μM O2 Research Articles

Characterisation of a microelectrochemical biosensor for real-time detection of brain extracellular d-serine

A modified development protocol and concomitant characterisation of a first generation biosensor for the detection of brain extracellular d-serine is reported. Functional parameters important for neurochemical monitoring, including sensor sensitivity, O2 interference, selectivity, shelf-life and biocompatibility were examined. Construction and development involved the enzyme d-amino acid oxidase (DAAO), utilising a dip-coating immobilisation method employing a new extended drying approach. The resultant Pt-based polymer enzyme composite sensor achieved high sensitivity to d-serine (0.76 ± 0.04 nA mm−2. μM−1) and a low μM limit of detection (0.33 ± 0.02 μM). The in-vitro response time was within the solution stirring time, suggesting potential sub-second in-vivo response characteristics. Oxygen interference studies demonstrated a 1 % reduction in current at 50 μM O2 when compared to atmospheric O2 levels (200 μM), indicating that the sensor can be used for reliable neurochemical monitoring of d-serine, free from changes in current associated with physiological O2 fluctuations. Potential interference signals generated by the principal electroactive analytes present in the brain were minimised by using a permselective layer of poly(o-phenylenediamine), and although several d-amino acids are possible substrates for DAAO, their physiologically relevant signals were small relative to that for d-serine. Additionally, changing both temperature and pH over possible in vivo ranges (34–40 °C and 7.2–7.6 respectively) resulted in no significant effect on performance. Finally, the biosensor was implanted in the striatum of freely moving rats and used to monitor physiological changes in d-serine over a two-week period.

Construction of a super large Stokes shift near-infrared fluorescent probe for detection and imaging of superoxide anion in living cells, zebrafish and mice

As one of the major reactive oxygen species (ROS), superoxide anion (O2•−) is engaged in maintaining redox homeostasis in the cell microenvironment. To identify the pathological roles in related disorders caused by abnormal expression of O2•−, it is of great significance to monitor and track the fluctuation of O2•− concentration in vivo. However, the low concentration of O2•− and the interference caused by tissue autofluorescence make the development of an ideal detection methodology full of challenges. Herein, a “Turn-On” chemical response near-infrared (NIR) fluorescence probe Dcm-Cu-OTf for O2•− detection in inflamed models, was constructed by conjugating the NIR fluorophore (dicyanisophorone derivative) with an O2•− sensing moiety (trifluoromethanesulfonate). Dcm-Cu-OTf exerted about 140-fold fluorescence enhancement after reacting 200 μM O2•− with an excellent limited of detection (LOD) as low as 149 nM. Additionally, Dcm-Cu-OTf exhibited a super large Stokes shift (260 nm) and high selectivity over other bio-analytes in stimulated conditions. Importantly, Dcm-Cu-OTf showed low toxicity and enabled imaging of the generation of O2•− in the Lipopolysaccharide (LPS)-stimulated HeLa cells, zebrafish, and LPS-induced inflamed mice. The present study provided a potential and reliable detection tool to inspect the physiological and pathological progress of O2•− in living biosystems.

Superoxide, nitric oxide and peroxynitrite production by macrophages under different physiological oxygen tensions

Macrophages count on two O2-consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O2•−) and nitric oxide (•NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O2•− and •NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO2) normally ranges within 2–14 %, with a typical average pO2 value for most tissues ca. 5 %. Given that O2 is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O2•− and •NO production. Also, O2 is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula, analyzing the formation and effects of O2•− and •NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO2; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O2) which are far from the physiological pO2. Here, the role of O2 as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO2 and O2•− and •NO formation rates were measured, obtaining a KM of 26 and 42 μM O2 for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO2, reaching a maximum at ≥ 10 % O2, but even at levels as low as 2 % O2, a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO2 values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions.

Analysis of tumour oxygenation in model animals on a phosphorescence lifetime based macro-imager

Monitoring of tissue O2 is essential for cancer development and treatment, as hypoxic tumour regions develop resistance to radio- and chemotherapy. We describe a minimally invasive technique for the monitoring of tissue oxygenation in developing grafted tumours, which uses the new phosphorescence lifetime based Tpx3Cam imager. CT26 cells stained with a near-infrared emitting nanoparticulate O2 probe NanO2-IR were injected into mice to produce grafted tumours with characteristic phosphorescence. The tumours were allowed to develop for 3, 7, 10 and 17 days, with O2 imaging experiments performed on live and euthanised animals at different time points. Despite a marked trend towards decreased O2 in dead animals, their tumour areas produced phosphorescence lifetime values between 44 and 47 µs, which corresponded to hypoxic tissue with 5–20 μM O2. After the O2 imaging in animals, confocal Phosphorescence Lifetime Imaging Microscopy was conducted to examine the distribution of NanO2-IR probe in the tumours, which were excised, fixed and sliced for the purpose. The probe remained visible as bright and discrete ‘islands’ embedded in the tumour tissue until day 17 of tumour growth. Overall, this O2 macro-imaging method using NanO2-IR holds promise for long-term studies with grafted tumours in live animal models, providing quantitative 2D mapping of tissue O2.

Biofilm formation and cell plasticity drive diazotrophy in an anoxygenic phototrophic bacterium.

The contribution of non-cyanobacterial diazotrophs (NCDs) to total N2 fixation in the marine water column is unknown, but their importance is likely constrained by the limited availability of dissolved organic matter and low O2 conditions. Light could support N2 fixation and growth by NCDs, yet no examples from bacterioplankton exist. In this study, we show that the phototrophic NCD, Rhodopseudomonas sp. BAL398, which is a member of the diazotrophic community in the surface waters of the Baltic Sea, can utilize light. Our study highlights the significance of biofilm formation for utilizing light and fixing N2 under oxic conditions and the role of cell plasticity in regulating these processes. Our findings have implications for the general understanding of the ecology and importance of NCDs in marine waters.

The terminal oxidase cytochrome bd-I confers carbon monoxide resistance to Escherichia coli cells

Carbon monoxide (CO) plays a multifaceted role in the physiology of organisms, from poison to signaling molecule. Heme proteins, including terminal oxidases, are plausible CO targets. Three quinol oxidases terminate the branched aerobic respiratory chain of Escherichia coli. These are the heme‑copper cytochrome bo3 and two copper-lacking bd-type cytochromes, bd-I and bd-II. All three enzymes generate a proton motive force during the four-electron oxygen reduction reaction that is used for ATP production. The bd-type oxidases also contribute to mechanisms of bacterial defense against various types of stresses. Here we report that in E. coli cells, at the enzyme concentrations tested, cytochrome bd-I is much more resistant to inhibition by CO than cytochrome bd-II and cytochrome bo3. The apparent half-maximal inhibitory concentration values, IC50, for inhibition of O2 consumption of the membrane-bound bd-II and bo3 oxidases by CO at ~150 μM O2 were estimated to be 187.1 ± 11.1 and 183.3 ± 13.5 μM CO, respectively. Under the same conditions, the maximum inhibition observed with the membrane-bound cytochrome bd-I was 20 ± 10% at ~200 μM CO.

Polarized Signatures of a Habitable World: Comparing Models of an Exoplanet Earth with Visible and Near-infrared Earthshine Spectra

In the JWST, Extremely Large Telescopes, and LUVOIR era, we expect to characterize a number of potentially habitable Earth-like exoplanets. However, the characterization of these worlds depends crucially on the accuracy of theoretical models. Validating these models against observations of planets with known properties will be key for the future characterization of terrestrial exoplanets. Due to its sensitivity to the micro- and macro-physical properties of an atmosphere, polarimetry will be an important tool that, in tandem with traditional flux-only observations, will enhance the capabilities of characterizing Earth-like planets. In this paper we benchmark two different polarization-enabled radiative-transfer codes against each other and against unique linear spectropolarimetric observations of the earthshine that cover wavelengths from ∼0.4 to ∼2.3 μm. We find that while the results from the two codes generally agree with each other, there is a phase dependency between the compared models. Additionally, with our current assumptions, the models from both codes underestimate the level of polarization of the earthshine. We also report an interesting discrepancy between our models and the observed 1.27 μm O2 feature in the earthshine, and provide an analysis of potential methods for matching this feature. Our results suggest that only having access to the 1.27 μm O2 feature coupled with a lack of observations of the O2 A and B bands could result in a mischaracterization of an Earth-like atmosphere. Providing these assessments is vital to aid the community in the search for life beyond the solar system.

Deoxygenation enhances photosynthetic performance and increases N2 fixation in the marine cyanobacterium Trichodesmium under elevated pCO2.

Effects of changed levels of dissolved O2 and CO2 on marine primary producers are of general concern with respect to ecological effects of ongoing ocean deoxygenation and acidification as well as upwelled seawaters. We investigated the response of the diazotroph Trichodesmium erythraeum IMS 101 after it had acclimated to lowered pO2 (~60 μM O2) and/or elevated pCO2 levels (HC, ~32 μM CO2) for about 20 generations. Our results showed that reduced O2 levels decreased dark respiration significantly, and increased the net photosynthetic rate by 66 and 89% under the ambient (AC, ~13 μM CO2) and the HC, respectively. The reduced pO2 enhanced the N2 fixation rate by ~139% under AC and only by 44% under HC, respectively. The N2 fixation quotient, the ratio of N2 fixed per O2 evolved, increased by 143% when pO2 decreased by 75% under the elevated pCO2. Meanwhile, particulate organic carbon and nitrogen quota increased simultaneously under reduced O2 levels, regardless of the pCO2 treatments. Nevertheless, changed levels of O2 and CO2 did not bring about significant changes in the specific growth rate of the diazotroph. Such inconsistency was attributed to the daytime positive and nighttime negative effects of both lowered pO2 and elevated pCO2 on the energy supply for growth. Our results suggest that Trichodesmium decrease its dark respiration by 5% and increase its N2-fixation by 49% and N2-fixation quotient by 30% under future ocean deoxygenation and acidification with 16% decline of pO2 and 138% rise of pCO2 by the end of this century.

Early diagenetic processes in an iron-dominated marine depositional system

The early diagenetic interplay between reactive iron, sulfur, and organic matter in the bathymetrically isolated Santa Monica Basin (SMB) sediments are investigated in this study. We explore solid-phase and porewater profiles from the basin, supplemented with a transect from 71 to 907 m water depth that includes oxygenated (>60 μM O2) bottom waters near the coast and oxygen-deficient waters (∼4 µM O2) in the basin. The geochemical data of the basin sediments are further scrutinized by means of reactive transport modeling.The results show that the basin sediments do not follow the traditional geochemical signatures of oxygen-deficient settings. A lack of dissolved sulfide accumulation and sulfurized iron persists despite the sediments being deposited under reducing conditions (without bioturbation/bioirrigation), strong organic carbon input (TOC up to 5.0 wt%), and active dissimilatory sulfate reduction. Not only did we find an exceptional enrichment in highly reactive Fe in the surface sediments (∼45 % of total Fe), but the enrichment of reactive Fe, including ferrihydrite, persists downcore and coexists with high levels of dissolved Fe. The enhanced preservation of Fe oxides and lack of iron-sulfide precipitation is in part explained by detection via Mössbauer spectra of iron oxides bounded to organic matter (Fe[III]-OM coprecipitates).The modeled Fe budget shows that most of the Fe oxides in the surface sediments are internally recycled by upward diffusion and subsequent oxidation of Fe2+. Sulfide oxidation coupled to Fe reduction effectively precludes sulfide accumulation while enhancing build-up of dissolved Fe, fueling the Fe cycle within the first 5 cm depth. Continuous reoxidation of Fe2+ enhances the formation of Fe(III)-OM coprecipitates, limiting the amount of reactive organic matter. In the unavailability of labile organic matter, other than within the uppermost layers, the organic-rich sediment profiles are dominated by Fe cycling that limits the production and preservation of sulfides and enhances the preservation of Fe oxides and organic carbon. This study highlights key local controls on Fe availability in marginal basins and describes an intricate biogeochemical C-Fe-S cycling in modern and possibly ancient marine systems with important implications for Fe availability in the marine realm.

Microbial metabolic activity in two basins of the Gulf of Mexico influenced by mesoscale structures

Information on microbial metabolic activity is essential for quantifying carbon and energy flows through marine food webs. We quantified community (Rcom) and prokaryotic (Rpro) respiration rates, bacterial production (BP), bacterial abundance (BA), and bacterial growth efficiencies (BGE) in the Perdido and Coatzacoalcos basins of the Gulf of Mexico (GOM) during summer and winter conditions in 2016. Our results showed seasonal, regional, and mesoscale eddy influences on those metabolic variables. Rpro accounted for >60% of total respiration in both regions, being three times higher in stations influenced by a cyclonic eddy (CE) in September (24.1 μM O2 d−1) than in stations affected by an anticyclonic eddy in March (7.2 μM O2 d−1) within the Coatzacoalcos basin where the eddy-trapping mechanism advected biomass-enriched waters from the Bay of Campeche. The eddy-stirring mechanism produced horizontal and vertical dipole patterns of metabolic variables increasing up to one order of magnitude Rcom and Rpro while decreasing BGE to 25-fold from the southeastern to the northwestern edges in CEs. This finding indicates that dissolved organic matter is more actively taken up to build bacterial biomass on the eastern edge of CEs in the GOM, while Rpro and Rcom increase on the western edges. Satellite integrated primary production was coupled with surface Rpro and Rcom at CEs and no eddies. BP was mainly regulated by CEs and was about 50% higher in the Coatzacoalcos basin (~0.03–0.14 μmol C L−1 d−1). BP increased in zones with high Rpro and Rcom, suggesting that Rcom is associated with heterotrophic prokaryote activity in both basins. BGE was lower than 25% within the upper 500 m during both cruises, but the highest values were quantified in the euphotic zone and during the September cruise. Metabolic variables integrated over the water column showed that 40–80% of the microbial activity occurred between the base of the euphotic zone and 150 m depth. Our findings contribute to a better understanding of the metabolic activity of the microbial communities in two regions of the GOM influenced by mesoscale eddies.

Cellular Respiration in Thymic Fragments from Mice.

This report aims to detail the use of the phosphorescence oxygen analyzer for in vitro investigation of thymic responses to pharmaceutical agents, in particular immunosuppressants and immunomodulators. Sirolimus (a highly specific inhibitor of the 'molecular target of rapamycin', mTOR) and ozanimod (an agonist of the sphingosine 1-phosphate receptor, recently approved for treatment of multiple sclerosis and ulcerative colitis) are used for this purpose. Thymic fragments from mice were placed in glass vials containing phosphate-buffered saline, bovine albumin, and Pd(II) meso-tetra (sulfophenyl) tetrabenzoporphyrin. The vials were sealed from air, and the cellular oxygen consumption was monitored as function of time. The decline of dissolved oxygen concentration with time (d[O2]/dt) was linear; thus, its rate (thymocyte respiration) was expressed as μM O2 min-1. Cyanide inhibited respiration, confirming the oxygen consumption was in cytochrome oxidase. In age-matched mice, the rate of thymocyte respiration (mean ± SD, in μM O2 min-1 mg-1) was 0.046 ± 0.011 (median = 0.043, range = 0.028 to 0.062, n = 10). In thymic fragments from littermates, this rate was inhibited in the presence of sirolimus (16% lower) or ozanimod (29% lower). Thymocyte respiration can serve as a surrogate biomarker for studying the mode-of-action and the cytotoxicity of immunotoxins and immunosuppressants.

Insights into Prokaryotic Community and Its Potential Functions in Nitrogen Metabolism in the Bay of Bengal, a Pronounced Oxygen Minimum Zone.

ABSTRACTOcean oxygen minimum zones (OMZs) around the global ocean are expanding both horizontally and vertically. Multiple studies have identified the significant influence of anoxic conditions (≤1 μM O2) on marine prokaryotic communities and biogeochemical cycling of elements. However, little attention has been paid to the expanding low-oxygen zones where the oxygen level is still above the anoxic level. Here, we studied the abundance and taxonomic and functional profiles of prokaryotic communities in the Bay of Bengal (BoB), where the oxygen concentration is barely above suboxic level (5 μM O2). We found the sinking of Trichodesmium into deep water was far more efficient than that of Prochlorococcus, suggesting Trichodesmium blooms might be an essential carbon and nitrogen source for the maintenance of the BoB OMZ. In addition to the shift in the prokaryotic community composition, the abundance of some functional genes also changed with the change of oxygen concentration. Compared to oxic (>60 μM O2) Tara Ocean and high-hypoxic (>20 to ≤60 μM O2) BoB samples, we found more SAR11-nar sequences (responsible for reducing nitrate to nitrite) in low-hypoxic (>5 to ≤20 μM O2) BoB waters. This suggested SAR11-nar genes would be more widespread due to the expansion of OMZs. It seems that the nitrite-N was not further reduced to nitrogen through denitrification but likely oxidized to nitrate by Nitrospinae in the BoB OMZ and then accumulated in the form of nitrate-N. However, the lack of N2 production in the BoB would change if the BoB OMZ became anoxic. Together, these results suggested that reduction of oxygen concentration and OMZ expansion may increase the use of nitrate by SAR11 and N2 production in the BoB.IMPORTANCE Recognizing the prokaryotic community and its functions in hypoxic (>5 to ≤60 μM O2) environments before further expansion of OMZs is critical. We demonstrate the prokaryotic community and its potential functions in nitrogen metabolism in the Bay of Bengal (BoB), where oxygen concentration is barely above suboxic level. This study highlighted that Trichodesmium might be an essential carbon and nitrogen source in the maintenance of the BoB OMZ. Additionally, we suggest that the lack of N2 production in the BoB would change if the BoB OMZ became anoxic, and the expansion of OMZs in the global ocean may potentially increase the use of nitrate by SAR11.

Vanadium nitride@carbon nanofiber composite: Synthesis, cascade enzyme mimics and its sensitive and selective colorimetric sensing of superoxide anion

Nanozymes featuring with favorable activity, good stability and easy scale-up production, are promising to replace natural enzymes for various applications. However, it remains a challenge to explore the cascade reactions of multi-enzyme mimics, aiming at synergistic catalysis for various applications. Herein, vanadium nitride nanoparticles deposited on carbon nanofibers (VN@CNFs) composite was facilely prepared by typical electrospinning route with subsequently ammonia reduction process. The nanocomposite showed excellent peroxidase (POD)-like and superoxide dismutase (SOD)-like activities. Additionally, their catalytic mechanisms were systematically researched. Coupling of SOD-like with POD-like as cascade enzyme, a selective and sensitive colorimetric detection of superoxide anion (O2•−) was explored, which has two linear parts, 0.05–30 μM and 30–250 μM O2•− with the LOD of 0.0167 μM (S/N = 3). The as-proposed method was applicable to practical samples detection with satisfactory accuracy and recovery. Therefore, the VN@CNFs composite shows great prospect in biosensing, superoxide anion removal and biocatalysis.

Energetically Informed Niche Models of Hydrogenotrophs Detected in Sediments of Serpentinized Fluids of the Samail Ophiolite of Oman

AbstractA geochemical gradient established by mixing between reduced, hyperalkaline (pH > 11), H2‐rich fluids generated through the process of serpentinization and surrounding surface water (pH ∼ 8) in the Samail Ophiolite of Oman provides an opportunity to characterize the geochemical and biological factors that influence the distribution of H2 oxidizing chemotrophs, hydrogenotrophs. In this study, 16S rRNA gene amplicon sequencing was implemented to characterize hydrogenotrophs in sediments underlying surface expressed serpentinized fluids in Oman. Hydrogenotroph phylotype distribution was evaluated as functions of chemical energy supplies for their given metabolic redox reactions. Through this approach, it was discovered that hydrogenotrophic taxa are likely constrained to sediments with overlying fluids that have <∼60 μm O2, including microorganisms of the genus, Hydrogenophaga. Sulfate reducers of the family, Thermodesulfovibrionaceae, likely require >∼10 μm SO4−2 for survival. In sediments with fluids having >∼10 μm SO4−2, sulfate reducers likely outcompete microorganisms of the methanogen genus, Methanobacterium, for H2. Additionally, differences in distribution between Thermodesulfovibrionaceae and Methanobacterium may be driven by the availability of electron acceptors and the redox reaction that is most energy yielding in the fluid. Taken together, observations from the Oman geochemical gradient result in a hydrogenotroph niche model that can be used to evaluate global distribution patterns of hydrogenotrophs in continental serpentinized fluids. On a global scale, based on previous studies, Methanobacterium is constrained to fluids that have <∼10 μm SO4−2.

Tissue Pharmacokinetic Properties and Bystander Potential of Hypoxia-Activated Prodrug CP-506 by Agent-Based Modelling.

Hypoxia-activated prodrugs are bioactivated in oxygen-deficient tumour regions and represent a novel strategy to exploit this pharmacological sanctuary for therapeutic gain. The approach relies on the selective metabolism of the prodrug under pathological hypoxia to generate active metabolites with the potential to diffuse throughout the tumour microenvironment and potentiate cell killing by means of a “bystander effect”. In the present study, we investigate the pharmacological properties of the nitrogen mustard prodrug CP-506 in tumour tissues using in silico spatially-resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) modelling. The approach employs a number of experimental model systems to define parameters for the cellular uptake, metabolism and diffusion of both the prodrug and its metabolites. The model predicts rapid uptake of CP-506 to high intracellular concentrations with its long plasma half-life driving tissue diffusion to a penetration depth of 190 µm, deep within hypoxic activating regions. While bioreductive metabolism is restricted to regions of severe pathological hypoxia (<1 µM O2), its active metabolites show substantial bystander potential with release from the cell of origin into the extracellular space. Model predictions of bystander efficiency were validated using spheroid co-cultures, where the clonogenic killing of metabolically defective “target” cells increased with the proportion of metabolically competent “activator” cells. Our simulations predict a striking bystander efficiency at tissue-like densities with the bis-chloro-mustard amine metabolite (CP-506M-Cl2) identified as a major diffusible metabolite. Overall, this study shows that CP-506 has favourable pharmacological properties in tumour tissue and supports its ongoing development for use in the treatment of patients with advanced solid malignancies.

The Oxygen Gradient in Hypoxic Conditions Enhances and Guides Dictyostelium discoideum Migration

Spatiotemporal variations of oxygen concentration affect the cell behaviors that are involved in physiological and pathological events. In our previous study with Dictyostelium discoideum (Dd) as a model of cell motility, aggregations of Dd cells exhibited long-lasting and highly stable migration in a self-generated hypoxic environment, forming a ring shape that spread toward the outer higher oxygen region. However, it is still unclear what kinds of changes in the migratory properties are responsible for the observed phenomena. Here, we investigated the migration of Dd to clarify the oxygen-dependent characteristics of aerokinesis and aerotaxis. Migratory behaviors of Dd cells were analyzed under various oxygen concentration gradients and uniform oxygen conditions generated in microfluidic devices. Under hypoxic conditions below 2% O2, corresponding to less than 25 µM O2 in the culture medium, the migration of Dd cells was enhanced (aerokinesis) and the oxygen gradient guided the cells toward the oxygen-rich region (aerotaxis). The aerotaxis was attributed to the increase in the frequency of migration associated with the direction of higher O2, the acceleration of migration velocity, and the enhancement of migration straightness. Thus, aerokinesis and aerotaxis are dependent on both the oxygen level and possibly relative gradient and are essential mechanisms for the migration of Dd.

Early (5-Day) Onset of Diabetes Mellitus Causes Degeneration of Photoreceptor Cells, Overexpression of Incretins, and Increased Cellular Bioenergetics in Rat Retina.

The effects of early (5-day) onset of diabetes mellitus (DM) on retina ultrastructure and cellular bioenergetics were examined. The retinas of streptozotocin-induced diabetic rats were compared to those of non-diabetic rats using light and transmission electron microscopy. Tissue localization of glucagon-like-peptide-1 (GLP-1), exendin-4 (EXE-4), and catalase (CAT) in non-diabetic and diabetic rat retinas was conducted using immunohistochemistry, while the retinal and plasma concentration of GLP-1, EXE-4, and CAT were measured with ELISA. Lipid profiles and kidney and liver function markers were measured from the blood of non-diabetic and diabetic rats with an automated biochemical analyzer. Oxygen consumption was monitored using a phosphorescence analyzer, and the adenosine triphosphate (ATP) level was determined using the Enliten ATP assay kit. Blood glucose and cholesterol levels were significantly higher in diabetic rats compared to control. The number of degenerated photoreceptor cells was significantly higher in the diabetic rat retina. Tissue levels of EXE-4, GLP-1 and CAT were significantly (p = 0.002) higher in diabetic rat retina compared to non-diabetic controls. Retinal cellular respiration was 50% higher (p = 0.004) in diabetic (0.53 ± 0.16 µM O2 min−1 mg−1, n = 10) than in non-diabetic rats (0.35 ± 0.07 µM O2 min−1 mg−1, n = 11). Retinal cellular ATP was 76% higher (p = 0.077) in diabetic (205 ± 113 pmol mg−1, n = 10) than in non-diabetic rats (116 ± 99 pmol mg−1, n = 12). Thus, acute (5-day) or early onslaught of diabetes-induced hyperglycemia increased incretins and antioxidant levels and oxidative phosphorylation. All of these events could transiently preserve retinal function during the early phase of the progression of diabetes.

A microsensor‐based method for measuring respiration of individual nematodes

Abstract Meiofauna (invertebrates that pass through a 1‐mm mesh sieve, but are retained on a 40‐µm mesh) represent the most abundant and diverse animal group on Earth, but empirical evidence of their role in benthic respiration, production and carbon cycling across ecosystems is not well documented. Moreover, how meiofauna respond to changing oxygen conditions is poorly understood. We further developed an incubation system, in which oxygen and temperature conditions are easily controlled and single meiofaunal nematode respiration is resolved in glass capillary tubes, using Clark‐type oxygen microsensor. We performed the respiration measurements after exposing nematodes to different ambient oxygen concentrations, which resulted in 3–60 µM O2 during hypoxic and 80–210 µM O2 during oxic incubations in close proximity to the respective nematodes. Individual nematode respiration rates ranged from 0.02 to 1.30 nmol O2 ind.−1 day−1 and were 27% lower during hypoxic than oxic incubations. Rates derived from established allometric relations were on average fourfold higher than our direct measurements. The presented method is suitable for single nematode respiration measurements and can be adapted to a wide range of experimental conditions. Therefore, it can be used to assess meiofauna contribution to ecosystem processes and investigate species‐specific responses to changing environmental conditions, for example, oxygen stress, increasing water temperature.

Temperature Dependence of the Collision‐Induced Absorption Band of O2 Near 1.27 µm

AbstractThe 1.27 μm O2 band is increasingly called upon for air‐mass determination from ground‐based and space‐borne atmospheric spectra because of its spectral proximity to CO2, and CH4 bands at 1.6 μm, in contrast to the more distant A‐band at 0.76 μm. For this purpose, it is important to well characterize not only the narrow absorption lines but also the strong underlying broad collision‐induced absorption (CIA) structure and its temperature dependence. Spectra of pure O2, and of O2 in N2, were recorded by cavity ring down spectroscopy (CRDS) at 271 K and 332 K, with the help of a newly developed temperature regulated cell. The quality of the spectra allowed determining the small variations (at the few percent level) of the , , and binary coefficients with temperature. Together with the binary coefficients at 297 K reported previously, they allow characterizing the temperature dependence of the O2 CIA at 1.27 µm in atmospheric conditions. The obtained results are compared with previous measurements by Fourier transform spectroscopy (FTS) and recent theoretical calculations. In the wings of the O2‐O2 CIA, an increase with temperature is observed in agreement with calculations but contrary to these calculations, a decrease is observed near the maximum of the CIA band. The temperature dependence is found in good agreement with the theoretical values and the experimental values derived from the FTS measurements. Binary coefficients integrated over the entire band show almost no variation with temperature for O2‐N2 and a small increase for pure O2, in agreement with the theoretical predictions.

Active DNRA and denitrification in oxic hypereutrophic waters

Since the start of synthetic fertilizer production more than a hundred years ago, the coastal ocean has been exposed to increasing nutrient loading, which has led to eutrophication and extensive algal blooms. Such hypereutrophic waters might harbor anaerobic nitrogen (N) cycling processes due to low-oxygen microniches associated with abundant organic particles, but studies on nitrate reduction in coastal pelagic environments are scarce. Here, we report on 15N isotope-labeling experiments, metagenome, and RT-qPCR data from a large hypereutrophic lagoon indicating that dissimilatory nitrate reduction to ammonium (DNRA) and denitrification were active processes, even though the bulk water was fully oxygenated (> 224 µM O2). DNRA in the bottom water corresponded to 83% of whole-ecosystem DNRA (water + sediment), while denitrification was predominant in the sediment. Microbial taxa important for DNRA according to the metagenomic data were dominated by Bacteroidetes (genus Parabacteroides) and Proteobacteria (genus Wolinella), while denitrification was mainly associated with proteobacterial genera Pseudomonas, Achromobacter, and Brucella. The metagenomic and microscopy data suggest that these anaerobic processes were likely occurring in low-oxygen microniches related to extensive growth of filamentous cyanobacteria, including diazotrophic Dolichospermum and non-diazotrophic Planktothrix. By summing the total nitrate fluxes through DNRA and denitrification, it results that DNRA retains approximately one fifth (19%) of the fixed N that goes through the nitrate pool. This is noteworthy as DNRA represents thus a very important recycling mechanism for fixed N, which sustains algal proliferation and leads to further enhancement of eutrophication in these endangered ecosystems.