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In the race against COVID-19 for timely therapeutic developments, mass spectrometry-based high-throughput methods have been valuable. COVID-19 manifests an extremely diverse spectrum of phenotypes from asymptomatic to life-threatening, drastic elevations in immune response or cytokine storm, multiple organ failure and death. These observations warrant a detailed understanding of associated molecular mechanisms to develop therapies. In this direction, high-throughput methods that generate large datasets focusing on changes in protein interactions, lipid metabolism, transcription, and epigenetic regulation of gene expression are extremely beneficial sources of information. Hence, mass spectrometry-based methods have been employed in several studies to detect changes in interactions among host proteins, and between host and viral proteins in COVID-19 patients. The methods have also been used to characterize host and viral proteins, and analyze lipid metabolism in COVID-19 patients. Information obtained using the above methods are complemented by high-throughput analysis of transcriptomic and epigenomic changes associated with COVID-19, coupled with next-generation sequencing. Hence, this review discusses the most recent studies focusing on the methods described above. The results establish the importance of mass spectrometry-based studies towards understanding the infection process, immune imbalance, disease mechanism, and indicate the potential of the methods’ therapeutic developments and biomarker screening against COVID-19 and future outbreaks.

Cyclic nucleotides determine a spatiotemporally regulated system that modulates uterine myometrial contractility and parturition timing. Adenylate and guanylate cyclases synthesize cAMP and cGMP upon β-adrenergic, natriuretic peptide, and nitric oxide stimulation. Before parturition, cAMP suppresses contraction-associated protein synthesis via PKA, and cGMP activates hyperpolarizing K+ current via PKG. At term, cAMP stimulates contraction-associated protein expression via EPAC just as decreasing PKG attenuates cGMP tocolysis. The cAMP-specific phosphodiesterase 4 (PDE4) facilitates myometrial contraction by degrading cyclic nucleotides. The precise roles of cGMP on myometrial activation and the effect of other PDEs in parturition are not yet clear. Strategies to reduce preterm birth could benefit from a better understanding of the myometrial cyclic nucleotide system.

Adiponectin is secreted by adipose tissue and promotes insulin sensitivity. Low circulating adiponectin is associated with increased risk for preterm labor, but the influence of adiponectin on uterine myometrial physiology is unknown. We hypothesized that adiponectin receptors (AdipoRs) decrease myometrial contractility via AMPK to promote uterine quiescence in pregnancy. Using quantitative RT-PCR, we found that nonpregnant or pregnant human and mouse myometrium express AdipoR1 and AdipoR2 mRNAs. We confirmed AdipoR2 protein expression in human and mouse myometrium, with increased abundance in late mouse pregnancy. Both recombinant adiponectin and a pharmacologic AdipoR agonist, AdipoRon, potently inhibited uterine myometrial strip contractions in physiologic organ bath. The relaxation was independent of contractile stimulus (oxytocin, KCl, U46619). AdipoR agonists increased AMPK phosphorylation in pregnant mouse myometrium, and the direct AMPK activator A769662 also relaxed myometrial strips. However, the AMPK inhibitor dorsomorphin (compound C) blocked AMPK phosphorylation but did not abolish relaxation with either AdipoRon or A769662. In summary, adiponectin inhibits myometrial contractility consistent with the possibility that it is a previously unrecognized link between maternal metabolism and pregnancy maintenance. We also identify a separate role for AMPK regulating myometrial contractions that may influence labor onset.—Vyas, V., Guerra, D. D., Bok, R., Powell, T., Jansson, T., Hurt, K. J. Adiponectin links maternal metabolism to uterine contractility.

Nitric oxide (NO) is a major inhibitory neurotransmitter that mediates nonadrenergic noncholinergic (NANC) signaling. Neuronal NO synthase (nNOS) is activated by Ca(2+)/calmodulin to produce NO, which causes smooth muscle relaxation to regulate physiologic tone. nNOS serine1412 (S1412) phosphorylation may reduce the activating Ca(2+) requirement and sustain NO production. We developed and characterized a nonphosphorylatable nNOS(S1412A) knock-in mouse and evaluated its enteric neurotransmission and gastrointestinal (GI) motility to understand the physiologic significance of nNOS S1412 phosphorylation. Electrical field stimulation (EFS) of wild-type (WT) mouse ileum induced nNOS S1412 phosphorylation that was blocked by tetrodotoxin and by inhibitors of the protein kinase Akt but not by PKA inhibitors. Low-frequency depolarization increased nNOS S1412 phosphorylation and relaxed WT ileum but only partially relaxed nNOS(S1412A) ileum. At higher frequencies, nNOS S1412 had no effect. nNOS(S1412A) ileum expressed less phosphodiesterase-5 and was more sensitive to relaxation by exogenous NO. Under non-NANC conditions, peristalsis and segmentation were faster in the nNOS(S1412A) ileum. Together these findings show that neuronal depolarization stimulates enteric nNOS phosphorylation by Akt to promote normal GI motility. Thus, phosphorylation of nNOS S1412 is a significant regulatory mechanism for nitrergic neurotransmission in the gut.

Gasotransmitters are endogenous small gaseous messengers exemplified by nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S or sulfide). Gasotransmitters are implicated in myriad physiologic functions including many aspects of reproduction. Our objective was to comprehensively review basic mechanisms and functions of gasotransmitters during pregnancy from conception to uterine involution and highlight future research opportunities. We searched PubMed and Web of Science databases using combinations of keywords nitric oxide, carbon monoxide, sulfide, placenta, uterus, labor, and pregnancy. We included English language publications on human and animal studies from any date through August 2018 and retained basic and translational articles with relevant original findings. All gasotransmitters activate cGMP signaling. NO and sulfide also covalently modify target protein cysteines. Protein kinases and ion channels transduce gasotransmitter signals, and co-expressed gasotransmitters can be synergistic or antagonistic depending on cell type. Gasotransmitters influence tubal transit, placentation, cervical remodeling, and myometrial contractility. NO, CO, and sulfide dilate resistance vessels, suppress inflammation, and relax myometrium to promote uterine quiescence and normal placentation. Cervical remodeling and rupture of fetal membranes coincide with enhanced oxidation and altered gasotransmitter metabolism. Mechanisms mediating cellular and organismal changes in pregnancy due to gasotransmitters are largely unknown. Altered gasotransmitter signaling has been reported for preeclampsia, intrauterine growth restriction, premature rupture of membranes, and preterm labor. However, in most cases specific molecular changes are not yet characterized. Nonclassical signaling pathways and the crosstalk among gasotransmitters are emerging investigation topics.

BackgroundCOVID-19 pandemic mitigation requires evidence-based strategies. Because COVID-19 can spread via respired droplets, most US states mandated mask use in public settings. Randomized control trials have not clearly demonstrated mask efficacy against respiratory viruses, and observational studies conflict on whether mask use predicts lower infection rates. We hypothesized that statewide mask mandates and mask use were associated with lower COVID-19 case growth rates in the United States.MethodsWe calculated total COVID-19 case growth and mask use for the continental United States with data from the Centers for Disease Control and Prevention and Institute for Health Metrics and Evaluation. We estimated post-mask mandate case growth in non-mandate states using median issuance dates of neighboring states with mandates.ResultsEarlier mask mandates were not associated with lower total cases or lower maximum growth rates. Earlier mandates were weakly associated with lower minimum COVID-19 growth rates. Mask use predicted lower minimum but not lower maximum growth rates. Growth rates and total growth were comparable between US states in the first and last mask use quintiles during the Fall-Winter wave. These observations persisted for both natural logarithmic and fold growth models and when adjusting for differences in US state population density.ConclusionsWe did not observe association between mask mandates or use and reduced COVID-19 spread in US states. COVID-19 mitigation requires further research and use of existing efficacious strategies, most notably vaccination.

During pregnancy, estrogen (E2) stimulates uterine artery blood flow (UBF) by enhancing nitric oxide (NO)-dependent vasodilation. Cystathionine γ-lyase (CSE) promotes vascular NO signaling by producing hydrogen sulfide (H2S) and by maintaining the ratio of reduced-to-oxidized intracellular glutathione (GSH/GSSG) through l-cysteine production. Because redox homeostasis can influence NO signaling, we hypothesized that CSE mediates E2 stimulation of UBF by modulating local intracellular cysteine metabolism and GSH/GSSG levels to promote redox homeostasis. Using non-pregnant ovariectomized WT and CSE-null (CSE KO) mice, we performed micro-ultrasound of mouse uterine and renal arteries to assess changes in blood flow upon exogenous E2 stimulation. We quantified serum and uterine artery NO metabolites (NOx), serum amino acids, and uterine and renal artery GSH/GSSG. WT and CSE KO mice exhibited similar baseline uterine and renal blood flow. Unlike WT, CSE KO mice did not exhibit expected E2 stimulation of UBF. Renal blood flow was E2-insensitive for both genotypes. While serum and uterine artery NOx were similar between genotypes at baseline, E2 decreased NOx in CSE KO serum. Cysteine was also lower in CSE KO serum, while citrulline and homocysteine levels were elevated. E2 and CSE deletion additively decreased GSH/GSSG in uterine arteries. In contrast, renal artery GSH/GSSG was insensitive to E2 or CSE deletion. Together, these findings suggest that CSE maintenance of uterine artery GSH/GSSG facilitates nitrergic signaling in uterine arteries and is required for normal E2 stimulation of UBF. These data have implications for pregnancy pathophysiology and the selective hormone responses of specific vascular beds., Graphical abstract Image 1, Highlights • CSE-null mice exhibit abnormal estrogen augmentation of uterine artery blood flow. • Estrogen lowers uterine artery nitric oxide metabolites in CSE null mice. • CSE loss and estrogen additively impair uterine artery glutathione homeostasis. • Neither CSE loss nor estrogen influences renal artery blood flow or glutathione.

BACKGROUND AND PURPOSE: The enteric neurotransmitter nitric oxide (NO) regulates gastrointestinal motility by relaxing smooth muscle. Pharmacological cAMP induction also relaxes gastrointestinal smooth muscle, but it is uncertain whether cAMP augments or suppresses enteric NO signalling. In other organ systems, cAMP can increase neuronal NO production by stimulating protein kinase A (PKA) to phosphorylate neuronal NOS (nNOS) Serine‐1412 (S1412). We hypothesized that cAMP also increases nNOS S1412 phosphorylation by PKA in enteric neurons to augment nitrergic relaxation of mouse ileum. EXPERIMENTAL APPROACH: We measured contractile force and nNOS S1412 phosphorylation in ileal rings suspended in an organ bath. We used forskolin to induce cAMP‐dependent relaxation of wild type, nNOS(S1412A) knock‐in and nNOSα‐null ileal rings in the presence or absence of PKA, protein kinase B (Akt) and NOS inhibitors. KEY RESULTS: Forskolin stimulated phosphorylation of nNOS S1412 in mouse ileum. Forskolin relaxed nNOSα‐null and nNOS(S1412A) ileal rings less than wild‐type ileal rings. PKA inhibition blocked forskolin‐induced nNOS phosphorylation and attenuated relaxation of wild type but not nNOS(S1412A) ileum. Akt inhibition did not alter nNOS phosphorylation with forskolin but did attenuate relaxation of wild type and nNOS(S1412A). NOS inhibition with l‐NAME eliminated the effects of PKA and Akt inhibitors on relaxation. CONCLUSION AND IMPLICATIONS: PKA phosphorylation of nNOS S1412 augments forskolin‐induced nitrergic ileal relaxation. The relationship between cAMP/PKA and NO is therefore synergistic in enteric nitrergic neurons. Because NO regulates gut motility, selective modulation of enteric neuronal cAMP synthesis may be useful for the treatment of gastrointestinal motility disorders.

The free radical nitric oxide (NO(•)) regulates diverse physiological processes from vasodilation in humans to gas exchange in plants. S-Nitrosoglutathione (GSNO) is considered a principal nitroso reservoir due to its chemical stability. GSNO accumulation is attenuated by GSNO reductase (GSNOR), a cysteine-rich cytosolic enzyme. Regulation of protein nitrosation is not well understood since NO(•)-dependent events proceed without discernible changes in GSNOR expression. Because GSNORs contain evolutionarily conserved cysteines that could serve as nitrosation sites, we examined the effects of treating plant (Arabidopsis thaliana), mammalian (human), and yeast (Saccharomyces cerevisiae) GSNORs with nitrosating agents in vitro. Enzyme activity was sensitive to nitroso donors, whereas the reducing agent dithiothreitol (DTT) restored activity, suggesting that catalytic impairment was due to S-nitrosation. Protein nitrosation was confirmed by mass spectrometry, by which mono-, di-, and trinitrosation were observed, and these signals were sensitive to DTT. GSNOR mutants in specific non-zinc-coordinating cysteines were less sensitive to catalytic inhibition by nitroso donors and exhibited reduced nitrosation signals by mass spectrometry. Nitrosation also coincided with decreased tryptophan fluorescence, increased thermal aggregation propensity, and increased polydispersity-properties reflected by differential solvent accessibility of amino acids important for dimerization and the shape of the substrate and coenzyme binding pockets as assessed by hydrogen-deuterium exchange mass spectrometry. Collectively, these data suggest a mechanism for NO(•) signal transduction in which GSNOR nitrosation and inhibition transiently permit GSNO accumulation.

Recent studies suggest cysteine S-nitrosation of S-nitrosoglutathione reductase (GSNOR) could regulate protein redox homeostasis. "Switch" assays enable discovery of putatively S-nitrosated proteins. However, with few exceptions, researchers have not examined the kinetics and biophysical consequences of S-nitrosation. Methods to quantify protein S-nitrosothiol (SNO) abundance and formation kinetics would bridge this mechanistic gap and allow interpretation of the consequences of specific modifications, as well as facilitate development of specific S-nitrosation inhibitors. Here, we describe a rapid assay to estimate protein SNO abundance with intact protein electrospray ionization mass spectrometry. Originally designed using recombinant GSNOR, these methods are applicable to any purified protein to test for or further study nitrosatable cysteines.

Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.

Intercellular amino acid transport is essential for the growth of all multicellular organisms, and its dysregulation is implicated in developmental disorders. By an unknown mechanism, amino acid efflux is stimulated in plants by overexpression of a membrane-localized protein (GLUTAMINE DUMPER 1 (GDU1)) that requires a ubiquitin ligase (LOSS OF GDU 2 (LOG2). Here we further explore the physiological consequences of the interaction between these two proteins. LOG2 ubiquitin ligase activity is necessary for GDU1-dependent tolerance to exogenous amino acids, and LOG2 self-ubiquitination was markedly stimulated by the GDU1 cytosolic domain, suggesting that GDU1 functions as an adaptor or coactivator of amino acid exporter(s). However, other consequences more typical of a ligase-substrate relationship are observed: disruption of the LOG2 gene increased the in vivo half-life of GDU1, mass spectrometry confirmed that LOG2 ubiquitinates GDU1 at cytosolic lysines, and GDU1 protein levels decreased upon co-expression with active, but not enzymatically inactive LOG2. Altogether these data indicate LOG2 negatively regulates GDU1 protein accumulation by a mechanism dependent upon cytosolic GDU1 lysines. Although GDU1-lysine substituted protein exhibited diminished in vivo ubiquitination, overexpression of GDU1 lysine mutants still conferred amino acid tolerance in a LOG2-dependent manner, consistent with GDU1 being both a substrate and facilitator of LOG2 function. From these data, we offer a model in which GDU1 activates LOG2 to stimulate amino acid export, a process that could be negatively regulated by GDU1 ubiquitination and LOG2 self-ubiquitination.

Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1’s role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

Purpose One third of the world’s population suffers from gastrointestinal (GI) dysmotility, a common feature of which is disrupted nitric oxide (NO) signaling and spastic contractility. In the healthy gut, neuronal NO synthase (nNOS) induces cGMP-dependent GI smooth muscle relaxation, which facilitates digestion. However, regulation of nNOS activity is poorly understood. PKA or Akt phosphorylation of nNOS S1412 enhances hypothalamic and penile NO synthesis. We hypothesized that nNOS S1412 phosphorylation promotes GI motility. Methods We quantified the contractile force of ileal segments from C57Bl/6 wildtype and nNOSS1412A mutant mice in an organ bath under non-adrenergic, non-cholinergic (NANC) conditions. Relaxation stimuli—neuronal depolarization (EFS; 20V, 2msec pulsewidth) or adenylate cyclase activation (FSK)—were applied to ileal segments in the presence of kinase, nNOS, or cGMP pathway inhibitors. We measured nNOS S1412 phosphorylation by western blot. We determined non-NANC ileal contraction velocity with the gastrointestinal motility monitor. Results EFS (1Hz) and FSK (300nM) increased nNOS S1412 phosphorylation by 310% (p Conclusions nNOS S1412 phosphorylation promotes GI motility by decreasing spastic contractility in murine ileal segments. Neuronal depolarization elicits Akt-dependent phosphorylation, while adenylate cyclase activation elicits PKA-dependent phosphorylation of nNOS. Common GI motility disorders like irritable bowel syndrome may benefit from therapies that upregulate nNOS S1412 phosphorylation.

Phosphorylation of the C-terminal Akt/PKA consensus site activates endothelial NOS (eNOS) and decreases its calcium/calmodulin requirement, but no definite physiological role for phosphorylation of the homologous site in neuronal NOS (nNOS) has been described. Therefore, we created a knock-in mouse in which nNOS Serine-1412 (S1412) is mutated to non-phosphorylatable alanine (nNOSS1412A). We tested whether nNOSS1412A ileal rings have altered nitrergic inhibitory non-adrenergic, non-cholinergic (NANC) signaling in a physiological organ bath. Under NANC conditions, electrical field stimulation (EFS; 20V, 2msec, 1Hz, 30sec train) relaxed WT to a greater degree than nNOSS1412A ileum (73 ± 20% inhibition vs 31 ± 17%; N=3). Similarly, pharmacological treatment with forskolin (FSK) also relaxed WT more effectively than nNOSS1412A ileum (IC50 95% CI 35-44nM; N=42 vs 78-107nM; N=29). Application of selective nNOS (NANT) or pan-NOS antagonists (L-NAME) reduced FSK dependent relaxation identically in WT and nNOSS1412A ilea (IC50 ~150nM for both genotypes), confirming that a portion of FSK relaxation was due to nNOS activity. Akt and PKA are putative nNOS S1412 kinases. While there was no difference between WT and nNOSS1412A relaxation with FSK using the Akt inhibitor MK-2206, PKA inhibitors (H-89, Myr-PKI, and Rp-cAMPs) only decreased FSK relaxation in WT ileum (~3-fold increase in IC50) but not in nNOSS1412A ileum, suggesting a critical link between PKA and nNOS. Together, our data identify a novel NANC neuroinhibitory mechanism in which PKA phosphorylation of nNOS S1412 leads to calcium independent neuronal NO production and smooth muscle relaxation. This previously unrecognized NO physiology might be important in other cell types and organ systems as well.

S-nitrosoglutathione reductase (GSNOR) is believed to modulate effects of reactive oxygen and nitrogen species through catabolism of S-nitrosoglutathione (GSNO). We combined bioinformatics of plant GSNOR genes, localization of GSNOR in Arabidopsis thaliana, and microarray analysis of a GSNOR null mutant to gain insights into the function and regulation of this critical enzyme in nitric oxide (NO) homeostasis. GSNOR-encoding genes are known to have high homology across diverse eukaryotic taxa, but contributions of specific conserved residues have not been assessed. With bioinformatics and structural modeling, we show that plant GSNORs likely localize to the cytosol, contain conserved, solvent-accessible cysteines, and tend to be encoded by a single gene. Arabidopsis thaliana homozygous for GSNOR loss-of-function alleles exhibited defects in stem and trichome branching, and complementation with Green fluorescent protein (GFP) -tagged GSNOR under control of the native promoter quantitatively rescued these phenotypes. GSNOR-GFP showed fluorescence throughout Arabidopsis seedlings, consistent with ubiquitous expression of the protein, but with especially high fluorescence in the root tip, apical meristem, and flowers. At the cellular level we observed cytosolic and nuclear fluorescence, with exclusion from the nucleolus. Microarray analysis identified 99 up- and 170 down-regulated genes (≥2-fold; p ≤ 0.01) in a GSNOR null mutant compared to wild type. Six members of the plant specific, ROXY glutaredoxins and three BHLH transcription factors involved in iron homeostasis were strongly upregulated, supporting a role for GSNOR in redox and iron metabolism. One third of downregulated genes are linked to pathogen resistance, providing further basis for the reported pathogen sensitivity of GSNOR null mutants. Together, these findings indicate GSNOR regulates multiple developmental and metabolic programs in plants and offer insight into putative routes of post-translational GSNOR regulation.

Ubiquitin ligation to other proteins modulates the activity, longevity, and/or localization of the target proteins in eukaryotic systems. As components of the ubiquitin pathway, plant hormone receptors determine the abundance of key transcriptional regulators of auxin, GA, and jasmonate response