Oxygen Deficient Zones (ODZs) are the largest pelagic sinks of N containing nutrients in the ocean. The offshore Eastern Tropical North Pacific (ETNP) ODZ has been shown to be limited by organic matter. We propose zooplankton/forage fish as a key source of particulate and dissolved organic matter for N 2 production that has previously been ignored. We examined data sets from four cruises (April 2012, January 2017, April 2018, October 2019) at a station in the central ETNP. Backscattering data were used to determine zooplankton vertical migration depths (250–450 m, maximum at 270–280 m). Metazoan DNA concentrations, as measured by quantitative PCR, had a reproducible maximum at 270–280 m, confirming that these signals indicate the presence of zooplankton/forage fish. Additionally, a large maximum in sinking pteropod shells was found at 270 m, indicating that pteropods were part of the migrating community. While crustacean zooplankton have been shown to reduce respiration and excretion of ammonium under anoxia, we found intermittently measurable ammonium concentrations at 270 m. Here we show signatures consistent with organic matter of zooplankton/forage fish origin in the C:N and δ 13 C of suspended and sinking organic matter at the vertical migration depth that suggest transportation to these depths by migrating zooplankton/forage fish. Also coincident with the migration maximum was a reproducible‐between‐years maximum in the biological N 2 gas, and a repeatable shoulder on the nitrite maximum, which suggest that the migrating zooplankton partially fuels N loss. Thus, zooplankton/forage fish appear to be one source of organic matter which can fuel N 2 production in ODZs.

Peptides are identified using a de novo-discovery approach in suspended and sinking particles from the eastern tropical North Pacific oxygen-deficient zone (ODZ) and in a culture of a dominant autotroph from the region, the cyanobacterium Prochlorococcus. The benchmarking experiment with Prochlorococcus shows de novo peptides to be taxonomically specific, and thus of value in augmenting database-driven approaches. Analysis of the suspended and sinking particles using the de novo-discovery approach reveals the presence of fungal proteins in deep sinking particles that were not in our original search database, contributing to growing recognition that fungi may play important roles in marine organic matter cycling. Cyanobacterial peptides that have been post-translationally modified were tracked to depth, where they contribute similar to 1% of the phylum-level identifiable peptide pool in the sediment trap sample. The majority of peptides found at depth in the detrital pool are associated with membranes, indicating that cellular location is associated with early preservation within the detrital pool. Modified amino acids in sinking and suspended particles include high levels of deamidation, suggesting that partial extracellular degradation of protein could fuel observed anammox and contribute to pools of refractory organic nitrogen.

The gravitational biological pump is not large enough to account for microbial heterotrophic activity in the mesopelagic ocean. Migrating zooplankton may be a key source of organic matter transport to depth. Here we show signatures of zooplankton in the suspended organic matter at the zooplankton vertical migration depth in the Eastern Tropical North Pacific Oxygen Deficient Zone (ETNP ODZ). We examine the mesozooplankton community in metagenomic depth profiles using the mitochondrial cytochrome c oxidase (COI) gene as a marker in the ETNP and Eastern Tropical South Pacific (ETSP) ODZs and at the oxic Hawaii Ocean Timeseries (HOT). Additionally, eukaryotic transcripts (polyA-selected) were examined for zooplankton in the ETNP. While zooplankton eDNA increased in the ODZ, zooplankton eRNA decreased in the ODZ, similar to previous net-based data, implying that eDNA is better preserved under anoxia. At all stations, Cnidaria, often missed in net-based data, contributed greatly to the zooplankton eDNA/eRNA. SAR11 abundance, determined from the single-copy core gene ( rpoB ), significantly correlated with zooplankton eDNA, with R 2 values >0.8 at all stations. Strong correlations between SAR11 and zooplankton have not been previously reported, but are logical as SAR11 bacteria consume and zooplankton excrete simple dissolved organic compounds. SAR11 bacteria possessed genes to utilize urea and taurine in the ODZ, both compounds known to be excreted by zooplankton. In ODZs, SAR11 bacteria preferentially used the taurine degradation pathway leading to C and N assimilation, not the pathway for organic S assimilation, probably due to additional sources of organic S in ODZs.

Peptides and proteins were identified during a controlled laboratory degradation of the marine diatom Thalassiosira weissflogii by a surface seawater microbiome. Samples from each time point were processed both with and without the protease trypsin, allowing a partial differentiation between peptides produced naturally by microbial enzymatic degradation and peptides produced from the laboratory digestion of intact protein. Over the 12-day degradation experiment, 31% of the particulate organic carbon was depleted, and there was no preferential degradation of the overall protein pool. However, there was distinct differentiation in the cellular location, secondary structure and modifications between peptides produced by microbial vs. laboratory breakdown. During the initial period of rapid algal decay and bacterial growth, intracellular components from the cytoplasm were consumed first, resulting in the accumulation of membrane-associated proteins and peptides in the detrital pool. Accompanying the enrichment of membrane protein material was an increase in the importance of ɑ-helix motifs. Methylated arginine, a post-translational modification common in cell senescence, was found in high amounts within the microbially produced detrital peptide pool, suggesting a link between in-cell modification and resistance to immediate degradation. Another modification—asparagine deamidation—accumulated within the detrital peptides. Protein taxonomies showed the bacterial community decomposing the algal material was rich in Proteobacteria, and protein annotations showed abundant transportation of solubilized carbohydrates and small peptides across membranes. At this early stage of diagenesis, no changes in bulk amino acids (THAA) were observed, yet a proteomic approach allowed us to observe selective changes in diatom protein preservation by using amino acid sequences to infer subcellular location, secondary structures, and post-translational modifications (PTMs).

Earth and Space Science Open Archive This work has been accepted for publication in Global Biogeochemical Cycles. Version of RecordESSOAr is a venue for early communication or feedback before peer review. Data may be preliminary. Learn more about preprints. preprintOpen AccessYou are viewing an older version [v1]Go to new versionSlow particle remineralization, rather than suppressed disaggregation, drives efficient flux transfer through the Eastern Tropical North Pacific Oxygen Deficient ZoneAuthorsJacobCramiDClaraFuchsmaniDMeganDuffyiDJessicaPrettyiDRachelLekanoffJacquelynNeibaueriDShirleyLeungiDKlaus B.HuebertiDThomasWeberDanieleBianchiiDNatalyaEvansiDAllanDevoliDRichardKeiliDAndrewMcDonnelliDSee all authors Jacob CramiDCorresponding Author• Submitting AuthorHorn Point Laboratory, University of Maryland Center for Environmental Science, Cambridge, MD, USA.iDhttps://orcid.org/0000-0001-9546-1130view email addressThe email was not providedcopy email addressClara FuchsmaniDHorn Point Laboratory, University of Maryland Center for Environmental Science, Cambridge, MD, USA.iDhttps://orcid.org/0000-0002-9151-4984view email addressThe email was not providedcopy email addressMegan DuffyiDSchool of Oceanography, University of Washington Seattle, Seattle, WA, USA.iDhttps://orcid.org/0000-0002-3212-4927view email addressThe email was not providedcopy email addressJessica PrettyiDCollege of Fisheries and Ocean SciencesUniversity of Alaska FairbanksFairbanksAKUSA.iDhttps://orcid.org/0000-0001-6542-8540view email addressThe email was not providedcopy email addressRachel LekanoffCollege of Fisheries and Ocean Sciences, University of Alaska Fairbanks, Fairbanks, AK, USA.view email addressThe email was not providedcopy email addressJacquelyn NeibaueriDSchool of Oceanography, University of Washington Seattle, Seattle, WA, USA.iDhttps://orcid.org/0000-0001-9920-2558view email addressThe email was not providedcopy email addressShirley LeungiDSchool of Oceanography, University of Washington Seattle, Seattle, WA, USA.iDhttps://orcid.org/0000-0002-6659-6420view email addressThe email was not providedcopy email addressKlaus B. HuebertiDHorn Point Laboratory, University of Maryland Center for Environmental Science, Cambridge, MD, USA.iDhttps://orcid.org/0000-0002-2432-7337view email addressThe email was not providedcopy email addressThomas WeberSchool of Arts and Sciences, University of Rochester, Rochester, NY, USA.view email addressThe email was not providedcopy email addressDaniele BianchiiDDepartment of Atmospheric and Oceanic Sciences, University of California Los Angeles, Los Angeles, CA, USA.iDhttps://orcid.org/0000-0002-6621-0858view email addressThe email was not providedcopy email addressNatalya EvansiDDepartment of Biological Sciences, University of Southern California, Los Angeles, CA, USA.iDhttps://orcid.org/0000-0002-2726-8272view email addressThe email was not providedcopy email addressAllan DevoliDSchool of OceanographyUniversity of Washington SeattleSeattleWAUSA.iDhttps://orcid.org/0000-0003-4016-9399view email addressThe email was not providedcopy email addressRichard KeiliDSchool of Oceanography, University of Washington Seattle, Seattle, WA, USA.iDhttps://orcid.org/0000-0001-7483-1606view email addressThe email was not providedcopy email addressAndrew McDonnelliDUniversity of Alaska Fairbanks, College of Fisheries and Ocean Sciences Fairbanks, AK, USA.iDhttps://orcid.org/0000-0003-1408-4869view email addressThe email was not providedcopy email address

Models and observations suggest that particle flux attenuation is lower across the mesopelagic zone of anoxic environments compared to oxic environments. Flux attenuation is controlled by microbial metabolism as well as aggregation and disaggregation by zooplankton, all of which shape the relative abundance of differently sized particles. Observing and modeling particle spectra can provide information about the contributions of these processes. We measured particle size spectrum profiles at one station in the oligotrophic Eastern Tropical North Pacific Oxygen Deficient Zone (ETNP ODZ) using an underwater vision profiler (UVP), a high‐resolution camera that counts and sizes particles. Measurements were taken at different times of day, over the course of a week. Comparing these data to particle flux measurements from sediment traps collected over the same time‐period allowed us to constrain the particle size to flux relationship, and to generate highly resolved depth and time estimates of particle flux rates. We found that particle flux attenuated very little throughout the anoxic water column, and at some time points appeared to increase. Comparing our observations to model predictions suggested that particles of all sizes remineralize more slowly in the ODZ than in oxic waters, and that large particles disaggregate into smaller particles, primarily between the base of the photic zone and 500 m. Acoustic measurements of multiple size classes of organisms suggested that many organisms migrated, during the day, to the region with high particle disaggregation. Our data suggest that diel‐migrating organisms both actively transport biomass and disaggregate particles in the ODZ core.

Up to half of marine N losses occur in oxygen-deficient zones (ODZs). Organic matter flux from productive surface waters is considered a primary control on N 2 production. Here we investigate the offshore Eastern Tropical North Pacific (ETNP) where a secondary chlorophyll a maximum resides within the ODZ. Rates of primary production and carbon export from the mixed layer and productivity in the primary chlorophyll a maximum were consistent with oligotrophic waters. However, sediment trap carbon and nitrogen fluxes increased between 105 and 150 m, indicating organic matter production within the ODZ. Metagenomic and metaproteomic characterization indicated that the secondary chlorophyll a maximum was attributable to the cyanobacterium Prochlorococcus , and numerous photosynthesis and carbon fixation proteins were detected. The presence of chemoautotrophic ammonia-oxidizing archaea and the nitrite oxidizer Nitrospina and detection of nitrate oxidoreductase was consistent with cyanobacterial oxygen production within the ODZ. Cyanobacteria and cyanophage were also present on large (>30 μm) particles and in sediment trap material. Particle cyanophage-to-host ratio exceeded 50, suggesting that viruses help convert cyanobacteria into sinking organic matter. Nitrate reduction and anammox proteins were detected, congruent with previously reported N 2 production. We suggest that autochthonous organic matter production within the ODZ contributes to N 2 production in the offshore ETNP.

Ocean metaproteomics is an emerging field enabling discoveries about marine microbial communities and their impact on global biogeochemical processes. Recent ocean metaproteomic studies have provided insight into microbial nutrient transport, colimitation of carbon fixation, the metabolism of microbial biofilms, and dynamics of carbon flux in marine ecosystems. Future methodological developments could provide new capabilities such as characterizing long-term ecosystem changes, biogeochemical reaction rates, and in situ stoichiometries. Yet challenges remain for ocean metaproteomics due to the great biological diversity that produces highly complex mass spectra, as well as the difficulty in obtaining and working with environmental samples. This review summarizes the progress and challenges facing ocean metaproteomic scientists and proposes best practices for data sharing of ocean metaproteomic data sets, including the data types and metadata needed to enable intercomparisons of protein distributions and annotations that could foster global ocean metaproteomic capabilities.

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

The anterior pituitary is specialized for the synthesis, storage and release of peptide hormones. The activation of inactive peptide hormone precursors requires a specific set of proteases and other post-translational processing enzymes. High levels of peptidylglycine α-amidating monooxygenase (PAM), an essential peptide processing enzyme, occur in the anterior pituitary. PAM, which converts glycine-extended peptides into amidated products, requires copper and zinc to support its two catalytic activities and calcium for structure. We used X-ray fluorescence microscopy on rat pituitary sections and inductively coupled plasma mass spectrometry on subcellular fractions prepared from rat anterior pituitary to localize and quantify copper, zinc and calcium. X-ray fluorescence microscopy indicated that the calcium concentration in pituitary tissue was about 2.5 mM, 10-times more than zinc and 50-times more than copper. Although no higher than cytosolic levels, secretory granule levels of copper exceeded PAM levels by a factor of 10. Atp7a, which transports copper into the lumen of the secretory pathway, was enriched in endosomes and Golgi, not in secretory granules. If Atp7a transfers copper directly to PAM, this pH-dependent process is likely to occur in Golgi and endosomes.

The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

Nonalcoholic fatty liver disease (NAFLD) prevalence is increasing worldwide, with the affected US population estimated near 30%. Diet is a recognized risk factor in the NAFLD spectrum, which includes nonalcoholic steatohepatitis (NASH) and fibrosis. Low hepatic copper (Cu) was recently linked to clinical NAFLD/NASH severity. Simple sugar consumption including sucrose and fructose is implicated in NAFLD, while consumption of these macronutrients also decreases liver Cu levels. Though dietary sugar and low Cu are implicated in NAFLD, transcript-level responses that connect diet and pathology are not established. We have developed a mature rat model of NAFLD induced by dietary Cu deficiency, human-relevant high sucrose intake (30% w/w) or both factors in combination. Compared to the control diet with adequate Cu and 10% (w/w) sucrose, rats fed either high-sucrose or low-Cu diet had increased hepatic expression of genes involved in inflammation and fibrogenesis, including hepatic stellate cell activation, while the combination of diet factors also increased ATP citrate lyase and fatty acid synthase gene transcription (fold change >2, P<0.02). Low dietary Cu decreased hepatic and serum Cu (P≤0.05), promoted lipid peroxidation and induced NAFLD-like histopathology, while the combined factors also induced fasting hepatic insulin resistance and liver damage. Neither low Cu nor 30% sucrose in the diet led to enhanced weight gain. Taken together, transcript profiles, histological and biochemical data indicate that low Cu and high sucrose promote hepatic gene expression and physiological responses associated with NAFLD and NASH, even in the absence of obesity or severe steatosis.

Background The transition metal copper enhances amyloid β aggregation and neurotoxicity, and in models of concomitant amyloid and tau pathology, copper also promotes tau aggregation. Since it is not clear if the effects of environmental copper upon tau pathology are dependent on the presence of pathological amyloid β, we tested the effects of copper overload and complexing in disease models which lack pathological amyloid β. Methods We used cell culture and transgenic murine models to test the effects of environmental copper on tau phosphorylation. We used oral zinc acetate as a copper lowering agent in mice and examined changes in blood and brain metals through inductively coupled plasma mass spectroscopy. Behavioral effects of copper lowering were assessed with Morris water maze and novel object recognition tasks. Changes in tau phosphorylation were examined by phosphorylation specific antibodies on Western blots. Results In human neuroblastoma cells, excess copper promoted tau phosphorylation and a copper complexing agent, tetrathiomolybdate, attenuated tau phosphorylation. In a transgenic mouse model expressing wild type human tau, copper-lowering by oral zinc suppressed plasma and brain levels of copper, and resulted in a marked attenuation of tau phosphorylation. No significant changes in behavior were observed with copper lowering, but a trend to improved recognition of the novel object was observed in zinc acetate treated mice. Conclusions We propose that reduction of brain copper by blocking uptake of copper from the diet may be a viable strategy for modulating tau pathology in Alzheimer’s disease. The potential benefits of this approach are tempered by the absence of a behavioral benefit and by the health risks of excessive lowering of copper.