Exercise heat acclimation has minimal effects on left ventricular volumes, function and systemic hemodynamics in euhydrated and dehydrated trained humans.

Heat acclimation (HA) may improve the regulation of cardiac output (Q̇) through increased blood volume (BV) and left ventricular (LV) diastolic filling and attenuate reductions in Q̇ during exercise-induced dehydration; however, these hypotheses have never been directly tested. Before and following 10-days exercise HA, eight males completed two trials of submaximal exercise in 33°C and 50% relative humidity while maintaining preexercise euhydrated body mass (EUH; -0.6 ± 0.4%) or becoming progressively dehydrated (DEH; -3.6 ± 0.7%). Rectal (Tre) and skin (Tsk) temperatures, heart rate (HR), LV volumes and function, systemic hemodynamics and BV were measured at rest and during bouts of semirecumbent cycling (55% V̇o2max) at 20, 100 and 180 min, interspersed by periods of upright exercise. Tre, BV, HR, LV volumes, LV systolic and diastolic function, and systemic hemodynamics were similar between trials at rest and during the first 20 min of exercise (all P > 0.05). These responses were largely unaffected by HA at 180 min in either hydration state. However, DEH induced higher Tre (0.6 ± 0.3°C) and HR (16 ± 7 beats/min) and lower end-diastolic volume (29 ± 16 mL), stroke volume (26 ± 16 mL), and Q̇ (2.1 ± 0.8 L/min) compared with EUH at 180 min (all P < 0.05), yet LV twist and untwisting rate were increased or maintained (P = 0.028 and 0.52, respectively). Findings indicate HA has minimal effects on LV volumes, LV mechanical function, and systemic hemodynamics during submaximal exercise in moderate heat, where HR and BV are similar. In contrast, DEH evokes greater hyperthermia and tachycardia, reduces BV, and impairs diastolic LV filling, lowering Q̇, regardless of HA state.NEW & NOTEWORTHY This study demonstrates that 10 days of exercise heat acclimation has minimal effects on left ventricular volumes, intrinsic cardiac function, and systemic hemodynamics during prolonged, repeated semirecumbent exercise in moderate heat, where heart rate and blood volume are similar to preacclimation levels. However, progressive dehydration is consistently associated with similar degrees of hyperthermia and tachycardia and reductions in blood volume, diastolic filling of the left ventricle, stroke volume, and cardiac output, regardless of acclimation state.

Noncoding Variations in the Gene Encoding Ceramide Synthase 6 are Associated with Type 2 Diabetes in a Large Indigenous Australian Pedigree.

Type 2 diabetes (T2D) is a chronic disease that disproportionately affects Indigenous Australians. We have previously reported the localization of a novel T2D locus by linkage analysis to chromosome 2q24 in a large admixed Indigenous Australian pedigree (Busfield et al. (2002). American Journal of Human Genetics, 70, 349-357). Here we describe fine mapping of this region in this pedigree, with the identification of SNPs showing strong association with T2D: rs3845724 (diabetes p = 7 × 10-4), rs4668106 (diabetes p = 9 × 10-4) and rs529002 (plasma glucose p = 3 × 10-4). These associations were successfully replicated in an independent collection of Indigenous Australian T2D cases and controls. These SNPs all lie within the gene encoding ceramide synthase 6 (CERS6) and thus may regulate ceramide synthesis.

Identification of second-generation P2X3 antagonists for treatment of pain.

A second-generation small molecule P2X3 receptor antagonist has been developed. The lead optimization strategy to address shortcomings of the first-generation preclinical lead compound is described herein. These studies were directed towards the identification and amelioration of preclinical hepatobiliary findings, reducing potential for drug-drug interactions, and decreasing the projected human dose of the first-generation lead.

Transition-Metal Hydride Radical Cations.

Transition-metal hydride radical cations (TMHRCs) are involved in a variety of chemical and biochemical reactions, making a more thorough understanding of their properties essential for explaining observed reactivity and for the eventual development of new applications. Generally, these species may be treated as the ones formed by one-electron oxidation of diamagnetic analogues that are neutral or cationic. Despite the importance of TMHRCs, the generally sensitive nature of these complexes has hindered their development. However, over the last four decades, many more TMHRCs have been synthesized, characterized, isolated, or hypothesized as reaction intermediates. This comprehensive review focuses on experimental studies of TMHRCs reported through the year 2014, with an emphasis on isolated and observed species. The methods used for the generation or synthesis of TMHRCs are surveyed, followed by a discussion about the stability of these complexes. The fundamental properties of TMHRCs, especially those pertaining to the M-H bond, are described, followed by a detailed treatment of decomposition pathways. Finally, reactions involving TMHRCs as intermediates are described.

Cerastecins inhibit membrane lipooligosaccharide transport in drug-resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.

ATP citrate lyase inhibition can suppress tumor cell growth.

Many tumors display a high rate of glucose utilization, as evidenced by 18-F-2-deoxyglucose PET imaging. One potential advantage of catabolizing glucose through glycolysis at a rate that exceeds bioenergetic need is that the growing cell can redirect the excess glycolytic end product pyruvate toward lipid synthesis. Such de novo lipid synthesis is necessary for membrane production and lipid-based posttranslational modification of proteins. A key enzyme linking glucose metabolism to lipid synthesis is ATP citrate lyase (ACL), which catalyzes the conversion of citrate to cytosolic acetyl-CoA. ACL inhibition by RNAi or the chemical inhibitor SB-204990 limits in vitro proliferation and survival of tumor cells displaying aerobic glycolysis. The same treatments also reduce in vivo tumor growth and induce differentiation.

A New Tool to Aid the Differential Diagnosis of Physiological Remodelling from Cardiac Pathology When Assessing Left Ventricle, Left Atrial and Aortic Structure and Function in Male Arab and Black Paediatric Athletes.

Aim: To determine if published Z-scores for left ventricular (LV), left atrial (LA) and aortic structure as well as indices of LV function (Doppler and TDI) in paediatric athletes and non-athletes are appropriate for application in male Arab and black paediatric athletes. If inappropriate, we aim to provide new nomograms and Z-scores for clinical application. Methods: 417 (297 Arab, 120 black) male paediatric (11-18 years) athletes, were evaluated by 2D echocardiography as per British Society of Echocardiography recommendations, and biological age (by radiological X-ray) assessment. Z-scores were tested by residual and correlation analysis together with visual inspection. New Z-scores involved allometric (a*BSA(b+c*chronological age)) and second-order polynomial (y=a*chronological age2+b*chronological age+c) equations for measures of cardiac size and indices of LV function, respectively. Results: Residual linear regression, correlation analysis and visual inspection revealed published z-scores in white peri-pubertal footballers and paediatric non-athletes to be inappropriate for application in male Arab and black paediatric athletes. Residual linear regression revealed new Z-scores for measures of LV, LA and aortic root size to be independent of BSA, ethnicity, chronological and biological age. Residual linear regression revealed new Z-scores for measures of function to be independent of chronological age. Conclusion: Our new z-scores may aid differential diagnosis of suspected pathology versus physiology remodelling, in cardiac screening of the Arab and black paediatric athlete. Nomograms are provided to assist the tracking of the paediatric athlete necessitating annual follow-up and Excel z-score calculation to facilitate use in day-to-day practice.

Rapid HATU-mediated solution phase siRNA conjugation.

Conditions for facile solution-phase amide conjugation of amine-modified siRNA with a diverse set of carboxylic acid partners using the coupling reagent HATU are described. These conditions eliminate the need for isolated activated esters and allow for rapid access to conjugates with a wide range of lipophilicity and functionality in good yield.

Orally bioavailable imidazoazepanes as calcitonin gene-related peptide (CGRP) receptor antagonists: discovery of MK-2918.

In our ongoing efforts to develop CGRP receptor antagonists for the treatment of migraine, we aimed to improve upon telecagepant by targeting a compound with a lower projected clinical dose. Imidazoazepanes were identified as potent caprolactam replacements and SAR of the imidazole yielded the tertiary methyl ether as an optimal substituent for potency and hERG selectivity. Combination with the azabenzoxazinone spiropiperidine ultimately led to preclinical candidate 30 (MK-2918).

Exercise Heat Acclimation With Dehydration Does Not Affect Vascular and Cardiac Volumes or Systemic Hemodynamics During Endurance Exercise.

Permissive dehydration during exercise heat acclimation (HA) may enhance hematological and cardiovascular adaptations and thus acute responses to prolonged exercise. However, the independent role of permissive dehydration on vascular and cardiac volumes, ventricular-arterial (VA) coupling and systemic hemodynamics has not been systematically investigated. Seven males completed two 10-day exercise HA interventions with controlled heart rate (HR) where euhydration was maintained or permissive dehydration (-2.9 ± 0.5% body mass) occurred. Two experimental trials were conducted before and after each HA intervention where euhydration was maintained (-0.5 ± 0.4%) or dehydration was induced (-3.6 ± 0.6%) via prescribed fluid intakes. Rectal (Tre) and skin temperatures, HR, blood (BV) and left ventricular (LV) volumes, and systemic hemodynamics were measured at rest and during bouts of semi-recumbent cycling (55% V̇O2 peak) in 33°C at 20, 100, and 180 min. Throughout HA sweat rate (12 ± 9%) and power output (18 ± 7 W) increased (P < 0.05), whereas Tre was 38.4 ± 0.2°C during the 75 min of HR controlled exercise (P = 1.00). Neither HA intervention altered resting and euhydrated exercising Tre, BV, LV diastolic and systolic volumes, systemic hemodynamics, and VA coupling (P > 0.05). Furthermore, the thermal and cardiovascular strain during exercise with acute dehydration post-HA was not influenced by HA hydration strategy. Instead, elevations in Tre and HR and reductions in BV and cardiac output matched pre-HA levels (P > 0.05). These findings indicate that permissive dehydration during exercise HA with controlled HR and maintained thermal stimulus does not affect hematological or cardiovascular responses during acute endurance exercise under moderate heat stress with maintained euhydration or moderate dehydration.

Structural understanding of non-nucleoside inhibition in an elongating herpesvirus polymerase.

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

Discovery of an Anion-Dependent Farnesyltransferase Inhibitor from a Phenotypic Screen.

By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.

Pharmacological characterization of MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide], a potent and orally active calcitonin gene-related peptide receptor antagonist for the treatment of migraine.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide that plays a key role in the pathophysiology of migraine headache. CGRP levels in the cranial circulation are increased during a migraine attack, and CGRP itself has been shown to trigger migraine-like headache. The correlation between CGRP release and migraine headache points to the potential utility of CGRP receptor antagonists as novel therapeutics in the treatment of migraine. Indeed, clinical proof-of-concept in the acute treatment of migraine was demonstrated with an intravenous formulation of the CGRP receptor antagonist BIBN4096BS (olcegepant). Here we report on the pharmacological characterization of the first orally bioavailable CGRP receptor antagonist in clinical development, MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]. In vitro, MK-0974 is a potent antagonist of the human (K(i) = 0.77 nM) and rhesus (K(i) = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for the canine and rat receptors as determined via (125)I-human CGRP competition binding assays. A rhesus pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging was utilized to determine the in vivo activity of CGRP receptor antagonism. MK-0974 produced a concentration-dependent inhibition of dermal vasodilation, generated by capsaicin-induced release of endogenous CGRP, with plasma concentrations of 127 and 994 nM required to block 50 and 90% of the blood flow increase, respectively. In conclusion, MK-0974 is a highly potent, selective, and orally bioavailable CGRP receptor antagonist, which may be valuable in the acute treatment of migraine.

Synthesis of the (3R,6S)-3-amino-6-(2,3-difluorophenyl)azepan-2-one of telcagepant (MK-0974), a calcitonin gene-related peptide receptor antagonist for the treatment of migraine headache.

Two novel routes have been developed to the (3 R,6 S)-3-amino-6-(2,3-difluorophenyl)-1-(2,2,2-trifluoroethyl)azepan-2-one 2 of the CGRP receptor antagonist clinical candidate telcagepant (MK-0974, 1). The first employs a ring-closing metathesis of the styrene 7 as the key reaction, while the second makes use of a highly diastereoselective Hayashi-Miyaura Rh-catalyzed arylboronic acid addition to nitroalkene 16. The latter route has been implemented to produce multigram quantities of telcagepant for extensive preclinical evaluation.

Using a two-step hydride transfer to achieve 1,4-reduction in the catalytic hydrogenation of an acyl pyridinium cation.

The stoichiometric reduction of N-carbophenoxypyridinium tetraphenylborate (6) by CpRu(P-P)H (Cp = eta(5)-cyclopentadienyl; P-P = dppe, 1,2-bis(diphenylphosphino)ethane, or dppf, 1,1'-bis(diphenylphosphino)ferrocene), and Cp*Ru(P-P)H (Cp* = eta(5)-pentamethylcyclopentadienyl; P-P = dppe) gives mixtures of 1,2- and 1,4-dihydropyridines. The stoichiometric reduction of 6 by Cp*Ru(dppf)H (5) gives only the 1,4-dihydropyridine, and 5 catalyzes the exclusive formation of the 1,4-dihydropyridine from 6, H(2), and 2,2,6,6-tetramethylpiperidine. In the stoichiometric reductions, the ratio of 1,4 to 1,2 product increases as the Ru hydrides become better one-electron reductants, suggesting that the 1,4 product arises from a two-step (e(-)/H(*)) hydride transfer. Calculations at the UB3LYP/6-311++G(3df,3pd)//UB3LYP/6-31G* level support this hypothesis, indicating that the spin density in the N-carbophenoxypyridinium radical (13) resides primarily at C4, while the positive charge in 6 resides primarily at C2 and C6. The isomeric dihydropyridines thus result from the operation of different mechanisms: the 1,2 product from a single-step H(-) transfer and the 1,4 product from a two-step (e(-)/H(*)) transfer.

Calcitonin gene-related peptide (CGRP) receptor antagonists: investigations of a pyridinone template.

In our effort to find potent, orally bioavailable CGRP receptor antagonists for the treatment of migraine, a novel series based on a pyridinone template was investigated. After optimizing the privileged structure and the placement of the attached phenyl ring, systematic SAR was carried out on both the N-alkyl and C-5 aryl substituents. Several analogs with good potency and pharmacokinetic profiles were identified.

Determination of 2D poloidal maps of the intrinsic W density for transport studies in JET-ILW.

The experimental method developed at ASDEX Upgrade for the determination of the intrinsic tungsten (W) density profile coupling data from the soft X-ray (SXR) diagnostic and vacuum-ultra-violet (VUV) spectroscopy has been upgraded for application to JET plasmas. The strong poloidal asymmetries in the SXR emission are modeled assuming a ln ( ϵ ( ρ , R ) / ϵ ( ρ , R 0 ) ) = λ ( ρ ) ( R 2 - R 0 2 ) distribution, where ρ is the flux coordinate, R is the major radius, and λ is the fit parameter. The W density is calculated from the resulting 2D SXR emissivity maps accounting for contributions from a low-Z impurity (typically beryllium) and main ion with the assumption that their contributions are poloidally symmetric. Comparing the result with the independent W concentration measurement of VUV spectroscopy, a recalibration factor for the SXR emissivity is calculated making the method robust against the decrease in the sensitivity of the SXR diodes which has been observed across multiple campaigns. The final 2D W density map is checked for consistency versus the time-evolution of the W concentration measurement from VUV spectroscopy, toroidal rotation measurements from charge exchange recombination spectroscopy, and tomographic reconstructions of bolometry data. The method has been found to be robust for W concentrations above a few 10-5 and in cases where the contributions from other medium-Z impurities such as Ni are negligible.

Clinical expression of plakophilin-2 mutations in familial arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder characterized by loss of cardiomyocytes and their replacement by adipose and fibrous tissue. It is considered a disease of cell adhesion because mutations in desmosomal genes, desmoplakin and plakoglobin, have been implicated in the pathogenesis of ARVC. In a recent report, mutations in plakophilin-2, a gene highly expressed in cardiac desmosomes, have been shown to cause ARVC. METHODS AND RESULTS: We investigated 100 white patients with ARVC for mutations in plakophilin-2. Nine different mutations were identified by direct sequencing in 11 cases. Five of these mutations are novel (A733fsX740, L586fsX658, V570fsX576, R413X, and P533fsX561) and predicted to cause a premature truncation of the plakophilin-2 protein. Family studies showed incomplete disease expression in mutation carriers and identified a number of individuals who would be misdiagnosed with the existing International Task Force and modified diagnostic criteria for ARVC. CONCLUSIONS: In this study, we provide new evidence that mutations in the desmosomal plakophilin-2 gene can cause ARVC. A systematic clinical evaluation of mutation carriers within families demonstrated variable phenotypic expression, even among individuals with the same mutation, and highlighted the need for a more accurate set of diagnostic criteria for ARVC.

Idiopathic restrictive cardiomyopathy is part of the clinical expression of cardiac troponin I mutations.

Restrictive cardiomyopathy (RCM) is an uncommon heart muscle disorder characterized by impaired filling of the ventricles with reduced volume in the presence of normal or near normal wall thickness and systolic function. The disease may be associated with systemic disease but is most often idiopathic. We recognized a large family in which individuals were affected by either idiopathic RCM or hypertrophic cardiomyopathy (HCM). Linkage analysis to selected sarcomeric contractile protein genes identified cardiac troponin I (TNNI3) as the likely disease gene. Subsequent mutation analysis revealed a novel missense mutation, which cosegregated with the disease in the family (lod score: 4.8). To determine if idiopathic RCM is part of the clinical expression of TNNI3 mutations, genetic investigations of the gene were performed in an additional nine unrelated RCM patients with restrictive filling patterns, bi-atrial dilatation, normal systolic function, and normal wall thickness. TNNI3 mutations were identified in six of these nine RCM patients. Two of the mutations identified in young individuals were de novo mutations. All mutations appeared in conserved and functionally important domains of the gene. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Potent, orally bioavailable calcitonin gene-related peptide receptor antagonists for the treatment of migraine: discovery of N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1- (2,2,2-trifluoroethyl)azepan-3-yl]-4- (2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin- 1-yl)piperidine-1-carboxamide (MK-0974).

Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Herein we describe optimization of CGRP receptor antagonists based on an earlier lead structure containing a (3R)-amino-(6S)-phenylcaprolactam core. Replacement of the phenylimidazolinone with an azabenzimidazolone gave stable derivatives with lowered serum shifts. Extensive SAR studies of the C-6 aryl moiety revealed the potency-enhancing effect of the 2,3-difluorophenyl group, and trifluoroethylation of the N-1 amide position resulted in improved oral bioavailabilities, ultimately leading to clinical candidate 38 (MK-0974).