Blocking GPR15 counteracts integrin-dependent T cell gut homing in vivo.

BACKGROUND AND AIMS: The G protein coupled receptor GPR15 is expressed on and functionally important for T cells homing to the large intestine. However, the precise mechanisms by which GPR15 controls gut homing have been unclear. Thus, we aimed to elucidate these mechanisms as well as to explore the potential of targeting GPR15 for interfering with T cell recruitment to the colon in IBD. METHODS: We used dynamic adhesion and transmigration assays as well as a humanized in vivo model of intestinal cell trafficking to study GPR15-dependent effects on gut homing. Moreover, we analysed GPR15 and integrin expression in patients with and without IBD cross-sectionally and longitudinally. RESULTS: GPR15 controlled T cell adhesion to MAdCAM-1 and VCAM-1 upstream of α4ß7 and α4ß1 integrin, respectively. Consistently, high co-expression of these integrins with GPR15 was found on T cells from patients with IBD and GPR15 also promoted T cell recruitment to the colon in humanized mice. Anti-GPR15 antibodies effectively blocked T cell gut homing in vitro and in vivo. In vitro data as well as observations in a cohort of patients treated with vedolizumab suggest that this might be more effective than inhibiting α4ß7. CONCLUSIONS: GPR15 seems to have a broad, but organ-selective impact on T cell trafficking and is therefore a promising target for future therapy of IBD. Further studies are needed.

Low energy electron microscopy at cryogenic temperatures.

We describe a cryogenic sample chamber for low energy electron microscopy (LEEM), and present first experimental results. Modifications to our IBM/SPECS aberration-corrected LEEM instrument are presented first. These include incorporation of mechanisms for cooling the sample and its surroundings, and reduction of various sources of heat load. Using both liquid nitrogen and liquid helium, we have reached sample temperatures down to about 15 K. We also present first results for low-temperature LEEM, obtained on a three-monolayer pentacene film. Specifically, we observe a reduction of the electron beam irradiation damage cross-section at 15 eV by more than a factor of five upon cooling from 300 K down to 52 K. We also observe changes in the LEEM-IV spectra of the sample upon cooling, and discuss possible causes.

Species-Specific Variation in Abscisic Acid Homeostasis and Responses Impacts Important Traits in Crassocephalum Orphan Crops.

Crassocephalum rubens and Crassocephalum crepidioides are plant species native to Africa, but grow in most tropical and subtropical regions of the world. They are rich in vitamins, minerals, and essential oils and are traditional leafy vegetables and medicinal plants in Sub-Saharan Africa. The plants are still mainly collected from the wild but shall be taken into cultivation and an important aim in the domestication of these species is to improve traits that are relevant for crop production. Here, seed formation and germination capacities in C. crepidioides and C. rubens were investigated, and it was found that C. crepidioides exhibits a higher level of seed dormancy, which could be broken with light, and was correlated with higher amounts of abscisic acid (ABA), a plant hormone that promotes seed dormancy. ABA is also very well-known for its role in abiotic stress tolerance, and it is shown that tetraploid C. crepidioides exhibits a higher level of resistance against drought and heat stress than diploid C. rubens, traits that will benefit the cultivation of these plants, particularly in rain-fed cropping systems. The potential of Crassocephalum to improve nutrition and increase the resilience of marginal cropping systems in Africa is discussed.

The Orphan Crop Crassocephalum crepidioides Accumulates the Pyrrolizidine Alkaloid Jacobine in Response to Nitrogen Starvation.

Crassocephalum crepidioides is an African orphan crop that is used as a leafy vegetable and medicinal plant. Although it is of high regional importance in Sub-Saharan Africa, the plant is still mainly collected from the wild and therefore efforts are made to promote its domestication. However, in addition to beneficial properties, there was first evidence that C. crepidioides can accumulate the highly toxic pyrrolizidine alkaloid (PA) jacobine and here it was investigated, how jacobine production is controlled. Using ecotypes from Africa and Asia that were characterized in terms of their PA profiles, it is shown that the tetraploid C. crepidioides forms jacobine, an ability that its diploid close relative Crassocephalum rubens appears to lack. Evidence is provided that nitrogen (N) deficiency strongly increases jacobine in the leaves of C. crepidioides, that this capacity depends more strongly on the shoot than the root system, and that homospermidine synthase (HSS) activity is not rate-limiting for this reaction. A characterization of HSS gene representation and transcription showed that C. crepidioides and C. rubens possess two functional versions, one of which is conserved, that the HSS transcript is mainly present in roots and that its abundance is not controlled by N deficiency. In summary, this work improves our understanding of how environmental cues impact PA biosynthesis in plants and provides a basis for the development of PA-free C. crepidioides cultivars, which will aid its domestication and safe use.

Determination of the [15N]-Nitrate/[14N]-Nitrate Ratio in Plant Feeding Studies by GC⁻MS.

Feeding experiments with stable isotopes are helpful tools for investigation of metabolic fluxes and biochemical pathways. For assessing nitrogen metabolism, the heavier nitrogen isotope, [15N], has been frequently used. In plants, it is usually applied in form of [15N]-nitrate, which is assimilated mainly in leaves. Thus, methods for quantification of the [15N]-nitrate/[14N]-nitrate ratio in leaves are useful for the planning and evaluation of feeding and pulse-chase experiments. Here we describe a simple and sensitive method for determining the [15N]-nitrate to [14N]-nitrate ratio in leaves. Leaf discs (8 mm diameter, approximately 10 mg fresh weight) were sufficient for analysis, allowing a single leaf to be sampled multiple times. Nitrate was extracted with hot water and derivatized with mesitylene in the presence of sulfuric acid to nitromesitylene. The derivatization product was analyzed by gas chromatography-mass spectrometry with electron ionization. Separation of the derivatized samples required only 6 min. The method shows excellent repeatability with intraday and interday standard deviations of less than 0.9 mol%. Using the method, we show that [15N]-nitrate declines in leaves of hydroponically grown Crassocephalum crepidioides, an African orphan crop, with a biological half-life of 4.5 days after transfer to medium containing [14N]-nitrate as the sole nitrogen source.

Pyrrolizidine Alkaloids: Biosynthesis, Biological Activities and Occurrence in Crop Plants.

Pyrrolizidine alkaloids (PAs) are heterocyclic secondary metabolites with a typical pyrrolizidine motif predominantly produced by plants as defense chemicals against herbivores. They display a wide structural diversity and occur in a vast number of species with novel structures and occurrences continuously being discovered. These alkaloids exhibit strong hepatotoxic, genotoxic, cytotoxic, tumorigenic, and neurotoxic activities, and thereby pose a serious threat to the health of humans since they are known contaminants of foods including grain, milk, honey, and eggs, as well as plant derived pharmaceuticals and food supplements. Livestock and fodder can be affected due to PA-containing plants on pastures and fields. Despite their importance as toxic contaminants of agricultural products, there is limited knowledge about their biosynthesis. While the intermediates were well defined by feeding experiments, only one enzyme involved in PA biosynthesis has been characterized so far, the homospermidine synthase catalyzing the first committed step in PA biosynthesis. This review gives an overview about structural diversity of PAs, biosynthetic pathways of necine base, and necic acid formation and how PA accumulation is regulated. Furthermore, we discuss their role in plant ecology and their modes of toxicity towards humans and animals. Finally, several examples of PA-producing crop plants are discussed.

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-α4ß7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn's disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow. The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field. Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Quantification of the Pyrrolizidine Alkaloid Jacobine in Crassocephalum crepidioides by Cation Exchange High-Performance Liquid Chromatography.

INTRODUCTION: Pyrrolizidine alkaloids (PAs) are secondary plant metabolites with considerable hepatoxic, tumorigenic and genotoxic potential. For separation, reversed phase chromatography is commonly used because of its excellent compatibility with detection by mass spectrometry. However, reversed phase chromatography has a low selectivity for PAs. OBJECTIVE: The objective of this work was to investigate the suitability of cation exchange chromatography for separation of PAs and to develop a rapid method for quantification of jacobine in Crassocephalum crepidioides that is suitable for analysis of huge sample numbers as required for mutant screening procedures. RESULTS: We demonstrate that cation exchange chromatography offers excellent selectivity for PAs allowing their separation from most other plant metabolites. Due to the high selectivity, plant extracts can be directly analysed after simple sample preparation. Detection with UV at 200 nm instead of mass spectrometry can be applied, which makes the method very simple and cost-effective. The recovery rate of the method exceeded 95%, the intra-day and inter-day standard deviations were below 7% and the limit of detection and quantification were 1 mg/kg and 3 mg/kg, respectively. CONCLUSION: The developed method is sufficiently sensitive for reproducible detection of jacobine in C. crepidioides. Simple sample preparation and rapid separation allows for quantification of jacobine in plant material in a high-throughput manner. Thus, the method is suitable for genetic screenings and may be applicable for other plant species, for instance Jacobaea maritima. In addition, our results show that C. crepidioides cannot be considered safe for human consumption. Copyright © 2017 John Wiley & Sons, Ltd.

Density and confinement effects of glass forming m-toluidine in nanoporous Vycor investigated by depolarized dynamic light scattering.

We investigate the reorientational dynamics of supercooled m-toluidine contained in a matrix of nanoporous Vycor with depolarized dynamic light scattering. Under equilibrium conditions a clear sample is obtained and the dynamics of m-toluidine molecules from inside the nanopores can be accessed via light scattering. However, when supercooling the imbibed liquid at conventional cooling rates, strong non-equilibrium effects occur due to the mismatch of expansion coefficients and the sample gets turbid several tens of Kelvin above the bulk glass transition. Only at cooling rates as low as 0.02 K/min this can be avoided and the dynamics of m-toluidine in confinement can be followed even below the bulk glass transition temperature. In confinement a pronounced acceleration of the reorientational dynamics is observed and the characteristic correlation times follow an Arrhenius law close to T(g). However it seems likely that part of the observed differences to bulk behavior is due to density effects, which are reduced but cannot be fully avoided at low cooling rates.

Signature of a type-A glass transition and intrinsic confinement effects in a binary glass-forming system.

We study dynamically highly asymmetric binary mixtures comprised of small methyl tetrahydrofuran (MTHF) molecules and polystyrene. Combined use of dielectric spectroscopy, 2H nuclear magnetic resonance, incoherent quasielastic neutron scattering, and depolarized dynamic light scattering allows us to selectively probe the dynamics of the components in a broad dynamic range. It turns out that the mixtures exhibit two glass transitions in a wide concentration range although being fully miscible on a macroscopic scale. In between both glass transition temperatures, the dynamics of the small molecules show strong confinement effects, e.g., a crossover from Vogel-Fulcher to Arrhenius behavior of the time constants. Moreover, the dynamical behavior of small molecules close to the slow matrix is consistent with mode coupling theory predictions for a type-A glass transition, which was expected from recent theoretical and simulation studies in comparable systems.

Mismatch of arterial and central venous blood gas analysis during haemorrhage.

BACKGROUND AND OBJECTIVE: Arterial base excess and lactate levels are key parameters in the assessment of critically ill patients. The use of venous blood gas analysis may be of clinical interest when no arterial blood is available initially. METHODS: Twenty-four pigs underwent progressive normovolaemic haemodilution and subsequent progressive haemorrhage until the death of the animal. Base excess and lactate levels were determined from arterial and central venous blood after each step. In addition, base excess was calculated by the Van Slyke equation modified by Zander (BE(z)). Continuous variables were summarized as mean +/- SD and represent all measurements (n = 195). RESULTS: Base excess according to National Committee for Clinical Laboratory Standards for arterial blood was 2.27 +/- 4.12 versus 2.48 +/- 4.33 mmol(-l) for central venous blood (P = 0.099) with a strong correlation (r(2) = 0.960, P < 0.001). Standard deviation of the differences between these parameters (SD-DIFBE) did not increase (P = 0.355) during haemorrhage as compared with haemodilution. Arterial lactate was 2.66 +/- 3.23 versus 2.71 +/- 2.80 mmol(-l) in central venous blood (P = 0.330) with a strong correlation (r(2) = 0.983, P < 0.001). SD-DIFLAC increased (P < 0.001) during haemorrhage. BE(z) for central venous blood was 2.22 +/- 4.62 mmol(-l) (P = 0.006 versus arterial base excess according to National Committee for Clinical Laboratory Standards) with strong correlation (r(2) = 0.942, P < 0.001). SD-DIFBE(z)/base excess increased (P < 0.024) during haemorrhage. CONCLUSION: Central venous blood gas analysis is a good predictor for base excess and lactate in arterial blood in steady-state conditions. However, the variation between arterial and central venous lactate increases during haemorrhage. The modification of the Van Slyke equation by Zander did not improve the agreement between central venous and arterial base excess.

Concentration fluctuations in a binary glass former investigated by x-ray photon correlation spectroscopy.

We investigate structure and dynamics of concentration fluctuations in the binary glass former methyl-tetrahydrofuran and oligomeric methyl metacrylate by photon correlation spectroscopy with partially coherent x-rays from a synchrotron source. Although the system is macroscopically well miscible and optically clear in the full temperature range, calorimetric and dielectric measurements reveal two distinct glass transition temperatures. The relaxation of long range concentration fluctuations turns out to be diffusive and exponential only well above the upper glass transition temperature. As the characteristic time tau(cf) for concentration fluctuations shows a much weaker temperature dependence than the alpha-relaxation both traces finally intersect upon lowering the temperature. Thus, close to T(g), the concentration fluctuations show pronounced features of out-of-equilibrium dynamics such as compressed relaxation functions and a crossover to a ballistic wave vector dependence of tau(cf), like previously observed in various soft matter systems. Moreover, the analysis of time-resolved correlation functions reveals that the relaxation of concentration fluctuations around T(g) involves pronounced dynamic heterogeneities.

Validity of an arterial pressure waveform analysis device: does the puncture site play a role in the agreement with intermittent pulmonary artery catheter thermodilution measurements?

OBJECTIVE: The measurement of cardiac output is a key element in the assessment of cardiac function. Recently, a pulse contour analysis-based device without need for calibration became available (FloTrac/Vigileo, Edwards Lifescience, Irvine, CA). This study was conducted to determine if there is an impact of the arterial catheter site and to investigate the accuracy of this system when compared with the pulmonary artery catheter using the bolus thermodilution technique (PAC). DESIGN: Prospective study. SETTING: The operating room of 1 university hospital. PARTICIPANTS: Twenty patients undergoing cardiac surgery. INTERVENTIONS: CO was determined in parallel by the use of the Flotrac/Vigileo systems in the radial and femoral position (CO_rad and CO_fem) and by PAC as the reference method. Data triplets were recorded at defined time points. The primary endpoint was the comparison of CO_rad and CO_fem, and the secondary endpoint was the comparison with the PAC. MEASUREMENTS AND MAIN RESULTS: Seventy-eight simultaneous data recordings were obtained. The Bland-Altman analysis for CO_fem and CO_rad showed a bias of 0.46 L/min, precision was 0.85 L/min, and the percentage error was 34%. The Bland-Altman analysis for CO_rad and PAC showed a bias of -0.35 L/min, the precision was 1.88 L/min, and the percentage error was 76%. The Bland-Altman analysis for CO_fem and PAC showed a bias of 0.11 L/min, the precision was 1.8 L/min, and the percentage error was 69%. CONCLUSION: The FloTrac/Vigileo system was shown to not produce exactly the same CO data when used in radial and femoral arteries, even though the percentage error was close to the clinically acceptable range. Thus, the impact of the introduction site of the arterial catheter is not negligible. The agreement with thermodilution was low.

Layering of [BMIM]+-based ionic liquids at a charged sapphire interface.

The structure of two model room temperature ionic liquids, [BMIM](+)[PF(6)](-) and [BMIM](+)[BF(4)](-), near the solid/liquid interface with charged Al(2)O(3)(0001) (sapphire) was determined with subnanometer resolution by high energy (72.5 keV) x-ray reflectivity. [BMIM](+)[PF(6)](-) exhibits alternately charged, exponentially decaying, near-surface layering. By contrast, the smaller-anion compound, [BMIM](+)[BF(4)](-), shows only a single layer of enhanced electron density at the interface. The different layering behaviors, and their characteristic length scales, correspond well to the different bulk diffraction patterns, also measured in this study. Complementary measurements of the surface and interface energies showed no significant different between the two RTILs. The combined bulk-interface results support the conclusion that the interfacial ordering is dominated by the same electrostatic ion-ion interactions dominating the bulk correlations, with hydrogen bonding and dispersion interactions playing only a minor role.

Molecular layering of fluorinated ionic liquids at a charged sapphire (0001) surface.

Room-temperature ionic liquids (RTILs) are promising candidates for a broad range of "green" applications, for which their interaction with solid surfaces plays a crucial role. In this high-energy x-ray reflectivity study, the temperature-dependent structures of three ionic liquids with the tris(pentafluoroethyl)trifluorophosphate anion in contact with a charged sapphire substrate were investigated with submolecular resolution. All three RTILs show strong interfacial layering, starting with a cation layer at the substrate and decaying exponentially into the bulk liquid. The observed decay length and layering period point to an interfacial ordering mechanism, akin to the charge inversion effect, which is suggested to originate from strong correlations between the unscreened ions. The observed layering is expected to be a generic feature of RTILs at charged interfaces.

Impact of the C2/C6 ratio of high-molecular-weight hydroxyethyl starch on pharmacokinetics and blood coagulation in pigs.

BACKGROUND: High-molecular-weight, low-substituted hydroxyethyl starch (HES) may not affect blood coagulation more than low-molecular-weight, low-substituted HES. The authors assessed in vivo the effect of a lowered C2/C6 ratio on pharmacokinetic characteristics and the impact on blood coagulation of high-molecular-weight, low-substituted HES. METHODS: A prospective, randomized, parallel study in 30 pigs compared HES 650/0.42/2.8 with HES 650/0.42/5.6. Before, during, and after infusion of 30 ml/kg body weight HES, blood samples were collected over 630 min to measure HES concentrations and plasmatic coagulation and to assess blood coagulation in whole blood by Thrombelastography (TEG; Haemoscope Corporation, Niles, IL). Pharmacokinetic parameters were estimated using a two-compartment model. RESULTS: The elimination constant was 0.009 +/- 0.001 min(-1) for HES 650/0.42/2.8 and 0.007 +/- 0.001 min(-1) for HES 650/0.42/5.6 (P < 0.001); the area under the plasma concentration-time curve was 1,374 +/- 340 min x g/l for HES 650/0.42/2.8 and 1,697 +/- 411 min x g/l for HES 650/0.42/5.6 (P = 0.026). The measured plasma HES concentrations were not different between HES 650/0.42/2.8 and HES 650/0.42/5.6. Both HES solutions equally affected blood coagulation: Thrombelastographic coagulation index decreased similarly at the end of infusion of HES 650/0.42/2.8 and at the end of infusion of HES 650/0.42/5.6 (P = 0.293). Also, activated partial thromboplastin and prothrombin times increased similarly for HES 650/0.42/2.8 and HES 650/0.42/5.6 (P = 0.831). CONCLUSION: Reducing the C2/C6 ratio in high-molecular, low-substituted HES solutions results in a slightly faster HES elimination. However, the blood coagulation compromising effect was unaffected.