Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.

In-vivo quantification of electron flow through photosystem I - Cyclic electron transport makes up about 35% in a cyanobacterium.

Photosynthetic electron flow, driven by photosystem I and II, provides chemical energy for carbon fixation. In addition to a linear mode a second cyclic route exists, which only involves photosystem I. The exact contributions of linear and cyclic transport are still a matter of debate. Here, we describe the development of a method that allows quantification of electron flow in absolute terms through photosystem I in a photosynthetic organism for the first time. Specific in-vivo protocols allowed to discern the redox states of plastocyanin, P700 and the FeS-clusters including ferredoxin at the acceptor site of PSI in the cyanobacterium Synechocystis sp. PCC 6803 with the near-infrared spectrometer Dual-KLAS/NIR. P700 absorbance changes determined with the Dual-KLAS/NIR correlated linearly with direct determinations of PSI concentrations using EPR. Dark-interval relaxation kinetics measurements (DIRKPSI) were applied to determine electron flow through PSI. Counting electrons from hydrogen oxidation as electron donor to photosystem I in parallel to DIRKPSI measurements confirmed the validity of the method. Electron flow determination by classical PSI yield measurements overestimates electron flow at low light intensities and saturates earlier compared to DIRKPSI. Combination of DIRKPSI with oxygen evolution measurements yielded a proportion of 35% of surplus electrons passing PSI compared to PSII. We attribute these electrons to cyclic electron transport, which is twice as high as assumed for plants. Counting electrons flowing through the photosystems allowed determination of the number of quanta required for photosynthesis to 11 per oxygen produced, which is close to published values.

Restricted suitability of BODIPY for caging in biological applications based on singlet oxygen generation.

Recent studies report the boron-dipyrromethene (BODIPY) moiety to be interesting for caging applications in photopharmacology based on its response to irradiation with wavelengths in the biooptical window. Thus, in a model study, we investigated the meso-methyl-BODIPY caged CDK2 inhibitor AZD5438 and aimed to assess the usability of BODIPY as a photoremovable protecting group in photoresponsive kinase inhibitor applications. Photochemical analysis and biological characterisation in vitro revealed significant limitations of the BODIPY-caged inhibitor concept regarding solubility and uncaging in aqueous solution. Notably, we provide evidence for BODIPY-caged compounds generating singlet oxygen/radicals upon irradiation, followed by photodegradation of the caged compound system. Consequently, instead of caging, a non-specific induction of necrosis in cells suggests the potential usage of BODIPY derivatives for photodynamic approaches.

Analysis of radical formation by EPR in complex starch-protein-lipid model systems and corn extrudates.

The formation of short-lived and stable radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy and compared with hydroperoxides and hexanal in complex starch-protein-lipid model systems, as well as in corn extrudates. Stable radicals were detected directly in ground samples. Short-lived lipid radicals were measured ex situ in ethyl acetate extracts of model systems and extrudates by the use of the spin trap PBN. Significant adduct formation was found after 30 min at 50 °C. During storage, lipid radicals (PBN adducts) increased in model systems. Simulation of EPR spectra from bulk oil demonstrated that mainly alkoxyl radical adducts were detected, to which rapidly decomposing peroxyl radical adducts also contributed. Stable radicals in extrudates were attributed to protein radicals based on g-value of 2.00467 compared with 2.00474 found in model system prepared with zein. The signal intensity of the stable radical remained constant during storage, but increased during extrusion.

BioFlux™ 200 Microfluidic System to Study A. baumannii Biofilm Formation in a Dynamic Mode of Growth.

The ability of A. baumannii to develop biofilms on a wide range of surfaces can be associated to its persistence in hospital settings and the emergence of recalcitrant and chronic infections. Few compounds are available to eradicate A. baumannii biofilms, and most of them have been tested for their antibiofilm properties in static conditions. Microfluidics systems as BioFlux™ system are now available for studying A. baumannii biofilm formation in dynamic conditions. Here, we described the use of this system for studying the biofilm development of the reference strain A. baumannii ATCC 17978 in a dynamic mode. We showed how to test the activity of an antibiotic (colistin at the MIC concentration, 0.5 µg/mL) in these conditions of growth.

A stress recovery signaling network for enhanced flooding tolerance in Arabidopsis thaliana.

Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D, SENESCENCE-ASSOCIATED GENE113, and ORESARA1, which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis.

Composition of Quillaja saponin extract affects lipid oxidation in oil-in-water emulsions.

Quillaja saponin extract comprises both, surfactants and phenolic compounds, which makes it interesting, in particular, for the formulation of sensitive functional food ingredients and its protection against oxidation. The aim of this study was to investigate the antioxidant effect of Quillaja saponin extract in oil/water emulsions. Emulsions stabilised by Quillaja saponin showed decreased oxidation stability due to naturally occurring metals but stability increased to a great extent when a chelating agent was added. Antioxidant efficiency of the saponin extract was determined photometrically by 2,2'-diphenyl-1-picrylhydrazyl (DPPH) assay and by the use of electron paramagnetic resonance spectroscopy (EPR). EPR spectroscopy applying stable hydrophilic and hydrophobic radicals is advantageous, especially for characterisation of antioxidant efficiency at the interface. The extract showed antioxidant activity towards radicals in both environments, aqueous and hydrophobic, indicating the importance of phenolic compounds for the antioxidant properties of Quillaja saponin extract and their presence at the interface facilitated by saponin molecules.

Involvement of sulfates from cruzipain, a major antigen of Trypanosoma cruzi, in the interaction with immunomodulatory molecule Siglec-E.

In order to investigate the involvement of sulfated groups in the Trypanosoma cruzi host-parasite relationship, we studied the interaction between the major cysteine proteinase of T. cruzi, cruzipain (Cz), a sulfate-containing sialylated molecule and the sialic acid-binding immunoglobulin like lectin-E (Siglec-E). To this aim, ELISA, indirect immunofluorescence assays and flow cytometry, using mouse Siglec-E-Fc fusion molecules and glycoproteins of parasites, were performed. Competition assays verified that the lectins, Maackia amurensis II (Mal II) and Siglec-E-Fc, compete for the same binding sites. Taking into account that Mal II binding remains unaltered by sulfation, we established this lectin as sialylation degree control. Proteins of an enriched microsomal fraction showed the highest binding to Siglec-E as compared with those from the other parasite subcellular fractions. ELISA assays and the affinity purification of Cz by a Siglec-E column confirmed the interaction between both molecules. The significant decrease in binding of Siglec-E-Fc to Cz and to its C-terminal domain (C-T) after desulfation of these molecules suggests that sulfates contribute to the interaction between Siglec-E-Fc and these glycoproteins. Competitive ELISA assays confirmed the involvement of sulfated epitopes in the affinity between Siglec-E and Cz, probably modified by natural protein environment. Interestingly, data from flow cytometry of untreated and chlorate-treated parasites suggested that sulfates are not primary receptors, but enhance the binding of Siglec-E to trypomastigotic forms. Altogether, our findings support the notion that sulfate-containing sialylated glycoproteins interact with Siglec-E, an ortholog protein of human Siglec-9, and might modulate the immune response of the host, favoring parasitemia and persistence of the parasite.

Partitioning of nitroxides in dispersed systems investigated by ultrafiltration, EPR and NMR spectroscopy.

HYPOTHESIS: The partitioning behavior of paramagnetic nitroxides in dispersed systems can be determined by deconvolution of electron paramagnetic resonance (EPR) spectra giving equivalent results with the validated methods of ultrafiltration techniques (UF) and pulsed-field gradient nuclear magnetic resonance spectroscopy (PFG-NMR). EXPERIMENTS: The partitioning behavior of nitroxides with increasing lipophilicity was investigated in anionic, cationic and nonionic micellar systems and 10 wt% o/w emulsions. Apart from EPR spectra deconvolution, the PFG-NMR was used in micellar solutions as a non-destructive approach, while UF based on separation of very small volume of the aqueous phase. FINDINGS: As a function of their substituent and lipophilicity, the proportions of nitroxides that were solubilized in the micellar or emulsion interface increased with increasing nitroxide lipophilicity for all emulsifier used. Comparing the different approaches, EPR deconvolution and UF revealed comparable nitroxide proportions that were solubilized in the interfaces. Those proportions were higher than found with PFG-NMR. For PFG-NMR self-diffusion experiments the reduced nitroxides were used revealing a high dynamic of hydroxylamines and emulsifiers. Deconvolution of EPR spectra turned out to be the preferred method for measuring the partitioning behavior of paramagnetic molecules as it enables distinguishing between several populations at their individual solubilization sites.

Whey protein coating increases bilayer rigidity and stability of liposomes in food-like matrices.

Liposomes are suitable for encapsulating lipophilic bioactive compounds, enhancing compound solubility, stability and bioavailability. To enhance physical stability of liposomes in food-like matrices they were coated with positively charged whey protein isolate (WPI). WPI concentration, for a successful coating, was optimised by dynamic light scattering (DLS) and zeta potential measurements. Membrane properties of coated and uncoated vesicles were investigated by electron paramagnetic resonance (EPR) with site-directed and non-site-directed spin probes. Coexistence of two or three simulated spin probe populations indicated a less fluid membrane and higher concentration of water molecules in the phosphate/glycerol moiety with WPI coating. This relies on the insertion of WPI into the membrane, which is favoured by the molten globule state under investigated acidic conditions. Physical stability of liposomes benefits from WPI coating, as indicated by prolonged shelf-life, cancellation of osmotic effects in the presence of salts or sugars and a lower sensitivity towards low pH values during in vitro gastric digestion.

Reactive oxygen species mediate growth and death in submerged plants.

Aquatic and semi-aquatic plants are well adapted to survive partial or complete submergence which is commonly accompanied by oxygen deprivation. The gaseous hormone ethylene controls a number of adaptive responses to submergence including adventitious root growth and aerenchyma formation. Reactive oxygen species (ROS) act as signaling intermediates in ethylene-controlled submergence adaptation and possibly also independent of ethylene. ROS levels are controlled by synthesis, enzymatic metabolism, and non-enzymatic scavenging. While the actors are by and large known, we still have to learn about altered ROS at the subcellular level and how they are brought about, and the signaling cascades that trigger a specific response. This review briefly summarizes our knowledge on the contribution of ROS to submergence adaptation and describes spectrophotometrical, histochemical, and live cell imaging detection methods that have been used to study changes in ROS abundance. Electron paramagnetic resonance (EPR) spectroscopy is introduced as a method that allows identification and quantification of specific ROS in cell compartments. The use of advanced technologies such as EPR spectroscopy will be necessary to untangle the intricate and partially interwoven signaling networks of ethylene and ROS.

ACBP knockdown leads to down-regulation of genes encoding rate-limiting enzymes in cholesterol and fatty acid metabolism.

The human Acyl-CoA binding protein (ACBP) is a structural and functional highly conserved protein. As an intracellular pool former and carrier of acyl-CoAs, ACBP influences overall lipid metabolism. Its nuclear abundance and physical interaction with hepatocyte nuclear factor 4alpha suggested a gene regulatory function of ACBP. To identify ACBP target genes we performed genome-wide transcript profiling under siRNA-mediated ACBP knockdown in human liver HepG2 cells. Based on a single sided permutation T-test (p<0.05) we identified 256 down-regulated and 198 up-regulated transcripts with a minimal fold change of 1.32 (log 0.5). Gene annotation enrichment analysis revealed ACBP-mediated down-regulation of 18 genes encoding key enzymes in glycerolipid (i.e. mitochondrial glycerol-3-phosphate acyltransferase), cholesterol (i.e. HMG-CoA synthase and HMG-CoA reductase) and fatty acid (i.e. fatty acid synthase) metabolism. Integration of these genes in common pathways suggested decreased lipid biosynthesis. Accordingly, saturated (16:0) and monosaturated (16:1, 18:1) fatty acids were significantly reduced to 75% in ACBP-depleted cells. Taken together, we obtained evidence that ACBP functions in lipid metabolism at the level of gene expression. This effect seems to be translated into certain metabolites. The identified 454 ACBP regulated genes present a first reference for further studies to define the ACBP regulon in mammalian cells.

The partitioning of emulsifiers in o/w emulsions: a comparative study of SANS, ultrafiltration and dialysis.

The partitioning of SDS and CTAB in o/w emulsions was investigated by ultrafiltration (UF), dialysis and small-angle neutron scattering (SANS). It was possible to measure the monomeric and the micellar concentrations of the emulsifiers in the filtrate and permeate in the UF and dialysis experiments, respectively. In addition, the interfacial concentration was calculated as the difference to the initial concentration. SANS experiments provided data, from which the micellar concentrations were obtained, followed by the calculation of the interfacial concentrations. The three methods were compared on the basis of the area, which is occupied by each emulsifier molecule at the interface. Good agreement was shown for both emulsifiers studied. Micellation started at total emulsifier concentrations of approx. 10 mM in emulsions containing either CTAB or SDS. At saturation (>10 mM SDS in a 10% o/w emulsion), the area per SDS headgroup at the interface was between 48 and 64 A2, depending on the method. In emulsions with CTAB, saturation of the interface was not achieved. The minimum headgroup area was determined by UF to be 33 A2 at a concentration of 30 mM CTAB in a 10% o/w emulsion.

The location of phenolic antioxidants and radicals at interfaces determines their activity.

To characterize parameters influencing the antioxidant activity at interfaces a novel ESR approach was developed, which facilitates the investigation of the reaction stoichiometry of antioxidants towards stable radicals. To relate the activity of antioxidants towards the location of radicals at interfaces NMR experiments were conducted. Micellar solutions of SDS, Brij and CTAB were used to model interfaces of different chemical nature. The hydrophilic Fremy's radical was found to be solubilized exclusively in the aqueous phase of SDS micellar solution but partitioned partly into the hydrophilic headgroup area of Brij micelles. In contrast the hydrophobic galvinoxyl was exclusively located in the micellar phase with the increasing depth of intercalation in the order SDS < Brij < CTAB. Gallates revealed a higher stoichiometric factor towards galvinoxyl in CTAB systems, which is accounted to a concentration effect of antioxidant and radical being both solubilized in the palisade layer. In contrast, in SDS solutions hardly any reaction between galvinoxyl and gallates was found. SDS acted as a physical barrier between radical (palisade layer) and antioxidant (stern layer). The influence of the hydrophobic properties of the antioxidant was clearly seen in Brij micelles. Elongation of the alkyl chain in gallate molecule resulted in increasing stoichiometric factors in the presence of galvinoxyl being located in the deeper region of the bulky headgroup area. The reverse trend was found in the presence of Fremy's radical being located in the hydrated area of the micelles.

Investigating the location of propyl gallate at surfaces and its chemical microenvironment by (1)H NMR.

The location and the resulting chemical microenvironment of the antioxidant propyl gallate (PG) was studied in micellar solutions using the cationic emulsifier cetyl trimethyl ammonium bromide (CTAB), the anionic emulsifier sodium dodecyl sulphate (SDS) and the non-ionic emulsifier Brij 58 (polyoxyethylene-20-cetyl ester). T (1) relaxation time of the aromatic protons of PG was investigated in micellar solutions and compared with that in aqueous solution in the absence of emulsifier. The relaxation time of the PG portion that is solubilized in the micelle (T (1,eff)) was calculated from the partition behavior of PG in micellar solution. From the 1D-(1)H spectrum, the alteration in the electron density of the aromatic protons and the alteration in the peak shape of the emulsifier headgroup and alkyl chain proton signals were indicative of the location of propyl gallate in the different micelles. Nuclear Overhauser effects (NOE) made it possible to deduce the exact location of PG by calculation of the relative NOEs. Marked differences were found for the location of PG in CTAB, SDS and Brij 58 micelles. PG was found to be located in the palisade layer of CTAB micelles, in the region of the polyoxyethylene chain of Brij micelles and in the Stern layer of SDS micelles. For careful study of the location of antioxidants and therefore to be able to characterize the chemical microenvironment of the antioxidants is crucial for understanding differences in antioxidant activities as a function of lipid surfaces. The application of spectroscopic methods may help to optimize the antioxidant activity to inhibit lipid oxidation at surfaces that are formed in a wide range of foods (emulsions), cosmetics, pharmaceuticals (emulsions and carrier systems) and of biological membranes (LDL-particles).