Comparison of plant biostimulating properties of Chlorella sorokiniana biomass produced in batch and semi-continuous systems supplemented with pig manure or acetate.

Microalgae-derived biostimulants provide an eco-friendly biotechnology for improving crop productivity. The strategy of circular economy includes reducing biomass production costs of new and robust microalgae strains grown in nutrient-rich wastewater and mixotrophic culture where media is enriched with organic carbon. In this study, Chlorella sorokiniana was grown in 100 l bioreactors under sub-optimal conditions in a greenhouse. A combination of batch and semi-continuous cultivation was used to investigate the growth, plant hormone and biostimulating effect of biomass grown in diluted pig manure and in nutrient medium supplemented with Na-acetate. C. sorokiniana tolerated the low light (sum of PAR 0.99 ± 0.18 mol/photons/(m2/day)) and temperature (3.7-23.7° C) conditions to maintain a positive growth rate and daily biomass productivity (up to 149 mg/l/day and 69 mg/l/day dry matter production in pig manure and Na-acetate supplemented cultures respectively). The protein and lipid content was significantly higher in the biomass generated in batch culture and dilute pig manure (1.4x higher protein and 2x higher lipid) compared to the Na-acetate enriched culture. Auxins indole-3-acetic acid (IAA) and 2-oxindole-3-acetic acid (oxIAA) and salicylic acid (SA) were present in the biomass with significantly higher auxin content in the biomass generated using pig manure (> 350 pmol/g DW IAA and > 84 pmol/g DW oxIAA) compared to cultures enriched with Na-acetate and batch cultures (< 200 pmol/g DW IAA and < 27 pmol/g DW oxIAA). No abscisic acid and jasmonates were detected. All samples had plant biostimulating activity measured in the mungbean rooting bioassay with the Na-acetate supplemented biomass eliciting higher rooting activity (equivalent to 1-2 mg/l IBA) compared to the pig manure (equivalent to 0.5-1 mg/l IBA) and batch culture (equivalent to water control) generated biomass. Thus C. sorokiniana MACC-728 is a robust new strain for biotechnology, tolerating low light and temperature conditions. The strain can adapt to alternative nutrient (pig manure) and carbon (acetate) sources with the generated biomass having a high auxin concentration and plant biostimulating activity detected with the mungbean rooting bioassay.

Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.

Introduction: Chloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle. Methods: In the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress. Results: Altogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. Conclusions: These observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.

Synthetic and analytical routes to the L-amino acid conjugates of cis-OPDA and their identification and quantification in plants.

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the unambiguous identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic-lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels were estimated. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Our synthetic and analytical methodologies will support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

High-throughput interspecies profiling of acidic plant hormones using miniaturised sample processing.

BACKGROUND: Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. RESULTS: A high-throughput sample preparation method for liquid chromatography-tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. CONCLUSION: The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

Silver Nanoparticles Alter Microtubule Arrangement, Dynamics and Stress Phytohormone Levels.

The superior properties of silver nanoparticles (AgNPs) has resulted in their broad utilization worldwide, but also the risk of irreversible environment infestation. The plant cuticle and cell wall can trap a large part of the nanoparticles and thus protect the internal cell structures, where the cytoskeleton, for example, reacts very quickly to the threat, and defense signaling is subsequently triggered. We therefore used not only wild-type Arabidopsis seedlings, but also the glabra 1 mutant, which has a different composition of the cuticle. Both lines had GFP-labeled microtubules (MTs), allowing us to observe their arrangement. To quantify MT dynamics, we developed a new microscopic method based on the FRAP technique. The number and growth rate of MTs decreased significantly after AgNPs, similarly in both lines. However, the layer above the plasma membrane thickened significantly in wild-type plants. The levels of three major stress phytohormone derivatives-jasmonic, abscisic, and salicylic acids-after AgNP (with concomitant Ag+) treatment increased significantly (particularly in mutant plants) and to some extent resembled the plant response after mechanical stress. The profile of phytohormones helped us to estimate the mechanism of response to AgNPs and also to understand the broader physiological context of the observed changes in MT structure and dynamics.

Elevated CO2 modulates the effect of heat stress responses in Triticum aestivum by differential expression of isoflavone reductase-like (IRL) gene.

Two wheat genotypes forming high and low biomass (HB and LB), exhibiting differential expression of an isoflavone reductase-like (IRL) gene, and resulting in contrasting grain yield under heat stress field conditions, were analyzed in detail for their responses under controlled heat and elevated CO2 conditions. Significant differences in IRL expression between the two lines were hypothesized to be the basis of their differential performance under the tested conditions and their stress tolerance potential. By a holistic approach integrating advanced cell physiological phenotyping of the antioxidative and phytohormone system in spikes and leaves with measurements of ecophysiological and agronomic traits, the genetic differences of the genotypes in IRL expression were assessed. In response to heat and elevated CO2, the two genotypes showed opposite regulation of IRL expression, which was associated with cytokinin concentration, total flavonoid contents, activity of superoxide dismutase, antioxidant capacity and photosynthetic rate in leaves and cytokinin concentration and ascorbate peroxidase activity in spikes. Our study showed that IRL expression is associated with wheat yield performance under heat stress at anthesis, mediated by diverse physiological mechanisms. Hence, based on our results, the IRL gene is a promising candidate for developing genetic markers for breeding heat-tolerant wheat.

Is It Useful to Monitor Thiopurine Metabolites in Pediatric Patients with Crohn's Disease on Combination Therapy? A Multicenter Prospective Observational Study.

BACKGROUND: The additional value of azathioprine concomitant treatment on infliximab pharmacokinetics in children is not well described yet. AIMS: In the present study, we aimed to describe the relationship between thiopurine metabolite levels, infliximab trough levels, anti-IFX antibody formation, and clinical and laboratory markers of disease activity in pediatric patients with Crohn's disease, and to assess non-adherence. METHODS: Data were collected prospectively during repeated visits from pediatric patients followed for Crohn's disease in two Czech pediatric inflammatory bowel disease centers between January 2016 and June 2017. Thiopurine metabolites (6-thioguanine and 6-methylmercaptopurine) were measured by high-performance liquid chromatography. Infliximab trough levels and anti-IFX antibody serum levels were measured routinely by ELISA. The risk of loss of response to infliximab therapy was also assessed. RESULTS: A significant association between infliximab serum levels and 6-thioguanine erythrocyte levels was observed when tested as categorical variables (63 patients, 321 observations). To predict infliximab levels > 5 µg/mL, we propose a 6-thioguanine cutoff of 278 pmol/8 × 108 erythrocytes (sensitivity, 0.799; specificity, 0.347). A higher loss-of-response-to-infliximab rate (tested in a subgroup of 51 patients) was observed in patients with undetectable 6-thioguanine levels than in those with detectable levels (p = 0.026). Non-adherence to azathioprine therapy was suspected in 20% of patients. CONCLUSION: Thiopurine metabolite monitoring in pediatric patients with Crohn's disease is useful when optimizing combination therapy. Pediatric patients with undetectable 6-thioguanine levels are more likely to lose response to infliximab therapy. When targeting optimal infliximab levels, the 6-thioguanine cutoff levels in children appear to be higher than in adults.

In situ characterisation of phytohormones from wounded Arabidopsis leaves using desorption electrospray ionisation mass spectrometry imaging.

Phytohormones (plant hormones) are a group of small signalling molecules that act as important endogenous regulators in plant development and stress responses. Previous research has identified the phytohormone species, jasmonates, auxins and abscisic acid, and their related compounds in stressed leaf extracts. However, in situ visualisations of endogenous phytohormones from intact plant tissues remain elusive without the usage of labels or reporters. Mass spectrometry imaging is a label-free analytical technique that has been successfully applied for the direct detection of plant proteins, lipids, carbohydrates and many other biomolecules. In this study, desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) was used for high throughput visualisation and evaluation of wound-induced phytohormones inside Arabidopsis thaliana leaves. The results showed higher levels of jasmonates, salicylic acid, abscisic acid and indole-3-acetic acid in their ion intensity maps established from wounded leaves compared to control leaves, which have been validated in the parallel liquid chromatography-mass spectrometry quantification, and the untainted distributions of the identified phytohormones in leaves were confirmed by mass spectrometry imaging of instant leaf imprinted thin-layer chromatography plate samples. Further statistical analysis has not only demonstrated a significant increase of jasmonic acid and its precursor compounds in wounded leaves/regions but also highlighted a potential correlation in different phytohormone species. Our results suggest that DESI-MSI can be used to in situ characterise multiple phytohormone compounds from intact leaves with 200 µm spatial resolution to provide insight into phytohormone distributions in wounded leaves, along with their correlated precursors and metabolites under mechanical stress.

Prediction of Thiopurine Metabolite Levels Based on Haematological and Biochemical Parameters.

OBJECTIVES: Therapeutic drug monitoring of thiopurine erythrocyte levels is not available in all centers and it usually requires quite a long time to obtain the results. The aims of this study were to build a model predicting low levels of 6-thioguanine and 6-methylmercaptopurine in pediatric inflammatory bowel disease (IBD) patients and to build a model to predict nonadherence in patients treated with azathioprine (AZA). METHODS: The study consisted of 332 observations in 88 pediatric IBD patients. Low AZA dosing was defined as 6-thioguanine levels <125 pmol/8 × 10 erythrocytes and 6-methylmercaptopurine levels <5700 pmol/8 × 10 erythrocytes. Nonadherence was defined as undetectable levels of 6-thioguanine and 6-methylmercaptopurine <240 pmol/8 × 10 erythrocytes. Data were divided into training and testing part. To construct the model predicting low 6-thioguanine levels, nonadherence, and the level of 6-thioguanine, the modification of random forest method with cross-validation and resampling was used. RESULTS: The final models predicting low 6-thioguanine levels and nonadherence had area under the curve, 0.87 and 0.94; sensitivity, 0.81 and 0.82; specificity, 0.80 and 86; and distance, 0.31 and 0.21, respectively, when applied on the testing part of the dataset. When the final model for prediction of 6-thioguanine values was applied on testing dataset, a root-mean-square error of 110 was obtained. CONCLUSIONS: Using easily obtained laboratory parameters, we constructed a model with sufficient accuracy to predict patients with low 6-thioguanine levels and a model for prediction of AZA treatment nonadherence (web applications: https://hradskyo.shinyapps.io/6TG_prediction/ and https://hradskyo.shinyapps.io/Non_adherence/).

Oxidized glycerophosphatidylcholines in diabetes through non-targeted metabolomics: Their annotation and biological meaning.

Lipid oxidation is one of the most important processes occurring in living cells and has been investigated through stable end-products. Currently, new insights into many physiological and pathophysiological processes provide a measurement of the first products of oxidation, e.g., oxidized glycerophosphatidylcholines (oxGPCs). Here, we evaluate the capacity of untargeted global metabolomics to measure oxGPCs in serum samples. This evaluation covered analytical reproducibility and data quality as well as the ability to capture metabolic alterations in diverse conditions. The analytical evaluation was performed based on the quality control samples, while the comparative analysis was based on the model of the development of type 2 diabetes mellitus (T2DM). The novelty of this approach arises not only from the measurement of oxGPCs instead of lipid peroxide-derived aldehydes but also from the stratification of the patients according to body mass index (BMI). Such a scenario was dictated by the fact that, despite the well-known relationship between obesity and T2DM development, there are lean individuals suffering from T2DM as well as obese people with normal glucose homeostasis. Our results provided evidence to support the ability of nontargeted metabolomics to measure oxGPCs. Comparative analysis of measured oxGPCs revealed differences in the level of oxGPCs either between different stages of disease development (insulin resistance, prediabetes) or BMI groups (normal weight, overweight, obese). The obtained results provided new insights into the metabolic processes leading to the development of T2DM and opened new paths in the investigation of the impact of body mass in T2DM progress.

3,5,7-Substituted Pyrazolo[4,3- d]pyrimidine Inhibitors of Cyclin-Dependent Kinases and Their Evaluation in Lymphoma Models.

Cyclin-dependent kinases are therapeutic targets frequently deregulated in various cancers. By convenient alkylation of the 5-sulfanyl group, we synthesized 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2) H-pyrazolo[4,3- d]pyrimidines with various substitutions at position 5 with potent antiproliferative activity in non-Hodgkin lymphoma cell lines. The most potent derivative 4.35 also displayed activities across more than 60 cancer cell lines. The kinase profiling confirmed high selectivity of 4.35 toward cyclin-dependent kinases (CDKs) 2, 5, and 9, and the cocrystal with CDK2/cyclin A2 revealed its binding in the active site. Cultured lymphoma cell lines treated with 4.35 showed dephosphorylation of CDK substrates, cleavage of PARP-1, downregulation of XIAP and MCL-1, and activation of caspases, which collectively confirmed ongoing apoptosis. Moreover, 4.35 demonstrated significant activity in various cell line xenograft and patient-derived xenograft mouse models in vivo both as a monotherapy and as a combination therapy with the BCL2-targeting venetoclax. These findings support further studies of combinatorial treatment based on CDK inhibitors.

Characterization and annotation of oxidized glycerophosphocholines for non-targeted metabolomics with LC-QTOF-MS data.

The biological role of oxidized glycerophosphocholines (oxPCs) is a current topic of research importantly contributing to the understanding of health and disease. Global non-targeted metabolomics offers an interesting approach to expand current knowledge and link oxPCs to new biological functions. Although this strategy is successful, it also has some limitations which are clearly noticeable during the identification process. For this reason, clear rules related to the identification of each group of metabolites are needed. This work attempts to provide the reader with a guideline for the recognition and annotation of oxidation among phosphocholines (PCs). Using several chromatographic characteristics and spectral information from tandem mass spectrometry, rapid and reliable annotation of long and short chain oxPCs can be performed. Some of this knowledge has been implemented in the publicly available annotation tool 'CEU Mass Mediator' (CMM) for semi-automated assignment of oxidation. Additionally, this tool was supplemented with accurate monoisotopic masses of oxPCs, expanding current information in other databases. Moreover, the characterization of oxidization products of PC(16:0/20:4) known as PAPC has been performed, providing a list of accurate mass product ions and neutral losses.

Newborn foal with atypical myopathy.

The case of atypical myopathy (AM) in newborn Haflinger foal with clinical signs of depression and weakness appearing 6 hours after birth resulting in recumbency 12 hours after birth is described. The foal's dam was diagnosed with AM in the 6th month of gestation based on clinical signs of a myopathy, elevated serum activity of creatine kinase, metabolomic analysis and the presence of methylenecyclopropyl acetyl carnitine (MCPA-carnitine) in the blood. At the time of delivery, the mare was grazing on a pasture near sycamore trees but was free of clinical signs of AM. Metabolomic analysis of the foal's blood revealed increased concentrations of acylcarnitines and MCPA-carnitine consistent with metabolic profiles of blood from AM affected horses. Two theories could explain this observation (a) hypoglycin A or its metabolites accumulated in the mare's placenta with consequent transfer to fetus or (b) these compounds were secreted into mare's milk.

LC-MS/MS method for determination of cyclin-dependent kinase inhibitors, BP-14 and BP-20, and its application in pharmacokinetic study in rat.

N2-(4-Amino-cyclohexyl)-9-cyclopentyl-N6-(6-furan-2-yl-pyridine-3-ylmethyl)-9H-purine-2,6-diamine (BP-14) and 2-(5-{[2-(4-amino-cyclohexylamino)-9-cyclopentyl-9H-purine-6-ylamino]-methyl}-pyridine-2-yl)-phenol (BP-20) are novel cyclin-dependent kinase inhibitors, structurally related to roscovitine, with significant biological activity. A simple, selective and sensitive liquid chromatography - tandem mass spectrometry method for determining them in rat plasma, using roscovitine as an internal standard, was developed and validated. Chromatographic separation was performed in reversed phase mode on Acquity BEH C18 column (100 × 2.1 mm, 1.7 µm) by gradient elution with mobile phases composed of 15 mM ammonium formate pH 4.0 and methanol at flow rate 0.25 mL/min at 40 °C. The analytes were detected based on their characteristic multiple reaction monitoring transitions in positive electrospray ionization mode m/z 473.07 > 157.93 for BP-14, m/z 499.62 > 184.2 for BP-20 and m/z 355.5 > 90.86 for internal standard. In plasma the method provided good linearity within the entire concentration range: 1-10,000 nmol/L (r2 = 0.9989) for BP-14 and 10-25,000 nmol/L (r2 = 0.9994) for BP-20; the limit of detection was 0.6 nmol/L for BP-14 and 6.1 nmol/L for BP-20. Validation was also performed in bile and urine. The results of validation fit within the acceptance limits following European Medicines Agency guidelines. The method was applied in a pharmacokinetic study of BP-14 and BP-20 in vivo in rats following intravenous and intraduodenal administration including plasma pharmacokinetics, tissue distribution and excretion (renal and biliary). Both compounds showed low bioavailability after intraduodenal administration (0.630 and 1.58% for BP-14 and BP-20, respectively). Distribution into all the analyzed tissues (brain, lungs, liver, kidney, spleen, muscle, adipose tissue) was observed 3 h after single dose administration, the highest and lowest concentrations being reached in the adipose tissue and brain, respectively. The biliary excretion of the parent BP-14 and BP-20 compounds accounted for 4.81% and 10.6% of the doses, respectively, and renal excretion for <0.5% in both cases. The obtained results represent pilot knowledge for further development of a new generation of compounds with strong anticancer activities.

Plant Hormonomics: Multiple Phytohormone Profiling by Targeted Metabolomics.

Phytohormones are physiologically important small molecules that play essential roles in intricate signaling networks that regulate diverse processes in plants. We present a method for the simultaneous targeted profiling of 101 phytohormone-related analytes from minute amounts of fresh plant material (less than 20 mg). Rapid and nonselective extraction, fast one-step sample purification, and extremely sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry enable concurrent quantification of the main phytohormone classes: cytokinins, auxins, brassinosteroids, gibberellins, jasmonates, salicylates, and abscisates. We validated this hormonomic approach in salt-stressed and control Arabidopsis (Arabidopsis thaliana) seedlings, quantifying a total of 43 endogenous compounds in both root and shoot samples. Subsequent multivariate statistical data processing and cross-validation with transcriptomic data highlighted the main hormone metabolites involved in plant adaptation to salt stress.

Enhanced Secondary- and Hormone Metabolism in Leaves of Arbuscular Mycorrhizal Medicago truncatula.

Arbuscular mycorrhizas (AM) are the most common symbiotic associations between a plant's root compartment and fungi. They provide nutritional benefit (mostly inorganic phosphate [Pi]), leading to improved growth, and nonnutritional benefits, including defense responses to environmental cues throughout the host plant, which, in return, delivers carbohydrates to the symbiont. However, how transcriptional and metabolic changes occurring in leaves of AM plants differ from those induced by Pi fertilization is poorly understood. We investigated systemic changes in the leaves of mycorrhized Medicago truncatula in conditions with no improved Pi status and compared them with those induced by high-Pi treatment in nonmycorrhized plants. Microarray-based genome-wide profiling indicated up-regulation by mycorrhization of genes involved in flavonoid, terpenoid, jasmonic acid (JA), and abscisic acid (ABA) biosynthesis as well as enhanced expression of MYC2, the master regulator of JA-dependent responses. Accordingly, total anthocyanins and flavonoids increased, and most flavonoid species were enriched in AM leaves. Both the AM and Pi treatments corepressed iron homeostasis genes, resulting in lower levels of available iron in leaves. In addition, higher levels of cytokinins were found in leaves of AM- and Pi-treated plants, whereas the level of ABA was increased specifically in AM leaves. Foliar treatment of nonmycorrhized plants with either ABA or JA induced the up-regulation of MYC2, but only JA also induced the up-regulation of flavonoid and terpenoid biosynthetic genes. Based on these results, we propose that mycorrhization and Pi fertilization share cytokinin-mediated improved shoot growth, whereas enhanced ABA biosynthesis and JA-regulated flavonoid and terpenoid biosynthesis in leaves are specific to mycorrhization.

Metabolic Response of Visceral White Adipose Tissue of Obese Mice Exposed for 5 Days to Human Room Temperature Compared to Mouse Thermoneutrality.

Housing of laboratory mice at room temperature (22°C) might be considered a constant cold stress, which induces a thermogenic program in brown adipose tissue (BAT). However, the early adaptive response of white adipose tissue (WAT), the fat storage organ of the body, to a change from thermoneutrality to room temperature is not known. This was investigated here for various WAT depots, focusing on epididymal WAT (eWAT), widely used as reference depot. Male adult diet-induced obese (DIO) C57BL/6JOlaHsd mice housed at thermoneutrality (29°C), were for 5 days either switched to room temperature (22°C) or remained at thermoneutrality. Energy metabolism was continuously measured using indirect calorimetry. At the end of the study, serum metabolomics and WAT transcriptomics were performed. We confirmed activation of the thermogenic program in 22°C housed mice. Body weight and total fat mass were reduced. Whole body energy expenditure (EE) was increased, with a higher fatty acid to carbohydrate oxidation ratio and increased serum acylcarnitine levels, while energy intake was not significantly different between the two groups. Transcriptome analysis of eWAT identified tissue remodeling and inflammation as the most affected processes. Expression of pro-inflammatory M1 macrophage-related genes, and M1 over M2 macrophage ratio were decreased, which might be linked to an increased insulin sensitivity. Markers of thermogenesis were not altered in eWAT. Decreased expression of tryptophan hydroxylase 2 (Tph2) and cholecystokinin (Cck) might represent altered neuroendocrine signaling. eWAT itself does not show increased fatty acid oxidation. The three measured WATs, epididymal, mesenteric, and retroperitoneal, showed mainly similar responses; reduced inflammation (s100a8), decreased carbohydrate oxidation, and no or small differences in fatty acid oxidation. However, Ucp1 was only expressed and increased in rWAT in 22°C housed mice. Cck expression was decreased in the three WATs, significantly in eWAT and rWAT, in contrast to Tph2, which was decreased in eWAT while not expressed in mWAT and rWAT. Our data show that tissue remodeling, inflammation and neuroendocrine signaling are early responses in WAT to a moderate decrease in environmental temperature.

Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia Patients.

The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liquid chromatography mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metabolism. We observed differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an additional tool for the assessment of response/resistance to imatinib.

Detailed study of imatinib metabolization using high-resolution mass spectrometry.

Modern high resolution mass spectrometry offers unique identification capability in drug metabolism studies. In this work detailed imatinib metabolization in the plasma of patients with chronic myeloid leukemia is presented. The metabolites were separated by liquid chromatography on a C18 column with mass spectrometry detection via an Orbitrap Elite instrument (Thermo Scientific) based on exact mass measurement. A scan range of m/z 350-1200 resolution of 60,000 was applied (mass accuracy of 5ppm). The data were evaluated using the advanced software for mass spectrometry Mass Frontier and MetWorks. In all plasma samples, studied 90 metabolites in the concentration range of 0.0001-1µmol/L were identified by m/z values and confirmed by exact mass measurement of the MS(2) and MS(3) fragmentations. In order to achieve optimal clinical response and avoid toxicity, current therapeutic monitoring of parent drug is a useful tool for the individualization of treatment. Current high-resolution mass spectrometry possesses the potential to broaden this approach by monitoring number of potentially clinically relevant drug metabolites.

Assay of urinary 8-hydroxy-2'-deoxyguanosine by capillary electrophoresis with spectrophotometric detection using a high-sensitivity detection cell and solid-phase extraction.

A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z-cell was devised for the assay of 8-hydroxy-2'-deoxyguanosine (8OHdG) in human urine. Solid-phase extraction (SPE) based on hydrophilic-lipophilic-balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused-silica capillaries employing a Z-cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10-1000 ng/mL; R(2) = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.