Effects of sources and meteorology on particulate matter in the Western Mediterranean Basin: An overview of the DAURE campaign

DAURE (Determination of the Sources of Atmospheric Aerosols in Urban and Rural Environments in the Western Mediterranean) was a multidisciplinary international field campaign aimed at investigating the sources and meteorological controls of particulate matter in the Western Mediterranean Basin (WMB). Measurements were simultaneously performed at an urban-coastal (Barcelona, BCN) and a rural-elevated (Montseny, MSY) site pair in NE Spain during winter and summer. State-of-the-art methods such as 14C analysis, proton-transfer reaction mass spectrometry, and high-resolution aerosol mass spectrometry were applied for the first time in the WMB as part of DAURE. WMB regional pollution episodes were associated with high concentrations of inorganic and organic species formed during the transport to inland areas and built up at regional scales. Winter pollutants accumulation depended on the degree of regional stagnation of an air mass under anticyclonic conditions and the planetary boundary layer height. In summer, regional recirculation and biogenic secondary organic aerosols (SOA) formation mainly determined the regional pollutant concentrations. The contribution from fossil sources to organic carbon (OC) and elemental carbon (EC) and hydrocarbon-like organic aerosol concentrations were higher at BCN compared with MSY due to traffic emissions. The relative contribution of nonfossil OC was higher at MSY especially in summer due to biogenic emissions. The fossil OC/EC ratio at MSY was twice the corresponding ratio at BCN indicating that a substantial fraction of fossil OC was due to fossil SOA. In winter, BCN cooking emissions were identified as an important source of modern carbon in primary organic aerosol.

Mutation of a single residue in the ba 3 oxidase specifically impairs protonation of the pump site

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

Qualitative Distinction of Autotrophic and Heterotrophic Processes at the Leaf Level by Means of Triple Stable Isotope (C–O–H) Patterns

Foliar samples were harvested from two oaks, a beech, and a yew at the same site in order to trace the development of the leaves over an entire vegetation season. Cellulose yield and stable isotopic compositions (δ13C, δ18O, and δD) were analyzed on leaf cellulose. All parameters unequivocally define a juvenile and a mature period in the foliar expansion of each species. The accompanying shifts of the δ13C-values are in agreement with the transition from remobilized carbohydrates (juvenile period), to current photosynthates (mature phase). While the opponent seasonal trends of δ18O of blade and vein cellulose are in perfect agreement with the state-of-art mechanistic understanding, the lack of this discrepancy for δD, documented for the first time, is unexpected. For example, the offset range of 18 permil (oak veins) to 57 permil (oak blades) in δD may represent a process driven shift from autotrophic to heterotrophic processes. The shared pattern between blade and vein found for both oak and beech suggests an overwhelming metabolic isotope effect on δD that might be accompanied by proton transfer linked to the Calvin-cycle. These results provide strong evidence that hydrogen and oxygen are under different biochemical controls even at the leaf level.

Influence of muscle fiber orientation on water and metabolite relaxation times, magnetization transfer, and visibility in human skeletal muscle

PURPOSE To gain a deeper understanding of the influence of skeletal muscle fiber orientation on metabolite visibility, magnetization transfer from water, and water proton relaxation rates in (1) H MR spectra. METHODS Non-water-suppressed MR spectroscopy was performed in tibialis anterior muscle (TA) of 10 healthy adults, with the TA oriented either parallel or at the magic angle to the 3T field. Spectra were acquired with metabolite-cycled PRESS, and water inversion from 50 to 2510 ms before excitation. Water proton T2 relaxation was sampled with STEAM with echo times from 12 to 272 ms. RESULTS Apparent concentrations of total creatine (tCr), taurine, and trimethylammonium compounds were reduced by 29% to 67% when TA was parallel to B0 . Both tCr peak areas were strongly correlated to the methylene peak splitting. Magnetization transfer rates from water to tCr CH3 were not significantly different between orientations. Water T1 s were similar between orientations, but T2 s were statistically significantly shorter by 1 ms in the parallel orientation (P = 0.002). CONCLUSION Muscle metabolite visibilities in MR spectroscopy and water T2 times depend substantially on muscle fiber orientation relative to B0 . In contrast, magnetization transfer rates appear to depend on muscle composition, rather than fiber orientation. Magn Reson Med, 2015. © 2015 Wiley Periodicals, Inc.

Effect of Acute Psychological Stress on Prefrontal GABA Concentration Determined by Proton Magnetic Resonance Spectroscopy

Objective Impaired function of the central gamma-aminobutyric acid (GABA) system, which provides the brain’s major inhibitory pathways, is thought to play an important role in the pathophysiology of anxiety disorders. The effect of acute psychological stress on the human GABA-ergic system is still unknown, however. The purpose of this study was to determine the effect of acute stress on prefrontal GABA levels. Method A recently developed noninvasive magnetic resonance spectroscopy method was used to measure changes in the GABA concentration of the prefrontal cortex in 10 healthy human subjects during a threat-of-shock condition and during a safe condition (two sessions on different days). The main outcome measure was the mean GABA concentration within a 3×3×2-cm3 voxel selected from the medial prefrontal cortex. Results Prefrontal GABA decreased by approximately 18% in the threat-of-shock condition relative to the safe condition. This reduction was specific to GABA, since the concentrations of N-acetyl-aspartate, choline-containing compounds, and glutamate/glutamine levels obtained in the same spectra did not change significantly. Conclusions This result appeared compatible with evidence from preclinical studies in rodents, which showed rapid presynaptic down-regulation of GABA-ergic neurotransmission in response to acute psychological stress. The molecular mechanism and functional significance of this reduced inhibitory effect of acute psychological stress in relation to impaired GABA-ergic function in anxiety disorders merit further investigation.

Radical Chain Reduction of Alkylboron Compounds with Catechols

The conversion of alkylboranes to the corresponding alkanes is classically performed via protonolysis of alkylboranes. This simple reaction requires the use of severe reaction conditions, that is, treatment with a carboxylic acid at high temperature (>150 degrees C). We report here a mild radical procedure for the transformation of organoboranes to alkalies. 4-tert-Butylcatechol, a well-established radical inhibitor and antioxidant, is acting as a source of hydrogen atoms. An efficient chain reaction is observed due to the exceptional reactivity of phenoxyl radicals toward alkylboranes. The reaction has been applied to a wide range of organoboron derivatives such as B-alkylcatecholboranes, trialkylboranes, pinacolboronates, and alkylboronic acids. Furthermore, the so far elusive rate constants for the hydrogen transfer between secondary alkyl radical and catechol derivatives have been experimentally determined. Interestingly, they are less than 1 order of magnitude slower than that of tin hydride at 80 degrees C, making catechols particularly attractive for a wide range of transformations involving C-C bond formation.

Magnetization exchange observed in human skeletal muscle by non-water-suppressed proton magnetic resonance spectroscopy

Many metabolites in the proton magnetic resonance spectrum undergo magnetization exchange with water, such as those in the downfield region (6.0-8.5 ppm) and the upfield peaks of creatine, which can be measured to reveal additional information about the molecular environment. In addition, these resonances are attenuated by conventional water suppression techniques complicating detection and quantification. To characterize these metabolites in human skeletal muscle in vivo at 3 T, metabolite cycled non-water-suppressed spectroscopy was used to conduct a water inversion transfer experiment in both the soleus and tibialis anterior muscles. Resulting median exchange-independent T(1) times for the creatine methylene resonances were 1.26 and 1.15 s, and for the methyl resonances were 1.57 and 1.74 s, for soleus and tibialis anterior muscles, respectively. Magnetization transfer rates from water to the creatine methylene resonances were 0.56 and 0.28 s(-1) , and for the methyl resonances were 0.39 and 0.30 s(-1) , with the soleus exhibiting faster transfer rates for both resonances, allowing speculation about possible influences of either muscle fibre orientation or muscle composition on the magnetization transfer process. These water magnetization transfer rates observed without water suppression are in good agreement with earlier reports that used either postexcitation water suppression in rats, or short CHESS sequences in human brain and skeletal muscle.

A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.

Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.

Proton-coupled oligopeptide transporter family SLC15: physiological, pharmacological and pathological implications

Mammalian members of the proton-coupled oligopeptide transporter family (SLC15) are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs. The driving force for uphill electrogenic symport is the chemical gradient and membrane potential which favors proton uptake into the cell along with the peptide/mimetic substrate. The peptide transporters are responsible for the absorption and conservation of dietary protein digestion products in the intestine and kidney, respectively, and in maintaining homeostasis of neuropeptides in the brain. They are also responsible for the absorption and disposition of a number of pharmacologically important compounds including some aminocephalosporins, angiotensin-converting enzyme inhibitors, antiviral prodrugs, and others. In this review, we provide updated information on the structure-function of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4) and PhT2 (SLC15A3), and their expression and localization in key tissues. Moreover, mammalian peptide transporters are discussed in regard to pharmacogenomic and regulatory implications on host pharmacology and disease, and as potential targets for drug delivery. Significant emphasis is placed on the evolving role of these peptide transporters as elucidated by studies using genetically modified animals. Whenever possible, the relevance of drug-drug interactions and regulatory mechanisms are evaluated using in vivo studies.

Proton diffusion tensor spectroscopy of metabolites in human muscle in vivo

PURPOSE To study the apparent diffusivity and its directionality for metabolites of skeletal muscle in humans in vivo by (1) H magnetic resonance spectroscopy. METHODS The diffusion tensors were determined on a 3 Tesla MR system using optimized acquisition and processing methods including an adapted STEAM sequence with orientation-dependent diffusion weighting, pulse-triggering with individually adapted delays, eddy-current correction schemes, median filtering, and simultaneous prior-knowledge fitting of all related spectra. RESULTS The average apparent diffusivities, as well as the fractional anisotropies of taurine (ADCav  = 0.74 × 10(-3) s/mm(2) , FA = 0.46), creatine (ADCav  = 0.41 × 10(-3)  s/mm(2) , FA = 0.33), trimethylammonium compounds (ADCav  = 0.48 × 10(-3)  s/mm(2) , FA = 0.34), carnosine (ADCav  = 0.46 × 10(-3)  s/mm(2) , FA = 0.47), and water (ADCav  = 1.5 × 10(-3)  s/mm(2) , FA = 0.36) were estimated. The diffusivities of most metabolites and water were significantly different from each other. Diffusion was found to be anisotropic and the diffusion tensors showed tensor correlation coefficients close to 1 and were hence found to be essentially coaligned. The magnitudes of apparent metabolite diffusivities were largely ordered according to molecular weight, with taurine as the smallest molecule diffusing fastest, both along and across the fiber direction. CONCLUSION Diffusivities, directional dependence of diffusion and fractional anisotropies of (1) H MRS-visible muscle metabolites were presented. It was shown that metabolites share diffusion directionality with water and have similar fractional anisotropies, hinting at similar diffusion barriers. Magn Reson Med, 2014. © 2014 Wiley Periodicals, Inc.

Materno-fetal nutrient transfer across primary human trophoblast layer.

MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN TROPHOBLAST MONOLAYER Objectives: Polarized trophoblasts represent the transport and metabolic barrier between the maternal and fetal circulation. Currently human placental nutrient transfer in vitro is mainly investigated unidirectionallyon cultured primary trophoblasts, or bidirectionally on the Transwell® system using BeWo cells treated with forskolin. As forskolin can induce various gene alterations (e.g. cAMP response element genes), we aimed to establish a physiological primary trophoblast model for materno-fetal nutrient exchange studies without forskolin application. Methods: Human term cytotrophoblasts were isolated by enzymatic digestion and Percoll® gradient separation. The purity of the primary cells was assessed by flow cytometry using the trophoblast-specific marker cytokeratin-7. After screening different coating matrices, we optimized the growth conditions for the primary cytotrophoblasts on Transwell/ inserts. The morphology of 5 days cultured trophoblasts was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally transport studies were performed on the polarized trophoblasts in the Transwell® system. Results: During 5 days culture, the trophoblasts (>90% purity) developed a modest trans-epithelial electrical resistance (TEER) and a sizedependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ~400-70’000D). SEM analyses confirmed a confluent trophoblast layer with numerous microvilli at day six, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein ZO-1, and the membrane proteins ABCA1 and Na+/K+-ATPase. Vectorial glucose and cholesterol transport studies confirmed functionality of the cultured trophoblast barrier. Conclusion: Evidence from cell morphology, biophysical parameters and cell marker expressions indicate the successful and reproducible establishment of a primary trophoblast monolayer model suitable for transport studies. Application of this model to pathological trophoblasts will help to better understand the mechanism underlying gestational diseases, and to define the consequences of placental pathology on materno-fetal nutrient transport.

Increased levels of choline metabolites are an early marker of docetaxel treatment response in BRCA1-mutated mouse mammary tumors: an assessment by ex vivo proton magnetic resonance spectroscopy.

BACKGROUND Docetaxel is one of the most frequently used drugs to treat breast cancer. However, resistance or incomplete response to docetaxel is a major challenge. The aim of this study was to utilize MR metabolomics to identify potential biomarkers of docetaxel resistance in a mouse model for BRCA1-mutated breast cancer. METHODOLOGY High resolution magic angle spinning (HRMAS) (1)H MR spectroscopy was performed on tissue samples obtained from docetaxel-sensitive or -resistant BRCA1-mutated mammary tumors in mice. Measurements were performed on samples obtained before treatment and at 1-2, 3-5 and 6-7 days after a 25 mg/kg dose of docetaxel. The MR spectra were analyzed by multivariate analysis, followed by analysis of the signals of individual compounds by peak fitting and integration with normalization to the integral of the creatine signal and of all signals between 2.9 and 3.6 ppm. RESULTS The HRMAS spectra revealed significant metabolic differences between sensitive and resistant tissue samples. In particular choline metabolites were higher in resistant tumors by more than 50% with respect to creatine and by more than 30% with respect to all signals between 2.9 and 3.6 ppm. Shortly after treatment (1-2 days) the normalized choline metabolite levels were significantly increased by more than 30% in the sensitive group coinciding with the time of highest apoptotic activity induced by docetaxel. Thereafter, choline metabolites in these tumors returned towards pre-treatment levels. No change in choline compounds was observed in the resistant tumors over the whole time of investigation. CONCLUSIONS Relative tissue concentrations of choline compounds are higher in docetaxel resistant than in sensitive BRCA1-mutated mouse mammary tumors, but in the first days after docetaxel treatment only in the sensitive tumors an increase of these compounds is observed. Thus both pre- and post-treatment tissue levels of choline compounds have potential to predict response to docetaxel treatment.

Transfer and effects of 1,2,3,5,7-pentachloronaphthalene in an experimental food chain.

Polychlorinated naphthalenes are environmentally relevant compounds that are measured in biota at concentrations in the μg/kg lipid range. Despite their widespread occurrence, literature data on the accumulation and effects of these compounds in aquatic ecosystems are sparsely available. The goal of this study was to gain insights into the biomagnification and effects of 1,2,3,5,7-pentachloronaphthalene (PeCN52) in an experimental food chain consisting of benthic worms and juvenile rainbow trout. Worms were contaminated with PeCN52 by passive dosing from polydimethylsiloxane silicone. The contaminated worms were then used to feed the juvenile rainbow trout at 0.12, 0.25 or 0.50 μg/g fish wet weight/day, and the resulting internal whole-body concentrations of the individual fish were linked to biological responses. A possible involvement of the cellular detoxification system was explored by measuring PeCN52-induced expression of the phase I biotransformation enzyme gene cyp1a1 and the ABC transporter gene abcb1a. At the end of the 28-day study, biomagnification factors were similar for all dietary intake levels with values between 0.5 and 0.7 kg lipid(fish)/kg lipid(worm). The average uptake efficiency of 60% indicated that a high amount of PeCN52 was transferred from the worms to the fish. Internal concentrations of up to 175 mg/kg fish lipid in the highest treatment level did not result in effects on survival, behavior, or growth of the juvenile trout, but were associated with the induction of phase I metabolism which was evident from the significant up-regulation of cyp1a1 expression in the liver. In contrast, no changes were seen in abcb1a transcript levels.