Quantification of carbonic anhydrase gene expression in ventricle of hypertrophic and failing human heart

Background: Carbonic anhydrase enzymes (CA) catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+ /H+ exchange (NHE) activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide), a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH) in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms. Methods: We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14), or heart transplantation (HT, n = 13). CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls. Results: Expression of atrial and brain natriuretic peptide (ANP and BNP) were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles. Conclusions: These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac hypertrophy.Fil: Alvarez, Bernardo. Universidad Nacional de la Plata. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Quon, Anita L.. University Of Alberta. Faculty Of Medicine And Oral Health Sciences; CanadáFil: Mullen, John. University of Alberta; CanadáFil: Casey, Joseph R.. University Of Alberta. Faculty Of Medicine And Oral Health Sciences; Canad

Quantification of carbonic anhydrase gene expression in ventricle of hypertrophic and failing human heart

Background: Carbonic anhydrase enzymes (CA) catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE) activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide), a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH) in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms.Methods: We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14), or heart transplantation (HT, n = 13). CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls.Results: Expression of atrial and brain natriuretic peptide (ANP and BNP) were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles.Conclusions: These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac hypertrophy.Centro de Investigaciones Cardiovasculare

Three-Dimensional Model for the Human Cl−/HCO3− Exchanger, AE1, by Homology to the E. coli ClC Protein

AbstractAE1 mediates electroneutral 1:1 exchange of bicarbonate for chloride across the plasma membrane of erythrocytes and type A cells of the renal collecting duct. No high-resolution structure is available for the AE1 membrane domain, which alone is required for its transport activity. A recent electron microscopy structure of the AE1 membrane domain was proposed to have a similar protein fold to ClC chloride channels. We developed a three-dimensional homology model of the AE1 membrane domain, using the Escherichia coli ClC channel structure as a template. This model agrees well with a long list of biochemically established spatial constraints for AE1. To investigate the AE1 transport mechanism, we created point mutations in regions corresponding to E. coli ClC transport mechanism residues. When expressed in HEK293 cells, several mutants had Cl−/HCO3− exchange rates significantly different from that of wild-type AE1. When further assessed in Xenopus laevis oocytes, there were significant changes in the transport activity of several AE1 point mutants as assessed by changes in pH. None of the mutants, however, added an electrogenic component to AE1 transport activity. This indicates that the AE1 point mutants altered the transport activity of AE1, without changing its electrogenicity and stoichiometry. The homology model successfully identified residues in AE1 that are critical to AE1 transport activity. Thus, we conclude that AE1 has a similar protein fold to ClC chloride channels

Functional assessment of SLC4A11, an integral membrane protein mutated in corneal dystrophies

SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na⁺ independent. Studies in oocytes and HEK293 cells could not detect Na⁺⁻ coupled HCO3⁻ transport or Cl⁻/HCO3⁻ exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltagedependent OH⁻ or H⁺ movement was not measurable in SLC4A11- expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH⁻/H⁺ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H⁺.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Shadow of a Colossus: A z=2.45 Galaxy Protocluster Detected in 3D Ly-a Forest Tomographic Mapping of the COSMOS Field

Using moderate-resolution optical spectra from 58 background Lyman-break galaxies and quasars at $z\sim 2.3-3$ within a $11.5'\times13.5'$ area of the COSMOS field ($\sim 1200\,\mathrm{deg}^2$ projected area density or $\sim 2.4\,h^{-1}\,\mathrm{Mpc}$ mean transverse separation), we reconstruct a 3D tomographic map of the foreground Ly$\alpha$ forest absorption at $2.2<z<2.5$ with an effective smoothing scale of $\sigma_{3d}\approx3.5\,h^{-1}\,\mathrm{Mpc}$ comoving. Comparing with 61 coeval galaxies with spectroscopic redshifts in the same volume, we find that the galaxy positions are clearly biased towards regions with enhanced IGM absorption in the tomographic map. We find an extended IGM overdensity with deep absorption troughs at $z=2.45$ associated with a recently-discovered galaxy protocluster at the same redshift. Based on simulations matched to our data, we estimate the enclosed dark matter mass within this IGM overdensity to be $M_{\rm dm} (z=2.45) = (9\pm4)\times 10^{13}\,h^{-1}\,\mathrm{M_\odot}$, and argue based on this mass and absorption strength that it will form at least one $z\sim0$ galaxy cluster with $M(z=0) = (3\pm 2) \times 10^{14}\,h^{-1}\mathrm{M_\odot}$, although its elongated nature suggests that it will likely collapse into two separate clusters. We also point out a compact overdensity of six MOSDEF galaxies at $z=2.30$ within a $r\sim 1\,h^{-1}\,\mathrm{Mpc}$ radius and $\Delta z\sim 0.006$, which does not appear to have a large associated IGM overdensity. These results demonstrate the potential of Ly$\alpha$ forest tomography on larger volumes to study galaxy properties as a function of environment, as well as revealing the large-scale IGM overdensities associated with protoclusters and other features of large-scale structure.Comment: To be submitted to ApJ. Figure 3 can be viewed on Youtube: https://youtu.be/KeW1UJOPMY

Sleep Duration is Increased Following Muscle Damaging Exercise in Hot Environmental Conditions

Sleep and recovery measures are typically negatively affected by a muscle-damaging bout of exercise. However, it remains unknown if the additive effects of hot environmental conditions, resulting in increased core temperature and other thermoregulatory responses during the exercise bout, further progress changes in quantity and performance quality of sleep duration. PURPOSE: To investigate the effect of muscle-damaging exercise in the heat, compared to a thermoneutral condition, on sleep and recovery measures. METHODS: Ten healthy males (age: 23 ± 3yr; body mass: 78.7 ± 11.5kg; height: 176.9 ± 5cm; lactate threshold [LT]: 9.7 ± 1.0km.hr-1) performed two protocols in a randomized, counterbalanced order of downhill running (DHR) for 30-minutes at the LT in either a thermoneutral (ambient temperate [Tamb], 20°C; relative humidity [RH], 20%) or hot environmental condition (Tamb, 35°C; RH, 40%) at a -10% gradient. Sleep and recovery measures were collected from a wearable sleep device participants wore the night after the DHR. Differences in sleep and recovery measures following DHR in the heat compared to a thermoneutral condition were analyzed using paired samples T-tests. RESULTS: Sleep hours, restorative sleep hours, rapid eye movement (REM) sleep hours, and slow wave sleep (SWS) hours were all greater following the heat condition (mean ± SD; sleep hours: 6.70 ± 0.74hr, p = 0.040; restorative sleep hours: 3.31 ± 0.90hr, p = 0.012; REM sleep hours: 1.70 ± 0.64hr, p = 0.046; SWS hours: 1.61 ± 0.35hr, p = 0.015) compared to the thermoneutral condition (sleep hours: 5.24 ± 1.75hr; restorative sleep hours: 2.45 ± 1.11hr; REM sleep hours: 1.23 ± 0.68hr; SWS: 1.22 ± 0.53hr). Also, recovery was higher following the heat condition (recovery: 75.88 ± 15.31, p = 0.023) compared to the thermoneutral condition (recovery: 50.75 ± 21.46). Sleep efficiency, sleep disturbance, sleep deprivation, sleep score, %REM, %SWS, light sleep, resting heart rate, and heart rate variability were not different between conditions (ps \u3e 0.05). CONCLUSION: Following muscle-damaging exercise in the heat, sleep and recovery duration measures were increased compared to a thermoneutral condition. These findings suggest that performing muscle-damaging exercises in hot conditions may require a greater amount of sleep for optimal recovery

Relationships between Morning and Afternoon WUT (Weight, Urine Color, and Thirst) Criteria and Hydration Markers

A Venn diagram decision tool consisting of weight, urine color, and thirst (WUT) is suggested to measure hydration status. The WUT Venn diagram has been used as a practical hydration status assessment tool; however, this relationship has only been investigated using a first-morning urine sample. PURPOSE: To investigate relationships between morning and afternoon WUT criteria, blood and urine markers. METHODS: Eight men (age: 21 ± 3; mass: 76.3 ± 15.6 kg) and five women (age: 22 ± 2; mass: 60.5 ± 13.6 kg) completed the study. Body mass, urine color, urine specific gravity (USG), urine osmolality (UOSM), thirst level, and plasma osmolality (POSM) were collected as a first-morning and afternoon spot urine (2:00-4:00 CST) for 3 consecutive days in a free-living situation and 3 consecutive days in a euhydrated state. Body mass loss \u3e1%, urine color \u3e5, and thirst level ≥5 were used as dehydration thresholds. The number of markers that indicated dehydration levels were counted and categorized into either 3, 2, 1, or 0 WUT markers indicating dehydration (defined by either USG, UOSM, or POSM). One-way ANOVA with Tukey pairwise comparisons were used to assess differences in USG, UOSM, and POSM between different numbers of WUT markers. Receiver operating characteristics analysis was performed to calculate the predictive value of 0, 1, 2, or 3 hydration markers in detecting a dehydrated or euhydrated state. RESULTS: Morning and afternoon 1, 2, and 3 WUT markers were not significantly different (ps \u3e .05) for USG and POSM. Morning and afternoon 0, 2, and 3 WUT markers were not significantly different for UOSM. Morning and afternoon 3 WUT resulted in a specificity of 0.984 and 1.000, 0.984 and 1.000, and 0.956 and 0.981 for USG \u3e 1.020, UOSM \u3e 700mOsm, and POSM \u3e 290mOsm, respectively. Meeting at 2 WUT for morning and afternoon resulted in a specificity of 0.820 and 0.985, and 0.806 and 0.984 for USG and UOSM, respectively. Meeting at 1 WUT for morning and afternoon resulted in a sensitivity of 1.000 and 0.813 for UOSM. CONCLUSION: These results suggest that when 2 or 3 WUT markers are met, urine and blood hydration markers indicate dehydration, and when 1 WUT marker is met, UOSM indicates not dehydrated. The WUT Venn diagram can assess hydration status when an afternoon spot urine sample is used

Quantification of carbonic anhydrase gene expression in ventricle of hypertrophic and failing human heart

Background: Carbonic anhydrase enzymes (CA) catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE) activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide), a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH) in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms.Methods: We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14), or heart transplantation (HT, n = 13). CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls.Results: Expression of atrial and brain natriuretic peptide (ANP and BNP) were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles.Conclusions: These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac hypertrophy.Centro de Investigaciones Cardiovasculare

Power, Avionics and Software Communication Network Architecture

This document describes the communication architecture for the Power, Avionics and Software (PAS) 2.0 subsystem for the Advanced Extravehicular Mobile Unit (AEMU). The following systems are described in detail: Caution Warn- ing and Control System, Informatics, Storage, Video, Audio, Communication, and Monitoring Test and Validation. This document also provides some background as well as the purpose and goals of the PAS project at Glenn Research Center (GRC)

Power, Avionics and Software - Phase 1.0:

This report describes Power, Avionics and Software (PAS) 1.0 subsystem integration testing and test results that occurred in August and September of 2013. This report covers the capabilities of each PAS assembly to meet integration test objectives for non-safety critical, non-flight, non-human-rated hardware and software development. This test report is the outcome of the first integration of the PAS subsystem and is meant to provide data for subsequent designs, development and testing of the future PAS subsystems. The two main objectives were to assess the ability of the PAS assemblies to exchange messages and to perform audio testing of both inbound and outbound channels. This report describes each test performed, defines the test, the data, and provides conclusions and recommendations