaluation of Aortic Stenosis Severity.

BACKGROUND: To accurately assess aortic stenosis (AS) severity, multiple windows should be used to best align the Doppler beam with the flow direction of the stenotic jet. To evaluate: (1) the frequency at which the highest peak AV velocity is found in the right parasternal window (RPW), (2) the extent of correlation between aortic root angulation and the location of the highest peak AV jet velocity, and (3) which patients would benefit most from RPW-based assessment of AS severity. METHODS: We evaluated AS velocity in the apical window (AW) and RPW by continuous-wave Doppler (CWD) in 263 patients with more than moderate AS. If the AV was inaccessible by the left parasternal long-axis view, the aortoseptal angle was measured by echocardiography and a sagittal view of a computed tomography (CT). RESULTS: Patients were divided into two groups, the RPW group and the AW group. Significant differences were observed between the two groups regarding the sinus of Valsalva diameter, the aortoseptal angle as measured by echocardiography, and the type of valve. The aortoseptal angle as measured by echocardiography was larger in the AW group compared with the RPW group. Aortoseptal angle (P < 0.001) and valve type (P = 0.036) were independent predictors that the highest AV Vmax would be obtained from the RPW. CONCLUSIONS: The right parasternal window must be evaluated to achieve the most accurate assessment of AS severity, especially in patients with an aortoseptal angle <119° in the parasternal long-axis view as measured by echocardiography.

Intrinsic resistance triggered under acid loading within normal esophageal epithelial cells: NHE1- and ROS-mediated survival.

The transition to a pathological phenotype such as Barrett's esophagus occurs via induction of resistance upon repeated contact with gastric refluxate in esophagus. This study examined the molecular changes within normal esophageal epithelial cells (EECs) under short-term acid loading and the role of these changes in defensive resistance against acidic cytotoxicity. After primary cultured EECs were exposed to pH 4-acidified medium (AM4), cell viability was determined by the MTT assay. Reactive oxygen species (ROS) and NAD(P)H oxidase (NOX) activity were measured. Activation of the mitogen-activated protein kinases (MAPKs) MEK/ERK1/2, p38 and JNK; phosphoinositol-3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-κB) were detected by Western blot analysis or immunofluorescence staining. AM4 incubation induced intracellular ROS generation accompanied by increase in NOX activity, which was further increased by Na(+) /H(+) exchange-1 (NHE1)-dependent inhibition but was prevented by inhibition of NOX or mitochondria complex I. AM4 also induced phosphorylation of MEK/ERK1/2, p38 MAPK, PI3K/Akt, and nuclear translocation of NF-κB, and all these effects, except for p38 MAPK phosphorylation, were abolished by inhibition of ROS. ROS-dependent PI3K/Akt activation, which mediates NF-κB nuclear translocation, was inhibited by protein tyrosine kinase (PTK) inhibitors and NHE1-specific inhibitor. All inhibitors of NHE, ROS, PTK, PI3K, or NF-κB further decreased AM4-induced cell viability. Acid loading in the presence of NHE1-dependent protection induced ROS generation by activating NOX and mitochondria complex I, which stimulated PTK/PI3K/Akt/NF-κB-dependent survival in EEC. Our data indicate that normal EEC initially respond to acid loading through intrinsic survival activation.

Aggravating factors associated with the severity of aortic regurgitation and clinical characteristics of patients with quadricuspid aortic valve.

BACKGROUND AND AIM OF THE STUDY: Quadricuspid aortic valve (QAV) is a rare congenital anomaly. Aortic regurgitation (AR) has been suggested to be the major functional abnormality of QAV. The study aim was to define the clinical characteristics of patients with QAV through echocardiography, thereby identifying factors related to the severity of AR. METHODS: Patients diagnosed with QAV by echocardiography were enrolled from electronic medical records obtained between November 1994 and May 2012. RESULTS: Twenty-six patients diagnosed with QAV were identified. AR was present in 20 of these patients (77%), among which 12 (46%) exhibited significant AR. Aortic stenosis was rare (n = 1; 4%). Patient age and severity of AR were weakly correlated. The most frequent valve morphology type was three equally sized cusps and one smaller cusp (n = 12; 46%), followed by two larger, equally sized cusps and two smaller, equally sized cusps (n = 6; 23%). The latter morphology type was accompanied by a high prevalence of significant AR (5/6; 83%). The initial clinical symptoms of the patients included dyspnea and chest pain (n = 14; 54%), other congenital abnormalities (n = 4; 15%), and arrhythmias (n = 4; 15%). Among the patients with significant AR, 10 were symptomatic. CONCLUSION: Approximately half of the patients with QAV had significant AR at the time of initial diagnosis by echocardiography. The severity of AR appeared to be correlated with patient age and valve morphology. These factors should be considered during echocardiographic assessment and follow up treatment for QAV.

Reversible covalent inhibition of eEF-2K by carbonitriles.

eEF-2K is a potential target for treating cancer. However, potent specific inhibitors for this enzyme are lacking. Previously, we identified 2,6-diamino-4-(2-fluorophenyl)-4H-thiopyran-3,5-dicarbonitrile (DFTD) as an inhibitor of eEF-2K. Here we describe its mechanism of action against eEF-2K, on the basis of kinetic, mutational, and docking studies, and use chemoinformatic approaches to identify a similar class of carbonitrile-containing compounds that exhibit the same mechanism of action. We show that DFTD behaves as a reversible covalent inhibitor of eEF-2K with a two-step mechanism of inhibition: a fast initial binding step, followed by a slower reversible inactivation step. Molecular docking suggests that a nitrile group of DFTD binds within 4.5 Å of the active site Cys146 to form a reversible thioimidate adduct. Because Cys146 is not conserved amongst other related kinases, targeting this residue holds promise for the development of selective covalent inhibitors of eEF-2K.