The potency of selatogrel, a reversible antagonist of the P2Y12 receptor, is affected by calcium concentration.

Here, we report the in vitro characterization of the P2Y12 receptor antagonist selatogrel (ACT-246475). Binding studies with radiolabeled selatogrel demonstrated that selatogrel is a competitive antagonist of ADP binding to the P2Y12 receptor with a fast onset of action. Consequently, selatogrel was confirmed to be a potent inhibitor of P2Y12-mediated intra-platelet signaling and ADP-induced platelet activation. Characterization of selatogrel in platelet-rich plasma in vitro demonstrated that the mode of anti-coagulation affected the anti-platelet potency. Specifically, in platelet-rich plasma containing physiological calcium concentration (anticoagulated with a direct thrombin inhibitor), selatogrel achieved half-maximal inhibition of ADP-induced platelet aggregation at a 3-fold lower concentration than in conditions with low calcium concentration (anticoagulated with citrate). Furthermore, calcium-dependent reduction in selatogrel potency was observed in whole blood platelet aggregation using the VerifyNow™ system with a 3.7-fold potency loss in low calcium conditions. A comparable potency loss was also observed with the reversible P2Y12 receptor antagonists ticagrelor, cangrelor and elinogrel. Furthermore, receptor-binding experiments using radiolabeled selatogrel confirmed a 3-fold lowering of selatogrel binding affinity to the P2Y12 receptor in low calcium conditions. In conclusion, our data suggest that in low calcium conditions (i.e., citrate-anticoagulated blood), there is a risk of underestimating the potency of reversible P2Y12 receptor antagonists. To avoid overdosing, and a potential increase in bleeding risk, we propose that the ex vivo evaluation of reversible P2Y12 receptor antagonists should be performed with platelet assay systems containing physiological calcium concentration.

Large differences in leaf cuticle conductance and its temperature response among 24 tropical tree species from across a rainfall gradient.

More frequent droughts and rising temperatures pose serious threats to tropical forests. When stomata are closed under dry and hot conditions, plants lose water through leaf cuticles, but little is known about cuticle conductance (gmin ) of tropical trees, how it varies among species and environments, and how it is affected by temperature. We determined gmin in relation to temperature for 24 tropical tree species across a steep rainfall gradient in Panama, by recording leaf drying curves at different temperatures in the laboratory. In contrast with our hypotheses, gmin did not differ systematically across the rainfall gradient; species differences did not reflect phylogenetic patterns; and in most species gmin did not significantly increase between 25 and 50°C. gmin was higher in deciduous than in evergreen species, in species with leaf trichomes than in species without, in sun leaves than in shade leaves, and tended to decrease with increasing leaf mass per area across species. There was no relationship between stomatal and cuticle conductance. Large species differences in gmin and its temperature response suggest that more frequent hot droughts may lead to differential survival among tropical tree species, regardless of species' position on the rainfall gradient.

Leaf cuticle analyses: implications for the existence of cutan/non-ester cutin and its biosynthetic origin.

BACKGROUND AND AIMS: The cuticle of a limited number of plant species contains cutan, a chemically highly resistant biopolymer. As yet, the biosynthesis of cutan is not fully understood. Attempting to further unravel the origin of cutan, we analysed the chemical composition of enzymatically isolated cuticular membranes of Agave americana leaves. METHODS: Cuticular waxes were extracted with organic solvents. Subsequently, the dewaxed cuticular membrane was depolymerized by acid-catalysed transesterification yielding cutin monomers and cutan, a non-hydrolysable, cuticular membrane residue. The cutan matrix was analysed by thermal extraction, flash pyrolysis and thermally assisted hydrolysis and methylation to elucidate the monomeric composition and deduce a putative biosynthetic origin. KEY RESULTS: According to gas chromatography-mass spectrometry analyses, the cuticular waxes of A. americana contained primarily very-long-chain alkanoic acids and primary alkanols dominated by C32, whereas the cutin biopolyester of A. americana mainly consisted of 9,10-epoxy ω-hydroxy and 9,10,ω-trihydroxy C18 alkanoic acids. The main aliphatic cutan monomers were alkanoic acids, primary alkanols, ω-hydroxy alkanoic acids and alkane-α,ω-diols ranging predominantly from C28 to C34 and maximizing at C32. Minor contributions of benzene-1,3,5-triol and derivatives suggested that these aromatic moieties form the polymeric core of cutan, to which the aliphatic moieties are linked via ester and possibly ether bonds. CONCLUSIONS: High similarity of aliphatic moieties in the cutan and the cuticular wax component indicated a common biosynthetic origin. In order to exclude species-specific peculiarities of A. americana and to place our results in a broader context, cuticular waxes, cutin and cutan of Clivia miniata, Ficus elastica and Prunus laurocerasus leaves were also investigated. A detailed comparison showed compositional and structural differences, indicated that cutan was only found in leaves of perennial evergreen A. americana and C. miniata, and made clear that the phenomenon of cutan is possibly less present in plant species than suggested in the literature.

Effects of temperature on the cuticular transpiration barrier of two desert plants with water-spender and water-saver strategies.

The efficacy of the cuticular transpiration barrier and its resistance to elevated temperatures are significantly higher in a typical water-saver than in a water-spender plant growing in hot desert.

The desert plant Phoenix dactylifera closes stomata via nitrate-regulated SLAC1 anion channel.

Date palm Phoenix dactylifera is a desert crop well adapted to survive and produce fruits under extreme drought and heat. How are palms under such harsh environmental conditions able to limit transpirational water loss? Here, we analysed the cuticular waxes, stomata structure and function, and molecular biology of guard cells from P. dactylifera. To understand the stomatal response to the water stress phytohormone of the desert plant, we cloned the major elements necessary for guard cell fast abscisic acid (ABA) signalling and reconstituted this ABA signalosome in Xenopus oocytes. The PhoenixSLAC1-type anion channel is regulated by ABA kinase PdOST1. Energy-dispersive X-ray analysis (EDXA) demonstrated that date palm guard cells release chloride during stomatal closure. However, in Cl- medium, PdOST1 did not activate the desert plant anion channel PdSLAC1 per se. Only when nitrate was present at the extracellular face of the anion channel did the OST1-gated PdSLAC1 open, thus enabling chloride release. In the presence of nitrate, ABA enhanced and accelerated stomatal closure. Our findings indicate that, in date palm, the guard cell osmotic motor driving stomatal closure uses nitrate as the signal to open the major anion channel SLAC1. This initiates guard cell depolarization and the release of anions together with potassium.

Localization of the Transpiration Barrier in the Epi- and Intracuticular Waxes of Eight Plant Species: Water Transport Resistances Are Associated with Fatty Acyl Rather Than Alicyclic Components.

Plant cuticular waxes play a crucial role in limiting nonstomatal water loss. The goal of this study was to localize the transpiration barrier within the layered structure of cuticles of eight selected plant species and to put its physiological function into context with the chemical composition of the intracuticular and epicuticular wax layers. Four plant species (Tetrastigma voinierianum, Oreopanax guatemalensis, Monstera deliciosa, and Schefflera elegantissima) contained only very-long-chain fatty acid (VLCFA) derivatives such as alcohols, alkyl esters, aldehydes, and alkanes in their waxes. Even though the epicuticular and intracuticular waxes of these species had very similar compositions, only the intracuticular wax was important for the transpiration barrier. In contrast, four other species (Citrus aurantium, Euonymus japonica, Clusia flava, and Garcinia spicata) had waxes containing VLCFA derivatives, together with high percentages of alicyclic compounds (triterpenoids, steroids, or tocopherols) largely restricted to the intracuticular wax layer. In these species, both the epicuticular and intracuticular waxes contributed equally to the cuticular transpiration barrier. We conclude that the cuticular transpiration barrier is primarily formed by the intracuticular wax but that the epicuticular wax layer may also contribute to it, depending on species-specific cuticle composition. The barrier is associated mainly with VLCFA derivatives and less (if at all) with alicyclic wax constituents. The sealing properties of the epicuticular and intracuticular layers were not correlated with other characteristics, such as the absolute wax amounts and thicknesses of these layers.

The ecophysiology of leaf cuticular transpiration: are cuticular water permeabilities adapted to ecological conditions?

When the stomata are closed under drought, the only route for water loss from the leaf interior to the atmosphere is across the cuticle. Thus, the extent of cuticular transpiration in relation to the reservoirs of water in the plant and the water acquisition from the soil determines the fitness and survival of the plant. It is, therefore, widely assumed that the cuticular water permeability of plants regularly experiencing drought is comparatively low and, thus, adapted to the environment. To test this hypothesis, 382 measurements of cuticular permeability from 160 species were extracted from the literature published between 1996 and 2017. The data had been produced either by using isolated cuticles and astomatous leaf sides or by measuring the minimum leaf conductance under conditions assumed to induce maximum stomatal closure. The species were assigned to 11 life form groups. Except for two particular cases (epiphytes, and climbers and lianas), the cuticular permeabilities of all groups either did not differ significantly or the available data did not allow a statistical test. In conclusion, present knowledge either does not support the hypothesis that ecological adaptions of cuticular water permeability exist or the available data are insufficient to test it.

P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice.

Adenosine triphosphate (ATP) and its metabolite, adenosine, are key regulators of polymorphonuclear neutrophil (PMN) functions. PMNs have recently been implicated in the initiation of thrombosis. We investigated the role of ATP and adenosine in PMN activation and recruitment at the site of endothelial injury. Following binding to the injured vessel wall, PMNs are activated and release elastase. The recruitment of PMNs and the subsequent fibrin generation and thrombus formation are strongly affected in mice deficient in the P2X1-ATP receptor and in wild-type (WT) mice treated with CGS 21680, an agonist of the A2A adenosine receptor or NF449, a P2X1 antagonist. Infusion of WT PMNs into P2X1-deficient mice increases fibrin generation but not thrombus formation. Restoration of thrombosis requires infusion of both platelets and PMNs from WT mice. In vitro, ATP activates PMNs, whereas CGS 21680 prevents their binding to activated endothelial cells. These data indicate that adenosine triphosphate (ATP) contributes to polymorphonuclear neutrophil (PMN) activation leading to their adhesion at the site of laser-induced endothelial injury, a necessary step leading to the generation of fibrin, and subsequent platelet-dependent thrombus formation. Altogether, our study identifies previously unknown mechanisms by which ATP and adenosine are key molecules involved in thrombosis by regulating the activation state of PMNs.

Water loss from litchi (Litchi chinensis) and longan (Dimocarpus longan) fruits is biphasic and controlled by a complex pericarpal transpiration barrier.

MAIN CONCLUSION: In litchi and longan fruits, a specialised pericarp controls water loss by a protective system consisting of two resistances in series and two water reservoirs separated by a barrier. In the fruits of litchi (Litchi chinensis) and longan (Dimocarpus longan), the pericarp is solely a protective structure lacking functional stomata and completely enclosing the aril that is the edible part. Maintaining a high water content of the fruits is crucial for ensuring the economic value of these important fruit crops. The water loss rates from mature fruits were determined and analysed in terms of the properties of the pericarps. Water loss kinetics and sorption isotherms were measured gravimetrically. The pericarps were studied with microscopy, and cuticular waxes and cutin were analysed with gas chromatography and mass spectrometry. The kinetics of fruit water loss are biphasic with a high initial rate and a lower equilibrium rate lasting for many hours. The outer and inner surfaces of the pericarps are covered with cuticles. Litchi and longan fruits have a unique type of transpiration barrier consisting of two resistances in series (endo- and exocarp cuticles) and two reservoirs of water (aril and mesocarp). The exocarp permeability controls the water loss from fresh fruits while in fruits kept for an extended time at low relative humidity it is determined by the endo- and exocarp permeabilities. Permeances measured are within the range for typical fruit cuticles. The findings may be used to design optimal postharvest storage strategies for litchi and longan fruits.

Deficiency in a very-long-chain fatty acid ß-ketoacyl-coenzyme a synthase of tomato impairs microgametogenesis and causes floral organ fusion.

Previously, it was shown that ß-ketoacyl-coenzyme A synthase ECERIFERUM6 (CER6) is necessary for the biosynthesis of very-long-chain fatty acids with chain lengths beyond C28 in tomato (Solanum lycopersicum) fruits and C26 in Arabidopsis (Arabidopsis thaliana) leaves and the pollen coat. CER6 loss of function in Arabidopsis resulted in conditional male sterility, since pollen coat lipids are responsible for contact-mediated pollen hydration. In tomato, on the contrary, pollen hydration does not rely on pollen coat lipids. Nevertheless, mutation in SlCER6 impairs fertility and floral morphology. Here, the contribution of SlCER6 to the sexual reproduction and flower development of tomato was addressed. Cytological analysis and cross-pollination experiments revealed that the slcer6 mutant has male sterility caused by (1) hampered pollen dispersal and (2) abnormal tapetum development. SlCER6 loss of function provokes a decrease of n- and iso-alkanes with chain lengths of C27 or greater and of anteiso-alkanes with chain lengths of C28 or greater in flower cuticular waxes, but it has no impact on flower cuticle ultrastructure and cutin content. Expression analysis confirmed high transcription levels of SlCER6 in the anther and the petal, preferentially in sites subject to epidermal fusion. Hence, wax deficiency was proposed to be the primary reason for the flower fusion phenomenon in tomato. The SlCER6 substrate specificity was revisited. It might be involved in elongation of not only linear but also branched very-long-chain fatty acids, leading to production of the corresponding alkanes. SlCER6 implements a function in the sexual reproduction of tomato that is different from the one in Arabidopsis: SlCER6 is essential for the regulation of timely tapetum degradation and, consequently, microgametogenesis.

Optimization of 2-phenyl-pyrimidine-4-carboxamides towards potent, orally bioavailable and selective P2Y(12) antagonists for inhibition of platelet aggregation.

2-Phenyl-pyrimidine-4-carboxamide analogs were identified as P2Y12 antagonists. Optimization of the carbon-linked or nitrogen-linked substituent at the 6-position of the pyrimidine ring provided compounds with excellent ex vivo potency in the platelet aggregation assay in human plasma. Compound 23u met the objectives for activity, selectivity and ADMET properties.

Aqueous pathways dominate permeation of solutes across Pisum sativum seed coats and mediate solute transport via diffusion and bulk flow of water.

The permeability of seed coats to solutes either of biological or anthropogenic origin plays a major role in germination, seedling growth and seed treatment by pesticides. An experimental set-up was designed for investigating the mechanisms of seed coat permeation, which allows steady-state experiments with isolated seed coats of Pisum sativum. Permeances were measured for a set of organic model compounds with different physicochemical properties and sizes. The results show that narrow aqueous pathways dominate the diffusion of solutes across pea seed coats, as indicated by a correlation of permeances with the molecular sizes of the compounds instead of their lipophilicity. Further indicators for an aqueous pathway are small size selectivity and a small effect of temperature on permeation. The application of an osmotic water potential gradient across isolated seed coats leads to an increase in solute transfer, indicating that the aqueous pathways form a water-filled continuum across the seed coat allowing the bulk flow of water. Thus, the uptake of organic solutes across pea testae has two components: (1) by diffusion and (2) by bulk water inflow, which, however, is relevant only during imbibition.

Evolution of novel tricyclic CRTh2 receptor antagonists from a (E)-2-cyano-3-(1H-indol-3-yl)acrylamide scaffold.

(E)-2-(3-(3-((3-Bromophenyl)amino)-2-cyano-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)acetic acid (1) was discovered in a HTS campaign for CRTh2 receptor antagonists. An SAR around this hit could be established and representatives with interesting activity profiles were obtained. Ring closing tactics to convert this hit series into a novel 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole based CRTh2 receptor antagonist series is presented.

Abscisic acid mediates the formation of a suberized stem scar tissue in tomato fruits.

During harvest, fleshy berry tomato fruits (Solanum lycopersicum) were wounded at their stem scar. Within 3 d, this wound was rapidly sealed by a process covering the wound site with a membranous layer which effectively protects the tomato fruit from excessive water loss, nutrient elution and the entry of pathogens. Chemical analysis of the de novo synthesized stem scar tissue revealed the presence of aromatic and aliphatic components characteristic of the biopolyester suberin. Gene expression patterns associated with suberization were identified at the stem scar region. Changes in the relative abundance of different transcripts suggested a potential involvement of the plant hormone abscisic acid (ABA) in the wound-healing processes. The amount of ABA present in the stem scar tissue showed a significantly increased level during wound healing, whereas ABA-deficient mutants notabilis, flacca and sitiens were largely devoid of this rise in ABA levels. The mutant fruits showed a retarded and less efficient suberization response at the stem scar wound, whereas the rate and strength of this response were positively correlated with ABA content. These results clearly indicate in vivo the involvement of ABA in the suberization-based wound-healing processes at the stem scar tissue of tomato fruits.

Novel 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids: discovery and hit-to-lead evolution of a selective CRTh2 receptor antagonist chemotype.

Hit-to-lead evolution of 2-(2-((2-(4-chlorophenoxy)ethyl)thio)-1H-benzo[d]imidazol-1-yl)acetic acid (1), discovered in a high-throughput screening campaign as a novel chemotype of CRTh2 receptor antagonist, is presented. SAR development as well as in vitro and in vivo DMPK properties of selected representatives of substituted 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids are discussed.

Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves.

This study aimed to investigate whether the presence of trichomes as conspicuous physical attributes of the leaf surface affects the microbial community composition on Arabidopsis thaliana leaves. The A. thaliana ecotype Col-0 and its trichomeless gl1 mutant were grown in growth cabinets under climate-controlled conditions. The gl1 mutant showed a similar wax composition as the Col-0 wild type with slightly reduced amounts of C(29), C(31) and C(33) alkanes by GC/MS and GC/FID analyses. 120 bacterial isolates representing 39 bacterial genera were obtained from A. thaliana Col-0 leaf surfaces. Phylogenetic analysis of nearly full-length 16S rRNA sequences from 29 selected isolates confirmed their affiliation to the Proteobacteria (Alpha-, Beta-, Gamma-), Actinobacteria, Bacteroidetes and Firmicutes. The bacterial diversity on A. thaliana ecotype Col-0 and its gl1 mutant, devoid of trichomes, were further compared by denaturing gradient gel electrophoresis (DGGE). Banding patterns and sequencing of representative DGGE bands revealed the presence of phylotypes related to Sphingomonas (Alphaproteobacteria), Methylophilus (Betaproteobacteria) and Dyadobacter (Bacteroidetes) which are common phyllosphere inhabitants. Furthermore, wildtype and trichomeless mutant plants were exposed to outdoor conditions for 4-5 weeks. The DGGE gels showed only minor differences between the two plant lines, thus suggesting that trichomes per se do not affect bacterial diversity on Arabidopsis leaves under the experimental conditions tested.

Measurement setup and protocol for characterizing and testing radio frequency personal exposure meters.

Body-worn radiofrequency electromagnetic field (RF-EMF) personal exposure meters (PEMs) have been increasingly used for exposure assessment in epidemiological research. However, little research on the measurement accuracy of these devices is available. In this article a novel measurement setup and a measurement protocol are presented for characterizing and testing PEMs. The whole setup and procedure is tested using two EME SPY 120 devices. The performance of the PEM was analyzed for absolute measurements in an anechoic chamber. Modulated signals representing the different services as real signals generated by appropriate testers were used. Measurement results were evaluated with respect to a root mean square detector. We found that measurement accuracy depends strongly on the carrier frequency and also on the number of occupied time slots for Time Division Multiple Access (TDMA)-based services. Thus, correction factors can only be derived if the distribution of the network configuration over the measurement time for all measurement points is available. As a result of the simplicity of the measurement setup and the straightforward measurement protocol, the possibility of fast validation leads to a higher accuracy in the characterization and testing of PEMs.

Multifunctional Contribution of the Inflated Fruiting Calyx: Implication for Cuticular Barrier Profiles of the Solanaceous Genera Physalis, Alkekengi, and Nicandra.

Pivotal barrier properties of the hydrophobic plant cuticle covering aerial plant surfaces depend on its physicochemical composition. Among plant species and organs, compounds of this boundary layer between the plant interior and the environment vary considerably but cuticle-related studies comparing different organs from the same plant species are still scarce. Thus, this study focused on the cuticle profiles of Physalis peruviana, Physalis ixocarpa, Alkekengi officinarum, and Nicandra physalodes species. Inflated fruiting calyces enveloping fruits make Physalis, Alkekengi, and Nicandra highly recognizable genera among the Solanoideae subfamily. Although the inflation of fruiting calyces is well discussed in the literature still little is known about their post-floral functionalities. Cuticular composition, surface structure, and barrier function were examined and compared in fully expanded amphistomatous leaves, ripe astomatous fruits, and fully inflated hypostomatous fruiting calyces. Species- and organ-specific abundances of non-glandular and glandular trichomes revealed high structural diversity, covering not only abaxial and adaxial leaf surfaces but also fruiting calyx surfaces, whereas fruits were glabrous. Cuticular waxes, which limit non-stomatal transpiration, ranged from <1 µg cm-2 on P. peruviana fruiting calyces and N. physalodes fruits to 22 µg cm-2 on P. peruviana fruits. Very-long-chain aliphatic compounds, notably n-alkanes, iso-, and anteiso-branched alkanes, alkanols, alkanoic acids, and alkyl esters, dominated the cuticular wax coverages (≥86%). Diversity of cuticular wax patterns rose from leaves to fruiting calyces and peaked in fruits. The polymeric cutin matrix providing the structural framework for cuticular waxes was determined to range from 81 µg cm-2 for N. physalodes to 571 µg cm-2 for A. officinarum fruits. Cuticular transpiration barriers were highly efficient, with water permeabilities being ≤5 × 10-5 m s-1. Only the cuticular water permeability of N. physalodes fruits was 10 × 10-5 m s-1 leading to their early desiccation and fruits that easily split, whereas P. peruviana, P. ixocarpa, and A. officinarum bore fleshy fruits for extended periods after maturation. Regarding the functional significance, fruiting calyces establish a physicochemical shield that reduces water loss and enables fruit maturation within a protective microclimate, and promotes different seed dispersal strategies among plant species investigated.