Aircraft measurements of water vapor heavy isotope ratios in the marine boundary layer and lower troposphere during ORACLES

This paper presents the water vapor heavy isotope ratio measurement system developed for aircraft in-situ measurements and used in the NASA ObseRvations of Aerosols above CLouds and their intEractionS (ORACLES) project. The resultant dataset collected, which includes measurements of specific humidity and the heavy isotope ratios D / H and 18O / 16O, is also presented. Aircraft sampling took place in the southeast Atlantic marine boundary layer and lower troposphere (equator to 22° S) over the months of Sept. 2016, Aug. 2017, and Oct. 2018. Isotope measurements were made using cavity ring-down spectroscopic analyzers integrated into the Water Isotope System for Precipitation and Entrainment Research (WISPER). The water concentration and isotopic data accompanied a suite of other variables including standard meteorological quantities (wind, temperature, moisture), trace gas and aerosol concentrations, radar, and lidar remote sensing. From an isotope perspective, the 300+ hours of 1 Hz in-situ data at levels in the atmosphere ranging from 70 m to 6 km represents a remarkably large and vertically resolved dataset. This paper provides a brief overview of the ORACLES mission and describes how water vapor heavy isotope ratios fit within the experimental design. Overviews of the sampling region and WISPER system setup are presented, along with calibration details, measurement uncertainties, and suggested data usage. Characteristics in the spatial variability of the study region over the three sampling periods are highlighted with latitude-altitude curtains. A number of individual tropospheric profiles are presented to illustrate the fidelity with which a series of different hydrologic processes are captured by the observations. The curtains and profiles demonstrate the dataset’s potential to provide a comprehensive perspective on moisture transport and isotopic content in this region. Readers interested in a quick reference to data usage and uncertainty estimation can consult the beginning of section 5. Data for the Sept. 2016, Aug. 2017, and Oct. 2018 sampling periods can be accessed at https://doi.org/10.5067/Suborbital/ORACLES/P3/2016_V2, https://doi.org/10.5067/Suborbital/ORACLES/P3/2017_V2, and https://doi.org/10.5067/Suborbital/ORACLES/P3/2018_V2, respectively (see references for ORACLES Science Team, 2020 – 2016 P3 data, 2017 P3 data, and 2018 P3 data).

Hfe Gene Knock-Out in a Mouse Model of Hereditary Hemochromatosis Affects Bodily Iron Isotope Compositions

Hereditary hemochromatosis is a genetic iron overload disease related to a mutation within the HFE gene that controls the expression of hepcidin, the master regulator of systemic iron metabolism. The natural stable iron isotope composition in whole blood of control subjects is different from that of hemochromatosis patients and is sensitive to the amount of total iron removed by the phlebotomy treatment. The use of stable isotopes to unravel the pathological mechanisms of iron overload diseases is promising but hampered by the lack of data in organs involved in the iron metabolism. Here, we use Hfe−/− mice, a model of hereditary hemochromatosis, to study the impact of the knock-out on iron isotope compositions of erythrocytes, spleen and liver. Iron concentration increases in liver and red blood cells of Hfe−/− mice compared to controls. The iron stable isotope composition also increases in liver and erythrocytes, consistent with a preferential accumulation of iron heavy isotopes in Hfe−/− mice. In contrast, no difference in the iron concentration nor isotope composition is observed in spleen of Hfe−/− and control mice. Our results in mice suggest that the observed increase of whole blood isotope composition in hemochromatosis human patients does not originate from, but is aggravated by, bloodletting. The subsequent rapid increase of whole blood iron isotope composition of treated hemochromatosis patients is rather due to the release of hepatic heavy isotope-enriched iron than augmented iron dietary absorption. Further research is required to uncover the iron light isotope component that needs to balance the accumulation of hepatic iron heavy isotope, and to better understand the iron isotope fractionation associated to metabolism dysregulation during hereditary hemochromatosis.

Abstract P388: Extra-cardiac BCAA Catabolism Lowers Blood Pressure And Protects From Heart Failure

Pharmacologic activation of branched chain amino acid (BCAA) catabolism is protective in numerous models of heart failure (HF). How this protection occurs has remained unclear, although a causative block in cardiac BCAA oxidation has been proposed. We use here in vivo heavy isotope infusion studies to show that cardiac preference for BCAA oxidation increases, rather than decreases, in multiple models of HF. We use various genetic models to show that cardiac-specific activation of BCAA oxidation does not protect from HF, even though systemic activation of BCAA oxidation does. Lowering plasma and cardiac BCAAs by genetic means is also not sufficient to confer protection comparable to that conferred by pharmacologic activation of BCAA oxidation, suggesting alternative mechanisms of protection. Surprisingly, telemetry and invasive hemodynamic studies showed that pharmacological activation of BCAA catabolism lowers blood pressure, a well-established cardioprotective mechanism. The effects on blood pressure occurred independently of nitric oxide (NO), and reflected a vascular resistance to adrenergic constriction. Finally, mendelian randomization studies revealed that elevations in plasma BCAAs portend higher blood pressure in large human cohorts. Together, these data indicate that activation of BCAA oxidation lowers blood pressure and protects from heart failure independently of any direct effects on the heart itself.

Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs

Antibiotics of the β-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate β-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this unprecedented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a β-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.

Lactate oxidative phosphorylation by annulus fibrosus cells: evidence for lactate-dependent metabolic symbiosis in intervertebral discs

Background Intervertebral disc degeneration contributes to low back pain. The avascular intervertebral disc consists of a central hypoxic nucleus pulpous (NP) surrounded by the more oxygenated annulus fibrosus (AF). Lactic acid, an abundant end-product of NP glycolysis, has long been viewed as a harmful waste that acidifies disc tissue and decreases cell viability and function. As lactic acid is readily converted into lactate in disc tissue, the objective of this study was to determine whether lactate could be used by AF cells as a carbon source rather than being removed from disc tissue as a waste byproduct. Methods Import and conversion of lactate to tricarboxylic acid (TCA) cycle intermediates and amino acids in rabbit AF cells were measured by heavy-isotope (13C-lactate) tracing experiments using mass spectrometry. Levels of protein expression of lactate converting enzymes, lactate importer and exporter in NP and AF tissues were quantified by Western blots. Effects of lactate on proteoglycan (35S-sulfate) and collagen (3H-proline) matrix protein synthesis and oxidative phosphorylation (Seahorse XFe96 Extracellular Flux Analyzer) in AF cells were assessed. Results Heavy-isotope tracing experiments revealed that AF cells imported and converted lactate into TCA cycle intermediates and amino acids using in vitro cell culture and in vivo models. Addition of exogenous lactate (4 mM) in culture media induced expression of the lactate importer MCT1 and increased oxygen consumption rate by 50%, mitochondrial ATP-linked respiration by 30%, and collagen synthesis by 50% in AF cell cultures grown under physiologic oxygen (2-5% O2) and glucose concentration (1-5 mM). AF tissue highly expresses MCT1, LDH-H, an enzyme that preferentially converts lactate to pyruvate, and PDH, an enzyme that converts pyruvate to acetyl-coA. In contrast, NP tissue highly expresses MCT4, a lactate exporter, and LDH-M, an enzyme that preferentially converts pyruvate to lactate. Conclusions These findings support disc lactate-dependent metabolic symbiosis in which lactate produced by the hypoxic, glycolytic NP cells is utilized by the more oxygenated AF cells via oxidative phosphorylation for energy and matrix production, thus shifting the current research paradigm of viewing disc lactate as a waste product to considering it as an important biofuel. These scientifically impactful results suggest novel therapeutic targets in disc metabolism and degeneration.

Conditions and extent of volatile loss from the Moon during formation of the Procellarum basin

Rocks from the lunar interior are depleted in moderately volatile elements (MVEs) compared to terrestrial rocks. Most MVEs are also enriched in their heavier isotopes compared to those in terrestrial rocks. Such elemental depletion and heavy isotope enrichments have been attributed to liquid–vapor exchange and vapor loss from the protolunar disk, incomplete accretion of MVEs during condensation of the Moon, and degassing of MVEs during lunar magma ocean crystallization. New Monte Carlo simulation results suggest that the lunar MVE depletion is consistent with evaporative loss at 1,670 ± 129 K and an oxygen fugacity +2.3 ± 2.1 log units above the fayalite-magnetite-quartz buffer. Here, we propose that these chemical and isotopic features could have resulted from the formation of the putative Procellarum basin early in the Moon’s history, during which nearside magma ocean melts would have been exposed at the surface, allowing equilibration with any primitive atmosphere together with MVE loss and isotopic fractionation.

Ultrafiltration-based Extraction and LC-MS/MS Quantification of Phenylalanine in Human Blood Sample for Metabolite Target Analysis

Background: Phenylalanine is a significant biomarker for various diseases like phenylketonuria, gastric cancers, and ischemic stroke according to recent studies. Methods: In the present study; a simple, sensitive, selective and novel analytical method was validated by using an ultrafiltration-based extraction and LC-MS/MS quantification of phenylalanine in human plasma using 13C phenylalanine heavy isotope. Amicon® Ultra Centrifugal Filter was used for ultrafiltration. Parameters affecting LC separation and MS/MS detection were investigated and optimized. Chromatographic separation was achieved on a Merck SeQuant ZIC-HILIC (100x4.6 mm, 5 μm) at a column temperature of 40°C using a mobile phase of mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid (50:50 v/v) at a flow rate of 0.35 mL/min. The transitions m/z 167→121 for 13C phenylalanine, m/z 166→120 for phenylalanine itself were monitored using the MRM mode. Result: The assay was linear concentration range of 0.0025 μg/mL to 1.20 μg/mL (R2=0.999). The developed method was validated according to FDA guidelines. The method was found linear, sensitive, precise, accurate, and selective.

HCl in the atmosphere of Mars: chlorine isotopic ratio

<p>The ExoMars Trace Gas Orbiter (TGO) mission had started regular measurements in 2018. Primary goal of the mission is to quantify trace gases that could indicate geologic or biogenic activity on Mars (Vago et al., 2015). Atmospheric Chemistry Suite mid-infrared channel (ACS MIR) is a high resolution cross-dispersion spectrometer operating in solar occultation mode (Korablev et al., 2018). It was designed to make the most sensitive measurements of the atmosphere to date. During each occultation up to 20 diffraction orders are simultaneously recorded at different tangent altitudes. In 2020 ACS MIR reports the discovery of the gaseous hydrogen chloride (HCl). Absorption features are present in several consecutive diffraction orders, withal both isotopes H<sup>37</sup>Cl and H<sup>35</sup>Cl are clearly observed. HCl was observed by ACS simultaneously in both hemispheres after the main phase of the global dust storm. Though the formation mechanism is not fully clear, we believe that the presence of HCl is associated with the lifted dust and chlorine component in it.</p> <p>On Earth, in general, the chlorine isotope variations in nature are relatively small, ranging from ~-2 to +2 ‰. However, large variations are observed, e.g. in extraterrestrial materials and volcanic gases, due to kinetic fractionation. On Mars Farley et al. (2016) reported a range from -1 ‰ to −51 ‰ (5% reduction) for the δ37Cl in the samples drilled in the Gale Crater. ACS observations demonstrate enrichment of the 37Cl up to +250 ‰ on average in the atmospheric gaseous. In principle, most atmospheric elements on Mars have heavy isotope enrichments due to preferential loss of the light isotope to space (e.g. Vandaele et al., 2019). Early hydrodynamic escape during intense extreme ultraviolet radiation followed by prolonged atmospheric ‘erosion’ explains the heavy isotope enrichment. Chlorine loss, as HCl, would raise the δ37Cl value of the residual materials, involved in the dust-atmospheric exchange cycle.</p> <p> </p> <p>References</p> <p>Farleya K.A., Martina P., Archer P.D. , et al.: Light and variable 37Cl/35Cl ratios in rocks from Gale Crater, Mars: Possible signature of perchlorate, Earth and Planetary Science Letters 438:14-24, DOI: 10.1016/j.epsl.2015.12.013, 2016.</p> <p>Korablev, O., Montmessin, F., Trokhimovskiy, et al..: The Atmospheric Chemistry Suite (ACS) of Three Spectrometers for the ExoMars 2016 Trace Gas Orbiter, Space. Sci. Rev., 214(1), 7, doi:10.1007/s11214-017-0437-6, 2018.</p> <p>Vago, J., Witasse, O., Svedhem, et al.: ESA ExoMars program: The next step in exploring Mars, Sol. Syst. Res., 49(7), 518–528, doi:10.1134/S0038094615070199, 2015.</p> <p>Vandaele, A. C., Korablev, O., Daerden, F. et al.: Martian dust storm impact on atmospheric H<sub>2</sub>O and D/H observed by ExoMars Trace Gas Orbiter, Nature, 568, 521–525, doi:10.1038/s41586-019-1097-3, 2019.</p>

Exploring Extracellular Vesicles Biogenesis in Hypothalamic Cells through a Heavy Isotope Pulse/Trace Proteomic Approach

Studies have shown that the process of extracellular vesicles (EVs) secretion and lysosome status are linked. When the lysosome is under stress, the cells would secrete more EVs to maintain cellular homeostasis. However, the process that governs lysosomal activity and EVs secretion remains poorly defined and we postulated that certain proteins essential for EVs biogenesis are constantly synthesized and preferentially sorted to the EVs rather than the lysosome. A pulsed stable isotope labelling of amino acids in cell culture (pSILAC) based quantitative proteomics methodology was employed to study the preferential localization of the newly synthesized proteins into the EVs over lysosome in mHypoA 2/28 hypothalamic cell line. Through proteomic analysis, we found numerous newly synthesized lysosomal enzymes—such as the cathepsin proteins—that preferentially localize into the EVs over the lysosome. Chemical inhibition against cathepsin D promoted EVs secretion and a change in the EVs protein composition and therefore indicates its involvement in EVs biogenesis. In conclusion, we applied a heavy isotope pulse/trace proteomic approach to study EVs biogenesis in hypothalamic cells. The results demonstrated the regulation of EVs secretion by the cathepsin proteins that may serve as a potential therapeutic target for a range of neurological disorder associated with energy homeostasis.