N-loss isotope effects in the Peru oxygen minimum zone studied using a mesoscale eddy as a natural tracer experiment

Mesoscale eddies in Oxygen Minimum Zones (OMZ's) have been identified as important fixed nitrogen (N) loss hotspots that may significantly impact both the global rate of N-loss as well as the ocean's N isotope budget. They also represent ‘natural tracer experiments’ with intensified biogeochemical signals that can be exploited to understand the large-scale processes that control N-loss and associated isotope effects (ε; the ‰ deviation from 1 in the ratio of reaction rate constants for the light versus the heavy isotopologues). We observed large ranges in the concentrations and N and O isotopic compositions of nitrate (NO3−), nitrite (NO2−) and biogenic N2 associated with an anticyclonic eddy in the Peru OMZ during two cruises in November and December 2012. In the eddy's center where NO3− was nearly exhausted, we measured the highest δ15N values for both NO3− and NO2− (up to ~70‰ and 50‰) ever reported for an OMZ. Correspondingly, N deficit and biogenic N2-N concentrations were also the highest near the eddy's center (up to ~40 µmol L−1). δ15N-N2 also varied with biogenic N2 production, following kinetic isotopic fractionation during NO2− reduction to N2 and, for the first time, provided an independent assessment of N isotope fractionation during OMZ N-loss. We found apparent variable ε for NO3− reduction (up to ~30‰ in the presence of NO2−). However, the overall ε for N-loss was calculated to be only ~13-14‰ (as compared to canonical values of ~20-30‰) assuming a closed system and only slightly higher assuming an open system (16-19‰). Our results were similar whether calculated from the disappearance of DIN (NO3− + NO2−) or from the appearance of N2 and changes in isotopic composition. Further, we calculated the separate ε for NO3− reduction to NO2− and NO2− reduction to N2 of ~16-21‰ and ~12‰, respectively, when the effect of NO2− oxidation could be removed. These results, together with the relationship between N and O of NO3− isotopes and the difference in δ15N between NO3− and NO2-, confirm a role for NO2− oxidation in increasing the apparent ε associated with NO3− reduction. The lower ε for NO3− and NO2− reduction as well as N-loss calculated in this study could help reconcile the current imbalance in the global N budget if they are representative of OMZ N-loss

Label-Free Phenotypic Profiling Identified D-Luciferin as a GPR35 Agonist

Fluorescent and luminescent probes are essential to both in vitro molecular assays and in vivo imaging techniques, and have been extensively used to measure biological function. However, little is known about the biological activity, thus potential interferences with the assay results, of these probe molecules. Here we show that D-luciferin, one of the most widely used bioluminescence substrates, is a partial agonist for G protein-coupled receptor-35 (GPR35). Label-free phenotypic profiling using dynamic mass redistribution (DMR) assays showed that D-luciferin led to a DMR signal in native HT-29 cells, whose characteristics are similar to those induced by known GPR35 agonists including zaprinast and pamoic acid. DMR assays further showed that D-luciferin is a partial agonist competitive to several known GPR35 agonists and antagonists. D-luciferin was found to cause the phosphorylation of ERK that was suppressed by known GPR35 antagonists, and also result in β-arrestin translocation signal but with low efficacy. These results not only suggest that D-luciferin is a partial agonist of GPR35, but also will evoke careful interpretation of biological data obtained using molecular and in vivo imaging assays when these probe molecules are used

Transcriptome Analysis Revealed Potential Neuro-Immune Interaction in Papillary Thyroid Carcinoma Tissues

Background: A recent study reported that papillary thyroid carcinoma (PTC) was associated with increased adrenergic nerve density. Meanwhile, emerging evidence suggested that tumor-innervating nerves might play a role in shaping the tumor microenvironment. We aimed to explore the potential interaction between neuronal markers and tumor microenvironmental signatures through a transcriptomic approach. Methods: mRNA sequencing was conducted using five pairs of PTC and adjacent normal tissues. The Gene Set Variation Analysis (GSVA) was performed to calculate enrichment scores of gene sets related to tumor-infiltrating immune cells and the tumor microenvironment. The potential interaction was tested using the expression levels of a series of neuronal markers and gene set enrichment scores. Results: PTC tissues were associated with increased enrichment scores of CD8 T cells, cancer-associated fibroblasts, mast cells, and checkpoint molecules. The neuronal marker for cholinergic neurons was positively correlated with CD8 T cell activation, while markers for serotonergic and dopaminergic neurons showed an inverse correlation. Conclusion: Distinct neuronal markers exerted different correlations with tumor microenvironmental signatures. Tumor-innervating nerves might play a role in the formation of the PTC microenvironment

Tyrphostin analogs are GPR35 agonists

AbstractGPR35 is an orphan G protein-coupled receptor that is not well-characterized. Here we employ dynamic mass redistribution (DMR) assays to discover new GPR35 agonists. DMR assays identified tyrphostin analogs as GPR35 agonists, which were confirmed with receptor internalization, Tango β-arrestin translocation, and extracellular-signal-regulated kinase phosphorylation assays. These agonists provide pharmacological tools to study the biology and function of GPR35

The DMR arising from co-stimulation with two agonists.

<p>(a) The dose-dependent responses of pamoic acid in the absence and presence of D-luciferin at three different fixed doses; the cell seeding density was 25000 cells per well; and (b) the dose-dependent response of D-luciferin in the absence and presence of pamoic acid at three fixed doses, the cell seeding density was 32000 cells per well. The ligand P-DMR amplitudes were used to calculate all dose responses. All data represent mean ± s.d. from 2 independent measurements, each in duplicate (n = 4).</p

D-Luciferin triggered ERK phosphorylation via GPR35.

<p>(a) Western blot of phosphorylated ERK1/2 (pERK1/2) after treated with 32 µM D-luciferin for different times; (b) Western blot of phosphorylated ERK 30 min after treated with three different known GPR35 agonists, YE210 (1 µM), zaprinast (1 µM) and pamoic acid (1 µM); (c) Western blot of phosphorylated ERK 5 min after treated with 32 µM in the presence of SPB05142 at different doses; (d) Western blot of phosphorylated ERK 5 min after treated with 32 µM in the presence of ML145 at different doses. The phosphorylated ERK and total ERK were blotted, and actin was used as control. Representative images obtained from 2 independent measurements were used.</p

The desensitization of HT-29 to 250 nM zaprinast after 1 hr pretreatment with D-luciferin at different doses.

<p>Only the zaprinast-induced DMR signals were shown: (a) the real time kinetic response of zaprinast; and (b) the zaprinast P-DMR amplitudes as a function of D-luciferin concentration. All data represent mean ± s.d. from 2 independent measurements, each in duplicate (n = 4).</p