Dynamic regulation of extracellular superoxide production by the coccolithophore Emiliania huxleyi (CCMP 374)

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Plummeer, S., Taylor, A. E., Harvey, E. L., Hansel, C. M., & Diaz, J. M. Dynamic regulation of extracellular superoxide production by the coccolithophore Emiliania huxleyi (CCMP 374). Frontiers in Microbiology, 10, (2019): 1546, doi: 10.3389/fmicb.2019.01546.In marine waters, ubiquitous reactive oxygen species (ROS) drive biogeochemical cycling of metals and carbon. Marine phytoplankton produce the ROS superoxide (O2−) extracellularly and can be a dominant source of O2− in natural aquatic systems. However, the cellular regulation, biological functioning, and broader ecological impacts of extracellular O2− production by marine phytoplankton remain mysterious. Here, we explored the regulation and potential roles of extracellular O2− production by a noncalcifying strain of the cosmopolitan coccolithophorid Emiliania huxleyi, a key species of marine phytoplankton that has not been examined for extracellular O2− production previously. Cell-normalized extracellular O2− production was the highest under presumably low-stress conditions during active proliferation and inversely related to cell density during exponential growth phase. Removal of extracellular O2− through addition of the O2− scavenger superoxide dismutase (SOD), however, increased growth rates, growth yields, cell biovolume, and photosynthetic efficiency (Fv/Fm) indicating an overall physiological improvement. Thus, the presence of extracellular O2− does not directly stimulate E. huxleyi proliferation, as previously suggested for other phytoplankton, bacteria, fungi, and protists. Extracellular O2− production decreased in the dark, suggesting a connection with photosynthetic processes. Taken together, the tight regulation of this stress independent production of extracellular O2− by E. huxleyi suggests that it could be involved in fundamental photophysiological processes.This research was supported by a Junior Faculty Seed Grant from the University of Georgia Research Foundation (JD), a National Science Foundation (NSF) Graduate Research Fellowship (SP), and NSF grant OCE-1355720 (CH). The FlowCam® and FIRe were purchased through a NSF Equipment Improvement Grant (1624593)

NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Diaz, J. M., Plummer, S., Hansel, C. M., Andeer, P. F., Saito, M. A., & McIlvin, M. R. NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica. Proceedings of the National Academy of Sciences of the United States of America, 116 (33), (2019): 16448-16453, doi: 10.1073/pnas.1821233116.Reactive oxygen species (ROS) like superoxide drive rapid transformations of carbon and metals in aquatic systems and play dynamic roles in biological health, signaling, and defense across a diversity of cell types. In phytoplankton, however, the ecophysiological role(s) of extracellular superoxide production has remained elusive. Here, the mechanism and function of extracellular superoxide production by the marine diatom Thalassiosira oceanica are described. Extracellular superoxide production in T. oceanica exudates was coupled to the oxidation of NADPH. A putative NADPH-oxidizing flavoenzyme with predicted transmembrane domains and high sequence similarity to glutathione reductase (GR) was implicated in this process. GR was also linked to extracellular superoxide production by whole cells via quenching by the flavoenzyme inhibitor diphenylene iodonium (DPI) and oxidized glutathione, the preferred electron acceptor of GR. Extracellular superoxide production followed a typical photosynthesis-irradiance curve and increased by 30% above the saturation irradiance of photosynthesis, while DPI significantly impaired the efficiency of photosystem II under a wide range of light levels. Together, these results suggest that extracellular superoxide production is a byproduct of a transplasma membrane electron transport system that serves to balance the cellular redox state through the recycling of photosynthetic NADPH. This photoprotective function may be widespread, consistent with the presence of putative homologs to T. oceanica GR in other representative marine phytoplankton and ocean metagenomes. Given predicted climate-driven shifts in global surface ocean light regimes and phytoplankton community-level photoacclimation, these results provide implications for future ocean redox balance, ecological functioning, and coupled biogeochemical transformations of carbon and metals.This work was supported by a postdoctoral fellowship from the Ford Foundation (to J.M.D.), the National Science Foundation (NSF) under grants OCE 1225801 (to J.M.D.) and OCE 1246174 (to C.M.H.), a Junior Faculty Seed Grant from the University of Georgia Research Foundation (to J.M.D.), and a National Science Foundation Graduate Research Fellowship (to S.P.). The FIRe was purchased through a NSF equipment improvement grant (1624593).The authors thank Melissa Soule for assistance with LC/MS/MS analysis of peptide samples

Extracellular superoxide production by key microbes in the global ocean

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Sutherland, K. M., Coe, A., Gast, R. J., Plummer, S., Suffridge, C. P., Diaz, J. M., Bowman, J. S., Wankel, S. D., & Hansel, C. M. Extracellular superoxide production by key microbes in the global ocean. Limnology and Oceanography, (2019), doi:10.1002/lno.11247.Bacteria and eukaryotes produce the reactive oxygen species superoxide both within and outside the cell. Although superoxide is typically associated with the detrimental and sometimes fatal effects of oxidative stress, it has also been shown to be involved in a range of essential biochemical processes, including cell signaling, growth, differentiation, and defense. Light‐independent extracellular superoxide production has been shown to be widespread among many marine heterotrophs and phytoplankton, but the extent to which this trait is relevant to marine microbial physiology and ecology throughout the global ocean is unknown. Here, we investigate the dark extracellular superoxide production of five groups of organisms that are geographically widespread and represent some of the most abundant organisms in the global ocean. These include Prochlorococcus, Synechococcus, Pelagibacter, Phaeocystis, and Geminigera. Cell‐normalized net extracellular superoxide production rates ranged seven orders of magnitude, from undetectable to 14,830 amol cell−1 h−1, with the cyanobacterium Prochlorococcus being the lowest producer and the cryptophyte Geminigera being the most prolific producer. Extracellular superoxide production exhibited a strong inverse relationship with cell number, pointing to a potential role in cell signaling. We demonstrate that rapid, cell‐number–dependent changes in the net superoxide production rate by Synechococcus and Pelagibacter arose primarily from changes in gross production of extracellular superoxide, not decay. These results expand the relevance of dark extracellular superoxide production to key marine microbes of the global ocean, suggesting that superoxide production in marine waters is regulated by a diverse suite of marine organisms in both dark and sunlit waters.The authors would like to acknowledge their funding sources including NASA NESSF NNX15AR62H (K.M.S.), NASA Exobiology grant NNX15AM04G to S.D.W. and C.M.H., NSF‐OCE grant 1355720 to C.M.H., NSF‐OPP 1641019 (J.S.B), and Simons Foundation SCOPE Award ID 329108 (Sallie W. Chisholm). The authors would also like to thank the Harvey lab (Skidaway Institute of Oceanography) for use of their flow cytometer in this study. We thank Stephen Giovannoni and Sallie Chisholm for providing bacteria strains and laboratory facilities. Additional thanks to Marianne Acker, Rogier Braakman, and Aldo Arellano for assistance in lab and helpful conversations

Spatial heterogeneity in particle-associated, light-independent superoxide production within productive coastal waters

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Sutherland, K. M., Grabb, K. C., Karolewski, J. S., Plummer, S., Farfan, G. A., Wankel, S. D., Diaz, J. M., Lamborg, C. H., & Hansel, C. M. Spatial heterogeneity in particle-associated, light-independent superoxide production within productive coastal waters. Journal of Geophysical Research: Oceans, 125(10), (2020): e2020JC016747, https://doi.org/10.1029/2020JC016747.In the marine environment, the reactive oxygen species (ROS) superoxide is produced through a diverse array of light‐dependent and light‐independent reactions, the latter of which is thought to be primarily controlled by microorganisms. Marine superoxide production influences organic matter remineralization, metal redox cycling, and dissolved oxygen concentrations, yet the relative contributions of different sources to total superoxide production remain poorly constrained. Here we investigate the production, steady‐state concentration, and particle‐associated nature of light‐independent superoxide in productive waters off the northeast coast of North America. We find exceptionally high levels of light‐independent superoxide in the marine water column, with concentrations ranging from 10 pM to in excess of 2,000 pM. The highest superoxide concentrations were particle associated in surface seawater and in aphotic seawater collected meters off the seafloor. Filtration of seawater overlying the continental shelf lowered the light‐independent, steady‐state superoxide concentration by an average of 84%. We identify eukaryotic phytoplankton as the dominant particle‐associated source of superoxide to these coastal waters. We contrast these measurements with those collected at an off‐shelf station, where superoxide concentrations did not exceed 100 pM, and particles account for an average of 40% of the steady‐state superoxide concentration. This study demonstrates the primary role of particles in the production of superoxide in seawater overlying the continental shelf and highlights the importance of light‐independent, dissolved‐phase reactions in marine ROS production.This work was funded by grants from the Chemical Oceanography program of the National Science Foundation (OCE‐1355720 to C. M. H. and C. H. L.), NASA Earth and Space Science Fellowship (Grant NNX15AR62H to K. M. S.), Agouron Institute Postdoctoral Fellowship (K. M. S.), NSF GRFPs (2016230268 to K. C. G. and 2017250547 to S. P.), and a Sloan Research Fellowship (J. M. D.). The Guava flow cytometer was purchased through an NSF equipment improvement grant (1624593)

Energy flux couples sulfur isotope fractionation to proteomic and metabolite profiles in Desulfovibrio vulgaris

Funding Information: We thank S. Moore and D. Fike for bulk sulfur isotope analyses (WashU); M. Seuss for assistance with lipid\u2010H isotope analyses (Bradley lab, WashU); X. Feng (Dartmouth) and M. Osburn (Northwestern) for water H\u2010isotope analyses; and A. Sessions and J. Adkins (CalTech) for access to HPLC\u2010ICP\u2010MS. Metabolite analyses were performed by the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center (St. Louis, MO, USA). Funding was provided: by NASA Exobiology Award 13\u2010EXO13\u20100082 (ASB, WDL, JW), NSF\u2010EAR Award 1928309 (WDL), Washington University in St. Louis Department of Earth & Planetary Sciences Fossett Fellowship (WDL), the Walter and Constance Burke Fund at Dartmouth College (WDL), and the Fulbright\u2014Bunge & Born\u2014Williams Foundation Scholarship Program (FJB), Funda\u00E7\u00E3o para a Ci\u00EAncia e Tecnologia (Portugal) through R&D unit MOSTMICRO\u2010ITQB (UIDB/04612/2020 and UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) (IACP), NSF GRFP [2017250547] (SP). Funding Information: We thank S. Moore and D. Fike for bulk sulfur isotope analyses (WashU); M. Seuss for assistance with lipid-H isotope analyses (Bradley lab, WashU); X. Feng (Dartmouth) and M. Osburn (Northwestern) for water H-isotope analyses; and A. Sessions and J. Adkins (CalTech) for access to HPLC-ICP-MS. Metabolite analyses were performed by the Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center (St. Louis, MO, USA). Funding was provided: by NASA Exobiology Award 13-EXO13-0082 (ASB, WDL, JW), NSF-EAR Award 1928309 (WDL), Washington University in St. Louis Department of Earth & Planetary Sciences Fossett Fellowship (WDL), the Walter and Constance Burke Fund at Dartmouth College (WDL), and the Fulbright\u2014Bunge & Born\u2014Williams Foundation Scholarship Program (FJB), Funda\u00E7\u00E3o para a Ci\u00EAncia e Tecnologia (Portugal) through R&D unit MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) (IACP), NSF GRFP [2017250547] (SP). Publisher Copyright: © 2024 The Authors. Geobiology published by John Wiley & Sons Ltd.Microbial sulfate reduction is central to the global carbon cycle and the redox evolution of Earth's surface. Tracking the activity of sulfate reducing microorganisms over space and time relies on a nuanced understanding of stable sulfur isotope fractionation in the context of the biochemical machinery of the metabolism. Here, we link the magnitude of stable sulfur isotopic fractionation to proteomic and metabolite profiles under different cellular energetic regimes. When energy availability is limited, cell-specific sulfate respiration rates and net sulfur isotope fractionation inversely covary. Beyond net S isotope fractionation values, we also quantified shifts in protein expression, abundances and isotopic composition of intracellular S metabolites, and lipid structures and lipid/water H isotope fractionation values. These coupled approaches reveal which protein abundances shift directly as a function of energy flux, those that vary minimally, and those that may vary independent of energy flux and likely do not contribute to shifts in S-isotope fractionation. By coupling the bulk S-isotope observations with quantitative proteomics, we provide novel constraints for metabolic isotope models. Together, these results lay the foundation for more predictive metabolic fractionation models, alongside interpretations of environmental sulfur and sulfate reducer lipid-H isotope data.publishersversionpublishe

Energy flux couples sulfur isotope fractionation to proteomicand metabolite profiles in Desulfovibrio vulgaris

Fil: Leavitt, William D. Dartmouth College. Department of Earth Sciences, Hanover, New Hampshire; United States of America.Fil: Leavitt, William D. Washington University in St. Louis. Department of Earth and Planetary Sciences, Missouri; United States of America.Fil: Waldbauer, Jacob. University of Chicago. Department of the Geophysical Sciences, Illinois; United States of America.Fil: Venceslau, Sofia S. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica António Xavier, Oeiras; Portugal.Fil: Sim, Min Sub. Seoul National University. School of Earth and Environmental Sciences; South Korea.Fil: Zhang, Lichun. University of Chicago. Department of the Geophysical Sciences, Illinois; United States of America.Fil: Flavia Jaquelina Boidi. Washington University in St. Louis. Department of Earth and Planetary Sciences; Missouri, United States of America.Fil: Flavia Jaquelina Boidi. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Investigaciones en Ciencias de la Tierra; Argentina.Fil: Flavia Jaquelina Boidi. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Plummer, Sydney. University of California, San Diego, La Jolla. Scripps Institution of Oceanography, California; United States of America.Fil: Díaz, Julia M. University of California, San Diego, La Jolla. Scripps Institution of Oceanography, California; United States of America.Fil: Pereira, Inês A. C. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica António Xavier, Oeiras; Portugal.Fil: Bradley, Alexander S. Washington University in St. Louis. Department of Earth and Planetary Sciences; Missouri, United States of America.Fil: Bradley, Alexander S. Washington University in St. Louis. Division of Biology and Biomedical Sciences, Missouri; United States of America.Abstract: Microbial sulfate reduction is central to the global carbon cycle and the redox evolu-tion of Earth's surface. Tracking the activity of sulfate reducing microorganisms overspace and time relies on a nuanced understanding of stable sulfur isotope fractiona-tion in the context of the biochemical machinery of the metabolism. Here, we link themagnitude of stable sulfur isotopic fractionation to proteomic and metabolite profilesunder different cellular energetic regimes. When energy availability is limited, cell-specific sulfate respiration rates and net sulfur isotope fractionation inversely covary. Beyond net S isotope fractionation values, we also quantified shifts in protein expres-sion, abundances and isotopic composition of intracellular S metabolites, and lipidstructures and lipid/water H isotope fractionation values. These coupled approachesreveal which protein abundances shift directly as a function of energy flux, those thatvary minimally, and those that may vary independent of energy flux and likely do notcontribute to shifts in S-isotope fractionation. By coupling the bulk S-isotope obser-vations with quantitative proteomics, we provide novel constraints for metabolic iso-tope models. Together, these results lay the foundation for more predictive metabolicfractionation models, alongside interpretations of environmental sulfur and sulfatereducer lipid-H isotope data.info:eu-repo/semantics/publishedVersionFil: Leavitt, William D. Dartmouth College. Department of Earth Sciences, Hanover, New Hampshire; United States of America.Fil: Leavitt, William D. Washington University in St. Louis. Department of Earth and Planetary Sciences, Missouri; United States of America.Fil: Waldbauer, Jacob. University of Chicago. Department of the Geophysical Sciences, Illinois; United States of America.Fil: Venceslau, Sofia S. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica António Xavier, Oeiras; Portugal.Fil: Sim, Min Sub. Seoul National University. School of Earth and Environmental Sciences; South Korea.Fil: Zhang, Lichun. University of Chicago. Department of the Geophysical Sciences, Illinois; United States of America.Fil: Flavia Jaquelina Boidi. Washington University in St. Louis. Department of Earth and Planetary Sciences; Missouri, United States of America.Fil: Flavia Jaquelina Boidi. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Investigaciones en Ciencias de la Tierra; Argentina.Fil: Flavia Jaquelina Boidi. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Plummer, Sydney. University of California, San Diego, La Jolla. Scripps Institution of Oceanography, California; United States of America.Fil: Díaz, Julia M. University of California, San Diego, La Jolla. Scripps Institution of Oceanography, California; United States of America.Fil: Pereira, Inês A. C. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica António Xavier, Oeiras; Portugal.Fil: Bradley, Alexander S. Washington University in St. Louis. Department of Earth and Planetary Sciences; Missouri, United States of America.Fil: Bradley, Alexander S. Washington University in St. Louis. Division of Biology and Biomedical Sciences, Missouri; United States of America

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

The biological role and enzymatic source of extracellular reactive oxygen species in marine phytoplankton

Within the marine environment, reactive oxygen species (ROS) are abundant and participate in geochemical reactions that shape the fate and availability of metals, carbon, and oxygen due to their reactive nature. Phytoplankton are major sources of the ROS superoxide (O2-) and hydrogen peroxide (H2O2). Indeed, by exporting electrons to surrounding oxygen via enzymes, phytoplankton generate extracellular O2- (eO2-) which can then dismutate to extracellular H2O2 (eH2O2). ROS are commonly associated with stress; however, they also serve beneficial biological functions. Despite the environmental importance of eROS, their enzymatic source and ecophysiological role in phytoplankton has remained mysterious. In phytoplankton, several biological functions have been proposed for eROS production, yet a consensus has not been reached. Additionally, a class of enzymes that catalyzes electron transfers called flavoenzymes mediates production of eROS in many organisms. However, pathways of eROS production by phytoplankton are poorly understood. Here, I interrogate the ecophysiological role(s) of eROS production and its enzymatic source in a diversity of phytoplankton in laboratory and field settings. In Chapter I, results demonstrate that eO2- production is stress-independent and dynamically regulated as a function of cell abundance and growth phase consistent with a signaling role, as well as light availability in the globally-relevant coccolithophore E. huxleyi. Chapter II reveals that eO2- production is light-driven, regulated by flavoenzymes, and promotes health by serving a photoprotective role in many phytoplankton. Further, results support my hypothesis that many phytoplankton form eO2- to dissipate excess energy from light stress. Also, I estimate that light-driven eO2- production by phytoplankton will increase in future ocean conditions where mixing layer light levels are predicted to increase due to climate change. In Chapter III, field results demonstrate that eH2O2 production is dynamically regulated consistent with a signaling role and influences phytoplankton growth and microzooplankton grazing. Indeed, eH2O2 production, phytoplankton growth, and grazing were inversely correlated. Moreover, incubations show that increasing eH2O2 production decreases phytoplankton growth and grazing. Overall, my work helps illuminate the ecophysiological role and enzymatic source of eROS production by phytoplankton, thereby advancing understanding of biogeochemical cycling, redox states, plankton web dynamics, and health of current and future oceans