Development and validation of a bioanalytical method for the quantification of methotrexate from serum and capillary blood using volumetric absorptive microsampling (VAMS) and on-line solid phase extraction (SPE) LC-MS.

High-dose methotrexate is part of the polychemotherapy protocols for the treatment of Acute lymphoblastic leukaemia (ALL) with therapeutic drug monitoring (TDM) to adjust leucovorin rescue. An immunoassay is commonly used to analyse serum samples collected via venous blood sampling. However, immunoassays cannot distinguish between the parent drug and its metabolites. Besides, the blood volume required by venous blood sampling is high. Therefore, the aim of this project was to develop a fast, simple, reliable and cost-efficient micro sampling bioanalytical method using capillary blood to minimize the harm of children and to analyse both methotrexate and its metabolites. To achieve this aim, a LC-MS method with on-line solid phase extraction (SPE) for the simultaneous detection of methotrexate and its metabolites from capillary blood using volumetric-absorptive-microsampling (VAMS) technology was developed and fully validated. Besides, the method was also validated and modified for serum samples to compare the results with the immunoassay. A single-quadrupole MS detector was used for detection. Through the use of on-line SPE technology, a lower limit of quantitation of 0.03 µM for MTX and 7-OH-MTX and of 0.05 µM for DAMPA from a 10 µL capillary blood sample was achieved. The accuracy is between 90.0 and 104% and the precision between 4.7 and 12% for methotrexate and its metabolites, respectively. Because of the cross reactivity of the immunoassay a cross-validation was not successful. Besides, a correlation factor of 0.46 for MTX between plasma and whole-blood was found. A fast, simple, reliable and cost-efficient extraction and analysis LC-MS method could be developed and validated, which is applicable in ambulatory and clinical care.

Tallo: A global tree allometry and crown architecture database.

Data capturing multiple axes of tree size and shape, such as a tree's stem diameter, height and crown size, underpin a wide range of ecological research-from developing and testing theory on forest structure and dynamics, to estimating forest carbon stocks and their uncertainties, and integrating remote sensing imagery into forest monitoring programmes. However, these data can be surprisingly hard to come by, particularly for certain regions of the world and for specific taxonomic groups, posing a real barrier to progress in these fields. To overcome this challenge, we developed the Tallo database, a collection of 498,838 georeferenced and taxonomically standardized records of individual trees for which stem diameter, height and/or crown radius have been measured. These data were collected at 61,856 globally distributed sites, spanning all major forested and non-forested biomes. The majority of trees in the database are identified to species (88%), and collectively Tallo includes data for 5163 species distributed across 1453 genera and 187 plant families. The database is publicly archived under a CC-BY 4.0 licence and can be access from: https://doi.org/10.5281/zenodo.6637599. To demonstrate its value, here we present three case studies that highlight how the Tallo database can be used to address a range of theoretical and applied questions in ecology-from testing the predictions of metabolic scaling theory, to exploring the limits of tree allometric plasticity along environmental gradients and modelling global variation in maximum attainable tree height. In doing so, we provide a key resource for field ecologists, remote sensing researchers and the modelling community working together to better understand the role that trees play in regulating the terrestrial carbon cycle.

Transcriptomic Profiling Reveals Shared Signalling Networks Between Flower Development and Herbivory-Induced Responses in Tomato.

Most flowering plants must defend themselves against herbivores for survival and attract pollinators for reproduction. Although traits involved in plant defence and pollinator attraction are often localised in leaves and flowers, respectively, they will show a diffuse evolution if they share the same molecular machinery and regulatory networks. We performed RNA-sequencing to characterise and compare transcriptomic changes involved in herbivory-induced defences and flower development, in tomato leaves and flowers, respectively. We found that both the herbivory-induced responses and flower development involved alterations in jasmonic acid signalling, suppression of primary metabolism and reprogramming of secondary metabolism. We identified 411 genes that were involved in both processes, a number significantly higher than expected by chance. Genetic manipulation of key regulators of induced defences also led to the expression changes in the same genes in both leaves and flowers. Targeted metabolomic analysis showed that among closely related tomato species, jasmonic acid and α-tomatine are correlated in flower buds and herbivory-induced leaves. These findings suggest that herbivory-induced responses and flower development share a common molecular machinery and likely have coevolved in nature.

Dopamine signaling regulates hematopoietic stem and progenitor cell function.

Hematopoietic stem and progenitor cell (HSPC) function in bone marrow (BM) is controlled by stroma-derived signals, but the identity and interplay of these signals remain incompletely understood. Here, we show that sympathetic nerve-derived dopamine directly controls HSPC behavior through D2 subfamily dopamine receptors. Blockade of dopamine synthesis, as well as pharmacological or genetic inactivation of D2 subfamily dopamine receptors, leads to reduced HSPC frequency, inhibition of proliferation, and low BM transplantation efficiency. Conversely, treatment with a D2-type receptor agonist increases BM regeneration and transplantation efficiency. Mechanistically, dopamine controls expression of the lymphocyte-specific protein tyrosine kinase (Lck), which, in turn, regulates MAPK-mediated signaling triggered by stem cell factor in HSPCs. Our work reveals critical functional roles of dopamine in HSPCs, which may open up new therapeutic options for improved BM transplantation and other conditions requiring the rapid expansion of HSPCs.

Evaluation of inhibitors of the arachidonic acid cascade with intact platelets using an on-line dilution and on-line solid phase extraction HPLC-MS method.

An automatic on-line dilution/on-line solid phase extraction (SPE) system has been developed for the detection of metabolites of the arachidonic acid cascade in platelets. The method allows the direct injection of larger quantities of centrifugates from cell suspensions previously treated with an equal volume of an acetonitrile/methanol mixture for protein precipitation. The method was used to study the effect of inhibitors of platelet arachidonic acid cascade enzymes (cytosolic phospholipase A2α, cyclooxygenase-1, thromboxane synthase, 12-lipoxygenase) and related targets (cyclooxygenase-2, microsomal prostaglandin E synthase-1, 5-lipoxygenase) in intact platelets after stimulation with calcium ionophore A23187. In addition to enzyme inhibition, the cell-damaging properties of the test compounds was determined by measuring the release of serotonin from the platelets into the incubation buffer.

Nanoparticle albumin-bound mTHPC for photodynamic therapy: Preparation and comprehensive characterization of a promising drug delivery system.

Nanoparticle albumin-bound (nab)-technology is an industrial applicable manufacturing method for the preparation of albumin-based drug carriers of poorly water-soluble drugs. In the present study the advantages of nanotechnology, albumin as an endogenous protein with the capability of high tumor enrichment and the selective light activation of the photosensitizer Temoporfin (mTHPC) were combined to a new delivery system for oncological use. The herewith provided well-established photodynamic therapy may enable a beneficial alternative for the treatment of solid tumors. In the present study a reproducible method for the preparation of stable mTHPC-albumin nanoparticles via nab-technology was established. The nanoparticles were physicochemically characterized with regard to particle size and size distribution and the impact of this preparation method on nanoparticle as well as mTHPC stability was investigated. Nanoparticles with improved colloidal stability over a broad pH range and in the presence of physiological NaCl concentrations were achieved in high yield. Due to high pressure homogenization a certain oxidative decay of mTHPC was observed. Cell culture experiments revealed an effective cellular uptake of mTHPC in a cholangiocarcinoma cell line (TFK-1). After light-activation high cytotoxicity was shown for photosensitizer loaded nanoparticles enabling the application of the proposed formulation in photodynamic therapy.

Slow rate of secondary forest carbon accumulation in the Guianas compared with the rest of the Neotropics.

Secondary forests are a prominent component of tropical landscapes, and they constitute a major atmospheric carbon sink. Rates of carbon accumulation are usually inferred from chronosequence studies, but direct estimates of carbon accumulation based on long-term monitoring of stands are rarely reported. Recent compilations on secondary forest carbon accumulation in the Neotropics are heavily biased geographically as they do not include estimates from the Guiana Shield. We analysed the temporal trajectory of aboveground carbon accumulation and floristic composition at one 25-ha secondary forest site in French Guiana. The site was clear-cut in 1976, abandoned thereafter, and one large plot (6.25 ha) has been monitored continuously since. We used Bayesian modeling to assimilate inventory data and simulate the long-term carbon accumulation trajectory. Canopy change was monitored using two aerial lidar surveys conducted in 2009 and 2017. We compared the dynamics of this site with that of a surrounding old-growth forest. Finally, we compared our results with that from secondary forests in Costa Rica, which is one of the rare long-term monitoring programs reaching a duration comparable to our study. Twenty years after abandonment, aboveground carbon stock was 64.2 (95% credibility interval 46.4, 89.0) Mg C/ha, and this stock increased to 101.3 (78.7, 128.5) Mg C/ha 20 yr later. The time to accumulate one-half of the mean aboveground carbon stored in the nearby old-growth forest (185.6 [155.9, 200.2] Mg C/ha) was estimated at 35.0 [20.9, 55.9] yr. During the first 40 yr, the contribution of the long-lived pioneer species Xylopia nitida, Goupia glabra, and Laetia procera to the aboveground carbon stock increased continuously. Secondary forest mean-canopy height measured by lidar increased by 1.14 m in 8 yr, a canopy-height increase consistent with an aboveground carbon accumulation of 7.1 Mg C/ha (or 0.89 Mg C·ha-1 ·yr-1 ) during this period. Long-term AGC accumulation rate in Costa Rica was almost twice as fast as at our site in French Guiana. This may reflect higher fertility of Central American forest communities or a better adaptation of the forest tree community to intense and frequent disturbances. This finding may have important consequences for scaling-up carbon uptake estimates to continental scales.

Improving plant allometry by fusing forest models and remote sensing.

Allometry determines how tree shape and function scale with each other, related through size. Allometric relationships help scale processes from the individual to the global scale and constitute a core component of vegetation models. Allometric relationships have been expected to emerge from optimisation theory, yet this does not suitably predict empirical data. Here we argue that the fusion of high-resolution data, such as those derived from airborne laser scanning, with individual-based forest modelling offers insight into how plant size contributes to large-scale biogeochemical processes. We review the challenges in allometric scaling, how they can be tackled by advances in data-model fusion, and how individual-based models can serve as data integrators for dynamic global vegetation models.

New formula and conversion factor to compute basic wood density of tree species using a global wood technology database.

PREMISE OF THE STUDY: Basic wood density is an important ecological trait for woody plants. It is used to characterize species performance and fitness in community ecology and to compute tree and forest biomass in carbon cycle studies. While wood density has been historically measured at 12% moisture, it is convenient for ecological purposes to convert this measure to basic wood density, i.e., the ratio of dry mass over green volume. Basic wood density can then be used to compute tree dry biomass from living tree volume. METHODS: Here, we derive a new exact formula to compute the basic wood density Db from the density at moisture content w denoted Dw , the fiber saturation point S, and the volumetric shrinkage coefficient R. We estimated a new conversion factor using a global wood technology database where values to use this formula are available for 4022 trees collected in 64 countries (mostly tropical) and representing 872 species. KEY RESULTS: We show that previous conversion factors used to convert densities at 12% moisture into basic wood densities are inconsistent. Based on theory and data, we found that basic wood density could be inferred from the density at 12% moisture using the following formula: Db = 0.828D12 . This value of 0.828 provides basic wood density estimates 4-5% smaller than values inferred from previous conversion factors. CONCLUSIONS: This new conversion factor should be used to derive basic wood densities in global wood density databases. Its use would prevent overestimating global forest carbon stocks and allow predicting better tree species community dynamics from wood density.

1-Heteroaryl-3-phenoxypropan-2-ones as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: Effect of the replacement of the ether oxygen with sulfur and nitrogen moieties on enzyme inhibition and metabolic stability.

Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that certain 3-phenoxy-substituted 1-heteroarylpropan-2-ones are inhibitors of cPLA2α and/or FAAH. Starting from 1-[2-oxo-3-(4-phenoxyphenoxy)propyl]indole-5-carboxylic acid (3) and 1-(1H-benzotriazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-one (4), the effect of the replacement of the oxygen in position 3 of the propan-2-one scaffold by sulfur and nitrogen containing moieties on inhibition of cPLA2α and fatty acid amide hydrolase as well as on metabolic stability in rat liver S9 fractions was investigated. As a result of these structure-activity relationship studies it was found that the ether oxygen is of great importance for enzyme inhibitory potency. Replacement by sulfur led to an about 100-fold decrease of enzyme inhibition, nitrogen and substituted nitrogen atoms at this position even resulted in inactivity of the compounds. The effect of the structural variations performed on metabolic stability of the important ketone pharmacophore was partly different in the two series of compounds. While introduction of SO and SO2 significantly increased stability of the ketone against reduction in case of the indole-5-carboxylic acid 3, it had no effect in case of the benzotriazole 4. Further analysis of the metabolism of 3 and 4 in rat liver S9 fractions revealed that the major metabolite of 3 was the alcohol 53 formed by reduction of the keto group. In contrast, in case of 4 beside keto reduction an excessive hydroxylation of the terminal phenoxy group occurred leading to the dihydroxy compound 50. Experiments with enzyme inhibitors showed that the phenylhydroxylation of 4 was catalyzed by tranylcypromine sensitive cytochrome P450 isoforms, while the reduction of the ketone function of 3 and 4 was mainly caused by cytosolic short chain dehydrogenases/reductases (cSDR).

Involvement of microsomal NADPH-cytochrome P450 reductase in metabolic reduction of drug ketones.

Recently, it was found that the carbonyl group of 1-[3-(4-phenoxyphenoxy)-2-oxopropyl]indole-5-carboxylic acid (5), an inhibitor of the pro-inflammatory enzyme cytosolic phospholipase A2 α, is easily reduced by rat liver S9 fractions in vitro. Determination of the inhibitory potency of certain putative inhibitors of carbonyl reducing enzymes on the transformation of the ketone derivative 5 to its alcohol 6 by recombinant microsomal NADPH-cytochrome P450 reductase and by recombinant cytosolic carbonyl reductase-1 now reveals that these compounds show a lack of specificity for these two enzymes in part. Thus, an assignment of the roles of different carbonyl reductases in metabolic keto reduction by the use of inhibitors is problematic. In addition, the ability of NADPH-cytochrome P450 reductase and carbonyl reductase-1 to reduce the ketone groups of the drugs haloperidol and daunorubicin was examined. Under the conditions applied, a pronounced reductive metabolism was only observed for daunorubicin in the presence of microsomal NADPH-cytochrome P450 reductase. Similarly, in rat liver S9 fractions a marked reduction of daunorubicin was seen, while haloperidol was only slightly metabolized to its alcohol. After separation of the S9 homogenate into a microsomal and a cytosolic fraction, it became evident that the ketone groups of daunorubicin, haloperidol and compound 5 were mainly reduced by cytosolic enzymes. However, since microsomes also catalysed these carbonyl reductions to some extent, it can be concluded that microsomal NADPH-cytochrome P450 reductase can contribute to metabolic keto reductions in xenobiotics. Copyright © 2015 John Wiley & Sons, Ltd.

Investigations on the metabolic stability of cytosolic phospholipase A2α inhibitors with 1-indolylpropan-2-one structure.

Cytosolic phospholipase A2α (cPLA2α) plays a key role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis, atopic dermatitis and Alzheimer's disease. Therefore, inhibition of this enzyme is assumed to provide a novel therapeutic option for the treatment of these maladies. In this study we investigated the metabolism of the potent cPLA2α inhibitors 1-[3-(4-phenoxyphenoxy)-2-oxopropyl]indole-5-carboxylic acid (1) and 3-isobutanoyl-1-[3-(4-phenoxyphenoxy)-2-oxopropyl]indole-5-carboxylic acid (2). Incubation of 1 with a mixture of human recombinant CYP1A2, 2C8, 2C9, 2C19, 2D6, 3A4 and NADPH-cytochrome P450 reductase enzymes led to reduction of its keto group and to hydroxylation at the terminal phenoxy residue. To identify the enzymes responsible for the observed reactions, experiments with isoform inhibitors were performed. In rat liver S9 fractions the only metabolite found was the alcohol 3 formed by the reduction of the keto group of 1. This reaction here was mainly catalyzed by cytosolic short-chain dehydrogenases/reductases (cSDR) as shown by inhibition experiments with different carbonyl reductase inhibitors. Furthermore, the metabolic stability of 2 in mouse brains was studied after intracerebroventricular application of this compound into the right brain hemispheres of mice. HPLC/MS analyses revealed that 2 is also readily reduced in the brain to an inactive alcohol metabolite most likely by carbonyl reductases.

Enantiomerically pure 1,3-dioxanes as highly selective NMDA and σ1 receptor ligands.

We synthesized and investigated the NMDA and σ1 receptor affinity of enantiomerically pure 2-(2-phenyl-1,3-dioxan-4-yl)ethanamines 17-26. The primary amines (R,R)-18-20 with an axially oriented phenyl moiety in position 2 interacted with high enantioselectivity (eudismic ratios 70-130) and high affinity (K(i)((R,R)-19) = 13 nM) with the PCP binding site of the NMDA receptor. Introduction of an N-benzyl moiety led to potent σ1 ligands including compound (S,R)-22 (K(i) = 6 nM) with an equatorially oriented phenyl moiety in position 2.

Pyrrole alkanoic acid derivatives as nuisance inhibitors of microsomal prostaglandin E2 synthase-1.

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) is an enzyme, which is induced during the inflammatory response. Therefore, inhibitors of this enzyme are considered to be potential anti-inflammatory drugs. We have identified 3-(4-dodecanoyl-1,3,5-trimethylpyrrol-2-yl)propionic acid (12) as submicromolar inhibitor of mPGES-1. Surprisingly, structural variations made around this lead only resulted in a relatively small change of enzyme inhibitory potency. Such flat structure-activity relationships are reported to be typical for so called nuisance inhibitors, which exert their action not by directly binding to the enzyme, but by forming colloid-like aggregates at micromolar and sometimes submicromolar concentrations, which somehow sequester and inhibit enzyme targets without specificity. Since aggregate-based inhibition is highly sensitive to non-ionic detergents such as Triton X-100, we investigated some of our compounds for inhibition of human recombinant mPGES-1 also in presence of this detergent. The pyrrole derivatives 12, 67 and 81, which exhibited IC(50) values in absence of Triton X-100 in the range of 0.1 and 1µM, were virtually inactive at the highest test concentration of 10µM when 0.1% of the detergent was added. In the same way, the published mPGES-1 inhibitor 2-[(4-{[(1,1'-biphenyl)-4-ylmethyl]amino}-6-chloropyrimidin-2-yl)thio]octanoic acid (Cay10589) (6) totally lost its activity under these conditions. Therefore, these compounds have to be judged as nuisance inhibitors of the enzyme. In contrast, the known indole derivative 3-[3-(tert-butylthio)-1-(4-chlorobenzyl)-5-isopropylindol-2-yl]-2,2-dimethylpropionic acid (MK-886) (2) showed a considerable activity (75% inhibition at 10µM) also in the presence of Triton X-100.

1-(5-carboxyindol-1-yl)propan-2-one inhibitors of human cytosolic phospholipase A(2)alpha with reduced lipophilicity: synthesis, biological activity, metabolic stability, solubility, bioavailability, and topical in vivo activity.

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 were previously reported to be potent inhibitors of human cytosolic phospholipase A(2)alpha (cPLA(2)alpha). In continuation of our attempts to develop clinical active cPLA(2)alpha inhibitors, a series of structurally related indole-5-carboxylic acids with reduced lipophilicity was synthesized and tested for cPLA(2)alpha-inhibitory potency. Furthermore, the thermodynamic solubility of these compounds and their metabolic stability in rat liver microsomes were evaluated. With an IC(50) of 0.012 microM against the isolated enzyme, compound 36 was one of the most potent cPLA(2)alpha inhibitors that emerged during the structure-activity relationship study. Concomitantly, 36 possessed the highest water solubility (212 microg/mL at pH 7.4) of all new target compounds. Despite these favorable properties, peroral application of 36 (100 mg/kg) in mice only led to low concentrations of the substance in blood plasma. A very high plasma clearance was observed after intravenous administration of 36 (10 mg/kg). However, in a topical murine model of contact dermatitis, 36 showed a pronounced anti-inflammatory in vivo activity.

Determination of prostaglandin E(2) by on-line solid-phase extraction-liquid chromatography with ultraviolet detection for microsomal prostaglandin E(2) synthase-1 inhibitor screening.

A rapid, robust and selective on-line solid-phase extraction-liquid chromatographic method with ultra-violet detection (on-line SPE-LC-UV) for microsomal prostaglandin E(2) synthase-1 (mPGES-1) inhibitor screening was developed and validated. Disrupted A549 cells were used as mPGES-1 source and the formation of prostaglandin E(2) (PGE(2)) out of the substrate prostaglandin H(2) (PGH(2)) was determined at 195 nm. Direct on-line sample clean up was achieved by automated column switch (C18 trap column) prior isocratic separation using a C18 analytical column. The on-line SPE-LC-UV method was accurate, precise and reproducible in the range of 71-1763 ng/ml for PGE(2) and met the generally accepted criteria for bioanalytical methods. The method was successfully applied to determine the IC(50) value of the known mPGES-1 inhibitor NS-398.

1-(2-Carboxyindol-5-yloxy)propan-2-ones as inhibitors of human cytosolic phospholipase A2alpha: synthesis, biological activity, metabolic stability, and solubility.

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 were previously reported to be potent inhibitors of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) isolated from human platelets. In continuation of our attempts to develop novel cPLA(2)alpha inhibitors, a series of structurally related indole-2-carboxylic acids containing 3-aryloxy-2-oxopropoxy residues in position 5 were synthesized and tested for their cPLA(2)alpha-inhibitory potency. Furthermore, the thermodynamic solubility of these compounds and their metabolic stability against rat liver microsomes were evaluated.

High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C.

A nonradioactive spectrometric assay for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC) is described. l-alpha-Phosphatidylinositol from bovine liver was used as substrate in the presence of the micelle-forming detergent deoxycholic acid. PI-PLC isolated from Bacillus cereus and crude cytosol fractions from porcine brain were used as enzyme sources. PI-PLC activity was determined by measuring the release of 1-stearoyl-2-arachidonoyl-sn-glycerol with reversed-phase HPLC and UV detection at 200 nm. PI-PLC from B. cereus was not inhibited by the putative PI-PLC inhibitors U-73122 and ET-18-OCH(3) at 100 microM, whereas the isobenzofuranone derivative 5 blocked the enzyme with an IC(50) of 75 microM. PI-PLC activity present in porcine brain cytosol was decreased by all three test compounds at 100 microM to approximately 30 to 50%.

Normal-phase HPLC and HPLC-MS studies of the metabolism of a cytosolic phospholipase A(2)alpha inhibitor with activated ketone group by rat liver microsomes.

Inhibition of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is assumed to provide a novel therapeutic approach for the treatment of many inflammatory diseases. 1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (2) is a potent inhibitor of cPLA(2)alpha. An important part of the pharmacophore of 2 is its activated electrophilic ketone moiety. Since it is known that activated ketones may be metabolically unstable, the metabolism of 2 by rat liver microsomes was investigated. For quantification of the metabolites normal-phase HPLC/UV on a cyano column was used, because under reversed-phase conditions with aqueous solvents 2 was partly transformed into its hydrate resulting in chromatograms with splitted peaks. Under the conditions applied about 30% of 2 were metabolized. The main metabolite was the alcohol 4 as shown by LC/MS(n).