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Liquid chromatography mass spectrometry (LC-MS) is the main workhorse of metabolomics owing to its high degree of analytical sensitivity and specificity when measuring diverse chemistry in complex biological samples. LC-MS-based metabolic profiling of human urine, a biofluid of primary interest for clinical and biobank studies, is not widely considered to be compromised by the presence of endogenous interferences and is often accomplished using a simple “dilute-and-shoot” approach. Yet, it is our experience that broad obscuring signals are routinely observed in LC-MS metabolic profiles and represent interferences which lack consideration in the relevant metabolomics literature. In this work we chromatographically isolated the interfering metabolites from human urine and unambiguously identified them via de novo structure elucidation as two separate proline-containing dipeptides: N,N,N-trimethyl-L-alanine-L-proline betaine (L,L-TMAP) and N,N-dimethyl-L-proline-L-proline betaine (L,L-DMPP), the latter reported here for the first time. Offline LC-MS/MS, MRMS, and NMR spectroscopy were essential components of this workflow for the full chemical and spectroscopic characterization of these metabolites and for establishing the co-existence of cis and trans isomers of both dipeptides in solution. Analysis of these definitive structures highlighted intramolecular ionic interactions as responsible for slow interconversion between these isomeric forms resulting in their unusually broad elution profiles. Proposed mitigation strategies, aimed at increasing the quality of LC-MS-based urine metabolomics data, include modification of the column temperature and mobile phase pH to reduce the chromatographic footprint of these dipeptides, thereby reducing their interfering effect on the underlying metabolic profiles. Alternatively, sample dilution and internal standardization methods may be employed to reduce or account for the observed effects of ionization suppression on the metabolic profile.

The inclusion of peptide retention time prediction promises to remove peptide identification ambiguity in complex LC-MS identification workflows. However, due to the way peptides are encoded in current prediction models, accurate retention times cannot be predicted for modified peptides. This is especially problematic for fledgling open modification searches, which will benefit from accurate retention time prediction for modified peptides to reduce identification ambiguity. We here therefore present DeepLC, a novel deep learning peptide retention time predictor utilizing a new peptide encoding based on atomic composition that allows the retention time of (previously unseen) modified peptides to be predicted accurately. We show that DeepLC performs similarly to current state-of-the-art approaches for unmodified peptides, and, more importantly, accurately predicts retention times for modifications not seen during training. Moreover, we show that DeepLC’s ability to predict retention times for any modification enables potentially incorrect identifications to be flagged in an open modification search of CD8-positive T-cell proteome data. DeepLC is available under the permissive Apache 2.0 open source license and comes with a user-friendly graphical user interface, as well as a Python package on PyPI, Bioconda, and BioContainers for effortless workflow integration.

The molecular diversity of the source substrate has been regarded as a significant controller of the proportion of plant material that is either mineralized or incorporated into soil organic matter (SOM). However, quantitative parameters to express substrate molecular diversity remain elusive. In this research, we fractionated leaves, twigs, bark, and root tissues of 13C-enriched eucalypt seedlings into hot water extractables (HWE), total solvent (acetone) extractables (TSE), a cellulosic fraction (CF), and the acid unhydrolyzable residue (AUR). We used 13C NMR spectroscopy to obtain a molecular diversity index (MDI) based on the relative abundance of carbohydrate, protein, lignin, lipid, and carbonyl functional groups within the biochemical fractions. Subsequently, we ob-tained artificial plant organs containing fixed proportions (25%) of their respective biochemical fractions to be incubated with soil material obtained from a Haplic Ferralsol for 200-days, under controlled temperature (25 +/- 1 degrees C) and moisture adjusted to 70-80% of the soil water holding capacity. Our experimental design was a randomized complete block design, arranged according to a factorial scheme including 4 plant organs, 4 biochemical fractions, and 3 blocks as replicates. During the incubation, we assessed the evolution of CO2 from the microcosms after 1, 2, 3, 4, 7, 10, 13, 21, 28, 38, 45, 70, 80, 92, 112, 148, 178 and 200 days from the start of the incubation. After the incubation, soil subsamples were submitted to a density fractionation to separate the light fraction of SOM (LFOM) i.e., with density

Post-translational modifications (PTMs) to the tails of the core histone proteins are critically involved in epigenetic regulation. Hypoxia affects histone modifications by altering the activities of histone-modifying enzymes and the levels of hypoxia-inducible factor (HIF) isoforms. Synthetic hypoxia mimetics promote a similar response, but how accurately the hypoxia mimetics replicate the effects of limited oxygen availability on the levels of histone PTMs is uncertain. Here we report studies on the profiling of the global changes to PTMs on intact histones in response to hypoxia/hypoxia-related stresses using liquid chromatography-mass spectrometry (LC-MS). We demonstrate that intact protein LC-MS profiling is a relatively simple and robust method for investigating potential effects of drugs on histone modifications. The results provide insights into the profiles of PTMs associated with hypoxia and inform on the extent to which hypoxia and hypoxia mimetics cause similar changes to histones. These findings imply chemically-induced hypoxia does not completely replicate the substantial effects of physiological hypoxia on histone PTMs, highlighting that caution should be used in interpreting data from their use.

Background Weaning is known to result in barrier dysfunction and villus atrophy in the immediate post-weaning phase, and the magnitude of these responses is hypothesized to correlate with changes in the glutathione (GSH) redox system. Therefore, these parameters were simultaneously measured throughout the weaning phase, in piglets differing in birth weight category and weaning age, as these pre-weaning factors are important determinants for the weaning transition. Low birth weight (LBW) and normal birth weight (NBW) littermates were assigned to one of three weaning treatments; i.e. weaning at 3 weeks of age (3w), weaning at 4 weeks of age (4w) and removal from the sow at 3 d of age and fed a milk replacer until weaning at 3 weeks of age (3d3w). For each of these treatments, six LBW and six NBW piglets were euthanized at 0, 2, 5, 12 or 28 d post-weaning piglets, adding up 180 piglets. Results Weaning increased the glutathione peroxidase activity on d 5 post-weaning in plasma, and duodenal and jejunal mucosa. Small intestinal glutathione-S-transferase activity gradually increased until d 12 post-weaning, and this was combined with a progressive rise of mucosal GSH up till d 12 post-weaning. Oxidation of the GSH redox status (GSH/GSSG Eh) was only observed in the small intestinal mucosa of 3d3w weaned piglets at d 5 post-weaning. These piglets also demonstrated increased fluorescein isothiocyanate dextran (FD4) and horseradish peroxidase fluxes in the duodenum and distal jejunum during the experiment, and specifically demonstrated increased FD4 fluxes at d 2 to d 5 post-weaning. On the other hand, profound villus atrophy was observed during the weaning transition for all weaning treatments. Finally, LBW and NBW piglets did not demonstrate notable differences in GSH redox status, small intestinal barrier function and histo-morphology throughout the experiment. Conclusion Although moderate changes in the GSH redox system were observed upon weaning, the GSH redox status remained at a steady state level in 3w and 4w weaned piglets and was therefore not associated with weaning induced villus atrophy. Conversely, 3d3w weaned piglets demonstrated GSH redox imbalance in the small intestinal mucosa, and this co-occurred with a temporal malfunction of their intestinal barrier function.

Oxidative stress is associated with a myriad of diseases including pregnancy pathologies with long-term cardiovascular repercussions for both the mother and baby. Aberrant redox signalling coupled with deficient antioxidant defence leads to chronic molecular impairment. Abnormal placentation has been considered the primary source for reactive species; however, placental dysfunction has been deemed secondary to maternal cardiovascular maladaptation in pregnancy. While various therapeutic interventions, aimed at combating deregulated oxidative stress during pregnancy have shown promise in experimental models, they often result as inconclusive or detrimental in clinical trials, warranting the need for further research to identify candidates. The strengths and limitations of current experimental methods in redox research are discussed. Assessment of redox status and oxidative stress in experimental models and in clinical practice remains challenging; the state-of-the-art of computational models in this field is presented in this review, comparing static and dynamic models which provide functional information such as protein-protein interactions, as well as the impact of changes in molecular species on the redox-status of the system, respectively. Enhanced knowledge of redox biology in during pregnancy through computational modelling such as generation of Systems Biology Markup Language model which integrates existing models to a larger network in the context of placenta physiology.

Drug-metabolizing enzymes (DMEs), a diverse group of enzymes responsible for the metabolic elimination of drugs and other xenobiotics, have been recognized as the critical determinants to drug safety and efficacy. Deciphering and understanding the key roles of individual DMEs in drug metabolism and toxicity, as well as characterizing the interactions of central DMEs with xenobiotics require reliable, practical and highly specific tools for sensing the activities of these enzymes in biological systems. In the last few decades, the scientists have developed a variety of optical substrates for sensing human DMEs, parts of them have been successfully used for studying target enzyme(s) in tissue preparations and living systems. Herein, molecular design principals and recent advances in the development and applications of optical substrates for human DMEs have been reviewed systematically. Furthermore, the challenges and future perspectives in this field are also highlighted. The presented information offers a group of practical approaches and imaging tools for sensing DMEs activities in complex biological systems, which strongly facilitates high-throughput screening the modulators of target DMEs and studies on drug/herb-drug interactions, as well as promotes the fundamental researches for exploring the relevance of DMEs to human diseases and drug treatment outcomes. (C) 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

A fast, accurate and reliable ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.

Resumen del póster presentado al International Scientific Association for Probiotics and Prebiotics Annual Meeting (ISAPP), celebrado en Sitges. Barcelona (España) del 15 al 17 de junio de 2022., Carbohydrates entering the complex microbial ecosystem resident in the human gut can be fermented to different degrees by the bacterial species present. Whilst insoluble carbohydrates may be fermented to a low degree, they may still be classed dietary fibres. More extensively fermented carbohydrates frequently display a selectivity towards particular microbial groups; if the end result of this process is an improvement in host health, then we would consider those carbohydrates to be prebiotics. It is a complex task to identify the structural basis of the fermentation selectivity underlying a prebiotic effect. Individuals vary in terms of the diversity of their gut microbiome and in the response of this microbiome to any given carbohydrate. The task is further complicated by the use of varying microbiology techniques across studies. Some generic understanding can be identified: It is clear that low molecular weight carbohydrates of a wide range of structure are very readily metabolised by bifidobacteria and are frequently seen to select for this genus in in vitro studies.. This selectivity is explained by the widespread presence of exo-glycosidase and sugar transport system genes in bifidobacterial genomes. Metabolism of higher molecular weight carbohydrates is dependant of organisms possessing the relevant polysaccharide utilisation genes like the bacteroides, rendering them highly adept at polysaccharide metabolism. To understand the fate of any given carbohydrate in the gut microbiome, however, the presence of cross-feeding networks must be taken into account. We know of some such cross feeding networks likely to operate in the gut but there is much to learn. ILSI Europe has commissioned an expert group to produce an authoritative view of the state of the art in terms of our understanding of structure function relationships in fermented carbohydrates. This presentation will highlight our current understanding of structure function relationships and point to the gaps in knowledge and the questions that arise in this area.

Obesity is becoming increasingly common all over the world and global strategies are accordingly being developed to prevent it. In order to support the strategies, the effects of green apple (Golden Delicious) and the consumption of its three major flavonols (quercetin-3-glucoside, quercetin-3-D-galactoside, and quercetin-3-rhamnoside) on body weight; the weight of liver, kidney, and spleen; some lipid parameters in serum; and total lipid ratios of liver and kidney and oxidative stress parameters of obese rats were studied. This study was conducted on two experimental groups: one of which was given an apple, and the other was given flavonols, in addition to their high-energy diet; along with a sham and a control rat group, for 4 weeks. According to results, there was no difference in body and organ weights between groups. The liver and kidney weights increased in obese rats, but there was no difference between the total lipid ratios in these organs. The addition of green apple and selected flavonols to the high-energy diet of rats was not sufficient to prevent the increase in body and organ weights, but it supported the reduction in some lipid fractions and in oxidative stress parameters of obese rats. Moreover, this study supported the argument that obesity causes most of the lipid fractions increase in serum and induces oxidative stress.

Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for clinical application is lacking. This present study describes a method to measure AA in the peripheral blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs and plasma were performed with HPLC with HILIC separation and UV detection. The sample preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation. A highly precise execution of the method was found with intra- and inter-assay variations at a maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108 cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for the quantitative determination of AA within PBMCs and plasma from human blood.

HPLC-PDA, LC-MS/MS methods were developed for simultaneous determination of a group of oxidative stress biomarkers (OSBs); 2dA, 2dC, 2dU, 3NLT, 5HMU and 8OHdG in 10 simulated artificial body fluids. O-phosphoric acid and methanol composed mobile phases A and B for gradient elution in HPLC-PDA using ODS-2 column. Linearity obtained for 1.0x10(-6)-1.0x10(-4)M range. LODs were 1.73x10(-6), 1.19x10(-6), 2.59x10(-6), 1.40x10(-6), 2.21x10(-6) and 4.07x10(-6)M for 2dU, 8OHdG, 2dA, 2dC, 5HMU and 3NLT, respectively. LOQs were 5.29x10(-6), 4.02x10(-6), 6.82x10(-6), 4.02x10(-6), 6.82x10(-6) and 9.92x10(-6)M. About 10 mM aqueous ammonium acetate solution and methanol containing 0.1% (v/v) formic acid composed mobile phases A and B for gradient elution in LC-MS/MS. Linearity obtained for 1.0x10(-8)-1.0x10(-6)M range. LODs were 2.88x10(-10), 1.01x10(-8), 3.38x10(-9), 1.36x10(-7), 1.81x10(-7) and 1.40x10(-8)M for 2dU, 8OHdG, 2dA, 2dC, 5HMU and 3NLT, respectively. LOQs were 9.37x10(-10), 3.22x10(-8), 1.91x10(-8), 4.53x10(-7), 5.90x10(-7) and 2.18x10(-8)M. Both methods were validated using ICH Q2(R1) guideline. Specificity, linearity, range, accuracy, precision, reproducibility, LOD, LOQ and recovery were achieved. Chemometric analysis was performed on raw PDA and MS data to check their significance for discrimination of OSBs. Sets of single and triple quadrupole fragmentations were evaluated for principle component analysis. Chosen number of PCs successfully distinguished OSBs of interest., Ege University Department of Scientific Research Projects (BAP) [2014-TIP086, 16/ECZ/022], This study was supported by Ege University Department of Scientific Research Projects (BAP, Project numbers: 2014-TIP086 and 16/ECZ/022).

Introduction: Right ventricular (RV) failure due to pressure load is an important determinant of clinical outcome in pulmonary hypertension, congenital heart disease and left ventricular failure. The last decades it has become clear that metabolic dysregulation is associated with the development of RV-failure. However, underlying mechanisms remain to be unraveled. Recently, disruption of intracardiac lipid content has been suggested as potential inducer of RV failure. In the present study, we used a rat model of RV-dysfunction and aimed to obtain insight in temporal changes in RV-function, -remodelling and -metabolism and relate this to RV lipid content.Methods and results: Male Wistar WU rats were subjected to pulmonary artery banding (n=25) or sham surgery (n=14) and cellular, hemodynamic and metabolic assessments took place after 2, 5 and 12 weeks. In this model RV dysfunction and remodelling occurred, including early upregulation of oxidative stress markers. After 12 weeks of pressure load, lipidomics revealed significant decreases of myocardial diglycerides and cardiolipins, driven by (poly-) unsaturated forms. The decrease of cardiolipins was driven by its most abundant form, tetralinoleoylcardiolipin. Mitochondrial capacity for fatty acid oxidation preserved, while the capacity for glucose oxidation increased.Conclusion: RV dysfunction due to pressure load, is associatedwith decreased intracardiac unsaturated lipids, especially tetralinoleoylcardiolipin. This was accompanied with preserved mitochondrial capacity regarding fatty acids oxidation, with increased capacity for glucose oxidation, and early activation of oxidative stress. We suggest that early interventions should be directed towards preservation of lipid availability as possible mean in order to prevent RV failure. (c) 2019 Published by Elsevier B.V.

The increasing demand by citizens and environmental organization for the protection, preservation, and possible restoration of the marine environment has made seawater protection one of the urgent priorities of the European Union. In this contest, extensive monitoring and surveillance are required to correctly assess the current status of marine environment, paying attention not only to traditional chemical parameters, but also to the so-called “emerging pollutants”. Among these, marine biotoxins represent a serious hazard, because they can accumulate in fish and mussels and enter the food chain. The detection of marine toxins released by algae has mostly been directed on the analysis of fish/shellfish homogenate rather than seawater samples. To fill this gap, in this work, we propose the re-modeling of ELISA kits to set-up non-automated direct competitive ELIMC (Enzyme-Linked Immuno-Magnetic Colorimetric) assays for the detection of Domoic Acid (DA), Okadaic Acid (OA) and Saxitoxin (STX) in seawater. For each toxin, linear working range, sensitivity and detection limit (0.03, 0.05, 0.01 ng/mL for DA, OA and STX, respectively) of the ELIMC assays were calculated. After evaluation of seawater matrix effect and recovery study, ELIMC assays were integrated within a novel automated networked system (ASMAT - Analytical System for Marine Algal Toxins) based on the micro Loop Flow Reactor (μLFR) technology, suitable for on-line monitoring of marine toxins. Once established the best operational conditions, ASMAT was calibrated towards DA, OA and STX verifying its capability to detect sub-ppb levels of the target toxins. Analysis of DA, OA and STX in real samples of marine water, sampled in Alonnisos (Greece) were carried out in laboratory using ELIMC assays, ASMAT and ELISA Kits, for a comparative evaluation. Finally, on-line suitability of ASMAT as an early warning alarm system was demonstrated by a field installation within a floating platform in the port of La Spezia (Italy). © 2018 Elsevier B.V.

A UPLC-MS/MS assay, employing a reversed-phase separation, has been applied to the analysis of a number of common amino acids and biogenic amines in rat urine. Analytes were derivatised, using 6‑aminoquinolyl‑N‑hydroxysuccinimidyl carbamate (AccQTag Ultra™). Derivatisation with this reagent, by increasing the hydrophobicity of the analytes, enables better retention by improving reversed-phase chromatographic properties and also improves ionisation efficiency to enhance MS-detection. The method allows for the determination of 38 amino compounds in 7.5 min, including baseline resolution of critical isomers. The assay has been validated for the absolute quantification of 29 amino compounds in rat urine, over a concentration range of 0.6–200 μM, for the purpose of exploratory metabolite phenotyping. Acceptable linearity (R2 > 0.995) and intra- and inter-day accuracy (

Made available in DSpace on 2019-10-06T17:00:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 An innovative method is validated for the analysis of ertapenem sodium by capillary electrophoresis using potassium phosphate buffer 10 mM pH 7 and 15 kV voltage, in the concentration range of 70 to 120 μg mL-1. Ertapenem had a migration time of 3.15 minutes and the linearity curve was y = 2281.7 x - 24495 with a R2 = 0.9994. Thus, we propose a routine analysis method that meets the principles of green analytical chemistry for the routine analysis of ertapenem sodium by capillary electrophoresis. UNESP-Univ Estadual Paulista Faculdade de Ciências Farmacêuticas KU Leuven-University of Leuven Department of Pharmaceutical and Pharmacological Sciences Pharmaceutical Analysis UNESP-Univ Estadual Paulista Faculdade de Ciências Farmacêuticas

Being traditionally utilized mainly as appetizers and herbal teas as well as used to ease abdominal pains, colds and gastrointestinal issues, the genusSalviaL. has gained significant consideration owing to its remarkable phytochemicals and industrial importance. The present study aimed to develop and validate an LC–MS/MS method for the qualitative and quantitative investigation of 19 fingerprint phytochemicals in six endemicSalviaspecies. The validation parameters of the developed LC–MS/MS method were repeatability (intermediate precision), recovery (accuracy), limits of detection and quantification, linearity and uncertainty (U% at 95% confidence level (k = 2)). Reversed-phase HPLC separation and mass spectrometry parameters were optimized for each analyte. Ethanol extracts of the studiedSalviaspecies collected in three consecutive years were screened for their fingerprint phytochemicals by using the developed and validated LC–MS/MS method. Moreover, studiedSalviaspecies were subjected to multivariate analysis such as principal component analysis techniques to demonstrate the variabilities in phytochemical contents by years and parts of the samples. Roots, flowers, leaves, branches and whole plant of theSalviaspecies collected in 2015, 2016 and 2017 were used for the analyses. It was observed that the roots and branches ofSalviaspecies were similar in terms of their salvianolic acid A, caffeic acid, and 6,7-dehydroroyleanone components. Accordingly, apigenin, rosmarinic acid, luteolin 7-O-glucoside, caffeic acid, salvianolic acid B, and 6,7-dehydroroyleanone were notable phytochemicals that were present in the studiedSalviaspecies.

Background: vegetative diversity is based on different climate and geographical origins. In terms of beekeeping, herbal diversity is strongly correlated to the production of a wide variety of honey. Therefore, based on the existing plant diversity in each country, multiple honey varieties are produced with different health characteristics. While beekeeping potential and consumption preferences are reflected in products' variety, this leads to an increase in the region's economy and extensive export. In the last years, monofloral honey has gained interest from consumers and especially in the medicinal field due to the presence of phytochemicals which are directly linked to health benefits, wound healing, antioxidant, anticancer and anti-inflammatory activities. Scope and approach: this review aims to highlight the physicochemical properties, mineral profiles and antioxidant activities of selected monofloral honeys based on their botanical and geographical origin. Moreover, this review focuses on the intercorrelation between monofloral honey's antioxidant compounds and in vitro and in vivo activities, focusing on the apoptosis and cell proliferation inhibition in various cell lines, with a final usage of honey as a potential therapeutic product in the fight towards reducing tumor growth. Key findings and conclusions: multiple studies have demonstrated that monofloral honeys have different physicochemical structures and bioactive compounds. Useful chemical markers to distinguish between monofloral honeys were evidenced, such as: 2-methoxybenzoic acid and trimethoxybenzoic acid are distinctive to Manuka honey while 4-methoxyphenylacetic acid is characteristic to Kanuka honey. Furthermore, resveratrol, epigallocatechin and pinostrobin are markers distinct to Sage honey, whereas carvacrol and thymol are found in Ziziphus honey. Due to their polyphenolic profile, monofloral honeys have significant antioxidant activity, as well as antidiabetic, antimicrobial and anticancer activities. It was demonstrated that Pine honey decreased the MDA and TBARS levels in liver, kidney, heart and brain tissues, whereas Malicia honey reduced the low-density lipoprotein level. Consumption of Clover, Acacia and Gelam honeys reduced the weight and adiposity, as well as trygliceride levels. Furthermore, the antiproliferative effect of chrysin, a natural flavone in Acacia honey, was demonstrated in human (A375) and murine (B16-F1) melanoma cell lines, whereas caffeic acid, a phenolic compound found in Kelulut honey, proves to be significant candidate in the chemoprevention of colon cancer. Based on these features, the use of hiney in the medicinal field (apitherapy), and the widespread usage of natural product consumption, is gaining interest by each year., [PN-III-P1-1.2-PCCDI-2017-0056-2PCCDI], The publication was funded by PN-III-P1-1.2-PCCDI-2017-0056-2PCCDI.

Graphical abstract, The recent approval of messenger RNA (mRNA)-based vaccines to combat the SARS-CoV-2 pandemic highlights the potential of both conventional mRNA and self-amplifying mRNA (saRNA) as a flexible immunotherapy platform to treat infectious diseases. Besides the antigen it encodes, mRNA itself has an immune-stimulating activity that can contribute to vaccine efficacy. This self-adjuvant effect, however, will interfere with mRNA translation and may influence the desired therapeutic outcome. To further exploit its potential as a versatile therapeutic platform, it will be crucial to control mRNA’s innate immune-stimulating properties. In this regard, we describe the mechanisms behind the innate immune recognition of mRNA and provide an extensive overview of strategies to control its innate immune-stimulating activity. These strategies range from modifications to the mRNA backbone itself, optimization of production and purification processes to the combination with innate immune inhibitors. Furthermore, we discuss the delicate balance of the self-adjuvant effect in mRNA vaccination strategies, which can be both beneficial and detrimental to the therapeutic outcome.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the relationship between alpha-lipoic acid (ALA) and the risk of insulin autoimmune syndrome (IAS). The Panel was also asked to advise on the dose below which ALA added to foods is not expected to cause IAS. A review of all possible adverse effects associated with consumption of ALA was not requested. This mandate refers to the procedure under Article 8(2) of Regulation (EC) No 1925/2006 on addition of vitamins, minerals and certain other substances to foods. No pre-established rule exists for the evaluation of the safety of foods when classical toxicity tests cannot be used, e.g. for autoimmune diseases. Published scientific evidence was retrieved through comprehensive literature searches, particularly 49 case reports in which IAS developed following ALA consumption. In all cases, IAS resolved after a few weeks to months when ALA was discontinued. No publication linking the intake of ALA naturally occurring in foods to IAS was identified. The Panel concludes that the consumption of ALA added to foods, including food supplements, is likely to increase the risk of developing IAS in individuals with certain genetic polymorphisms, who cannot be readily identified without genetic testing. The plausible mechanism of such an effect has not yet been fully elucidated. The incidence of IAS in Europe is low and likely lower than in Japan where it has been estimated to be 0.017 per 100,000 inhabitants in 2017-2018. Considering the limited data available, the risk associated with the development of IAS following ALA consumption cannot be quantified precisely. An ALA dose below which IAS is not expected to occur is likely to vary between individuals and cannot be determined from the available data. (c) 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2′-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2′-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease., Graphical abstract Image 1, Highlights • Oxidatively damaged nucleic acids are implicated in the pathogenesis of disease. • LC-MS/MS, comet assay and ELISA are often used to study oxidatively damaged DNA. • Urinary oxidatively damaged nucleic acids non-invasively reflect oxidative stress. • DNA adductomics will aid understanding the role of ROS damaged DNA in disease.

The present article examines recently published literature on lipids, mainly focusing on research involving glycero-, glycerophospho- and sphingo-lipids. The primary aim is identification of distinct profiles in biologic lipidomic systems by ultra-high-performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS, tandem MS) with multivariate data analysis. This review specifically targets lipid biomarkers and disease pathway mechanisms in humans and artificial targets. Different specimen matrices such as primary blood derivatives (plasma, serum, erythrocytes, and blood platelets), faecal matter, urine, as well as biologic tissues (liver, lung and kidney) are highlighted.

Amikacin, gentamicin and tobramycin are aminoglycoside antibiotics mostly used for the treatment of a wide range of aerobic Gram negative bacteria. Lack of a UV chromophore as well as their polar and non-volatile nature make direct determination of these aminoglycosides by conventional LC and GC very challenging. Existing analytical methodology is either expensive, complicated or time consuming. In this work, a thin layer chromatography – flame ionization method was developed for fast analysis of these aminoglycosides in pharmaceutical formulations. Paromomycin was used as internal standard for quantitation. Development of the rod with one single solvent mixture was sufficient to achieve the desired separation of target components. Good determination coefficients (>0.997) were achieved for all target analytes. Repeatability, recovery and detection limits were determined and reported. A total of 5 samples in duplicate can be analyzed simultaneously within 25 s. This method offers the advantage of being able to analyze these aminoglycosides without any need for derivatization or laborious coloration. The developed method was applied to the analysis of some pharmaceutical commercial samples.

Vincristine (VCR) is frequently used in pediatric oncology and can be administered intravenously through push injections or 1 h infusions. The effects of administration duration on population pharmacokinetics (PK) are unknown. We described PK differences related to administration duration and the relation between PK and VCR-induced peripheral neuropathy (VIPN). PK was assessed in 1&ndash, 5 occasions (1&ndash, 8 samples in 24 h per occasion). Samples were analyzed using high-performance liquid chromatography/tandem mass spectrometry. Population PK of VCR and its relationship with administration duration was determined using a non-linear mixed effect. We estimated individual post-hoc parameters: area under the concentration time curve (AUC) and maximum concentration (Cmax) in the plasma and peripheral compartment. VIPN was assessed using Common Terminology Criteria for Adverse Events (CTCAE) and the pediatric-modified total neuropathy score (ped-mTNS). Overall, 70 PK assessments in 35 children were evaluated. The population estimated that the intercompartmental clearance (IC-Cl), volume of the peripheral compartment (V2), and Cmax were significantly higher in the push group. Furthermore, higher IC-Cl was significantly correlated with VIPN development. Administration of VCR by push led to increased IC-Cl, V2, and Cmax, but were similar to AUC, compared to 1 h infusions. Administration of VCR by 1 h infusions led to similar or higher exposure of VCR without increasing VIPN.

Vitamin K and its essential role in coagulation (vitamin K [Koagulation]) have been well established and accepted the world over. Many countries have a Recommended Daily Intake (RDI) for vitamin K based on early research, and its necessary role in the activation of vitamin K-dependent coagulation proteins is known. In the past few decades, the role of vitamin K-dependent proteins in processes beyond coagulation has been discovered. Various isoforms of vitamin K have been identified, and vitamin K2 specifically has been highlighted for its long half-life and extrahepatic activity, whereas the dietary form vitamin K1 has a shorter half-life. In this review, we highlight the specific activity of vitamin K2 based upon proposed frameworks necessary for a bioactive substance to be recommended for an RDI. Vitamin K2 meets all these criteria and should be considered for a specific dietary recommendation intake.

Nutrients 12(6), 1852 (2020). doi:10.3390/nu12061852 special issue: "Special Issue "Physiology and Pathology of Vitamin K Intake" / Special Issue Editors: Dr. Maria Fusaro, Prof. Dr. Maurizio Gallieni", Published by MDPI, Basel

Oxidative stress is associated with a myriad of diseases including pregnancy pathologies with long-term cardiovascular repercussions for both the mother and baby. Aberrant redox signalling coupled with deficient antioxidant defence leads to chronic molecular impairment. Abnormal placentation has been considered the primary source for reactive species; however, placental dysfunction has been deemed secondary to maternal cardiovascular maladaptation in pregnancy. While various therapeutic interventions, aimed at combating deregulated oxidative stress during pregnancy have shown promise in experimental models, they often result as inconclusive or detrimental in clinical trials, warranting the need for further research to identify candidates. The strengths and limitations of current experimental methods in redox research are discussed. Assessment of redox status and oxidative stress in experimental models and in clinical practice remains challenging; the state-of-the-art of computational models in this field is presented in this review, comparing static and dynamic models which provide functional information such as protein-protein interactions, as well as the impact of changes in molecular species on the redox-status of the system, respectively. Enhanced knowledge of redox biology in during pregnancy through computational modelling such as generation of Systems Biology Markup Language model which integrates existing models to a larger network in the context of placenta physiology.

During the last years, supercritical fluid chromatography (SFC) has attracted a continuously growing number of users. Thanks to the introduction of state-of-the-art equipment, this technique has allowed to run three-to-five times faster separations than in high-performance liquid chromatography (HPLC) on columns packed with particles of comparable dimension, at lower pressure drops and without loss of efficiency. Thanks to its high versatility, its high-throughput screening capability, and “green” character of the mobile phase, SFC has become particularly attractive for the separation of chiral drugs in pharmaceutical industries. In this review, we will consider the latest applications of SFC for the analysis of compounds of pharmaceutical interest and/or with biological activity essentially covering main achievements of the last 3 years. We also focus on some very recent, remarkable applications of SFC in ultrafast enantioseparations on chiral columns of the latest generation. Technical improvements needed on commercial equipment to increase the competitiveness of SFC towards highly efficient enantioseparations are discussed.

Micronutrients are essential to health at every life stage and their deficiencies are associated with increased incidence of various pathophysiological states and poor quality of life. Efficient methods are therefore needed to monitor micronutrient status of individuals and to improve evidenced-based recommendations for populations. This review (i) reports current approaches to assess the vitamin and mineral status in humans, (ii) summarizes current analytical advantages and shortcomings and (iii) provides practical information for both nutrition research and nutritional status diagnostics. Future analytical perspectives are discussed in relation to micronutrient profiling, analytical sensitivity, and miniaturized technologies. (C) 2018 Elsevier B.V. All rights reserved.

Micronutrients are essential to health at every life stage and their deficiencies are associated with increased incidence of various pathophysiological states and poor quality of life. Efficient methods are therefore needed to monitor micronutrient status of individuals and to improve evidenced-based recommendations for populations. This review (i) reports current approaches to assess the vitamin and mineral status in humans, (ii) summarizes current analytical advantages and shortcomings and (iii) provides practical information for both nutrition research and nutritional status diagnostics. Future analytical perspectives are discussed in relation to micronutrient profiling, analytical sensitivity, and miniaturized technologies. (C) 2018 Elsevier B.V. All rights reserved.

Background: Therapeutic drug monitoring is useful in the treatment of tuberculosis to assure adequate exposure, minimize antibiotic resistance, and reduce toxicity. Salivary therapeutic drug monitoring could reduce the risks, burden, and costs of blood-based therapeutic drug monitoring. This systematic review compared human pharmacokinetics of antituberculosis drugs in saliva and blood to determine if salivary therapeutic drug monitoring could be a promising alternative.Methods: On December 2, 2016, PubMed and the Institute for Scientific Information Web of Knowledge were searched for pharmacokinetic studies reporting human salivary and blood concentrations of antituberculosis drugs. Data on study population, study design, analytical method, salivary Cmax, salivary area under the time-concentration curve, plasma/serum Cmax, plasma/serum area under the time-concentration curve, and saliva-plasma or saliva-serum ratio were extracted. All included articles were assessed for risk of bias.Results: In total, 42 studies were included in this systematic review. For the majority of antituberculosis drugs, including the first-line drugs ethambutol and pyrazinamide, no pharmacokinetic studies in saliva were found. For amikacin, pharmacokinetic studies without saliva-plasma or saliva-serum ratios were found.Conclusions: For gatifloxacin and linezolid, salivary therapeutic drug monitoring is likely possible due to a narrow range of saliva-plasma and saliva-serum ratios. For isoniazid, rifampicin, moxifloxacin, ofloxacin, and clarithromycin, salivary therapeutic drug monitoring might be possible; however, a large variability in saliva-plasma and saliva-serum ratios was observed. Unfortunately, sali-vary therapeutic drug monitoring is probably not possible for doripenem and amoxicillin/clavulanate, as a result of very low salivary drug concentrations.

Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, containing a cleavable N-terminal signal peptide and a C-terminal transmembrane helix that anchors the protein to the ER, followed by a short cytoplasmic tail. DcUGT2 is found to be heavily glycosylated. Truncated DcUGT2 proteins synthesized in yeast indicate the presence of an internal ER-targeting signal. The cleavable N-terminal signal peptide is shown to be essential for the activity of DcUGT2, whereas the transmembrane helix/cytoplasmic domains, although important, are not crucial for its catalytic function., Carminic acid is a widely applied red colorant that is still harvested from insects because its biosynthesis is not fully understood. Here, the authors identify and characterize a membrane-bound C-glucosyltransferase catalyzing the final step during carminic acid biosynthesis.

Membrane lipid composition varies greatly within submembrane compartments, different organelle membranes, and also between cells of different cell stage, cell and tissue types, and organisms. Environmental factors (such as diet) also influence membrane composition. The membrane lipid composition is tightly regulated by the cell, maintaining a homeostasis that, if disrupted, can impair cell function and lead to disease. This is especially pronounced in the brain, where defects in lipid regulation are linked to various neurological diseases. The tightly regulated diversity raises questions on how complex changes in composition affect overall bilayer properties, dynamics, and lipid organization of cellular membranes. Here, we utilize recent advances in computational power and molecular dynamics force fields to develop and test a realistically complex human brain plasma membrane (PM) lipid model and extend previous work on an idealized, "average" mammalian PM. The PMs showed both striking similarities, despite significantly different lipid composition, and interesting differences. The main differences in composition (higher cholesterol concentration and increased tail unsaturation in brain PM) appear to have opposite, yet complementary, influences on many bilayer properties. Both mixtures exhibit a range of dynamic lipid lateral inhomogeneities ("domains"). The domains can be small and transient or larger and more persistent and can correlate between the leaflets depending on lipid mixture, Brain or Average, as well as on the extent of bilayer undulations.

Solid phase microextraction (SPME) is a versatile, non-exhaustive sample preparation tool that has been demonstrated to be well-suited for facile and effective analysis of a broad range of compounds in a plethora of studies. A growing number of reports describing diverse SPME workflows for novel investigations in a variety of fields, such as flavor and fragrance investigations, environmental studies, and diverse bioanalytical applications, among others, corroborate the applicability of this microextraction tool in the analytical sciences

Terminalia laxiflora , Terminalia brownii and Anogeissus leiocarpus are used as decoctions, macerations, infusions and fumigations in East and West African traditional medicine for treatment of infectious diseases and their symptoms. Using this ethnopharmacological information as a guideline for our research and owing to the fact that these species have not been subjected to in depth antibacterial and phytochemical studies, thirty-nine extracts of various polarities of the stem bark, stem wood and roots were studied for growth inhibitory effects against the human pathogenic bacteria Staphylococcus epidermidis , Staphylococcus aureus , Micrococcus luteus and Pseudomonas aeruginosa . Our results indicate that the studied species contain antibacterial compounds of a wide range of polarities. All polar root extracts of T. laxiflora and various polar extracts of T. brownii roots, including hot water decoctions, gave broad-spectrum antibacterial effects and low MIC values of 39 μg/ml. The main ellagitannins in an ethyl acetate extract of the root of T. laxiflora were found to be corilagin and its derivative and punicalagin. A methanol extract of the roots of T. brownii contained methyl-(S)-flavogallonate and its derivative as the main identified ellagitannins. Moreover, both Terminalia species were found to contain ellagic acid xylopyranoside and methyl ellagic acid xyloside and pure ellagic acid was present in T. brownii . Pure punicalagin did not give as low MIC as an ethyl acetate extract of the roots of T. laxiflora , containing punicalagin as one of its main compounds, although this ellagitannin totally inhibited the growth of S. aureus at 125 μg/ml and P. aeruginosa at 500 μg/ml. Similarly, pure ellagic and gallic acid gave higher MIC values than the methanolic root extract of T. brownii against S. aureus and P. aeruginosa . Moreover, a Sephadex LH-20 fraction of the methanolic extract of the roots of T. brownii , enriched with methyl-(S)-flavogallonate and its isomer, gave higher MIC values than the crude methanolic extract. These results suggest that the polyphenols in the extracts might act synergistically with each other. A methanolic soxhlet extract of the roots of A. leiocarpus , containing ampelopsin, aromadendrin, taxifolin, pinosylvin and 4′-methylpinosylvin gave a low MIC value of 39 μg/ml against all bacterial strains used in this investigation. Our results demonstrate that the roots, stem bark and stem wood of T. brownii , T. laxiflora and A. leiocarpus are rich sources of (new) antimicrobial compounds and justify the uses of these plants for treatment of infections in African traditional medicine.

Paracetamol (acetaminophen, APAP) is the most frequently used analgesic drug worldwide. However, patients in several specific populations can have an increased exposure to toxic APAP metabolites. Therefore, APAP-protein adducts have been proposed as an alternative marker for the assessment of APAP intoxications and as an effective tool to study and steer APAP treatment in patients with an increased risk of APAP-induced liver damage. These adducts have been determined in plasma or serum as a matrix. Blood microsampling allows the determination of a variety of analytes, including protein adducts, in a drop of blood, facilitating convenient followup of patients in a home-sampling context, as well as repeated sampling of pediatric patients. We therefore evaluated the use of blood-based volumetric microsamples for the quantification of APAP-protein adducts. Quantitative methods for the determination of APAP-protein adducts in dried blood and dried plasma volumetric absorptive microsamples were developed and validated. Also a preliminary evaluation of pediatric patient dried blood microsamples was conducted. Method validation encompassed the evaluation of selectivity, carry over, calibration model, accuracy and precision, matrix effect, recovery and the effect of the hematocrit on the recovery, dilution integrity, and stability. All pre-set acceptance criteria were met, except for stability. Spiking of blank blood with APAP revealed a concentration-dependent ex vivo formation of APAP-protein adducts, resulting in a response for the measurand APAP-Cys, with an apparent role for the red blood cell fraction. Analysis of authentic samples, following intake of APAP at therapeutic dosing, revealed much higher APAP-Cys concentrations in dried blood vs. dried plasma samples, making interpretation of the results in the context of published intervals difficult. In addition, in contrast to what was observed during method validation, the data obtained for the patient samples showed a high and unacceptable variation. We conclude that, for a combination of reasons, dried blood is not a suitable matrix for the quantification of APAP-protein adducts via the measurement of the APAP-Cys digestion product. The collection of plasma or serum, either in the form of a liquid sample or a dried microsample for this purpose is advised.

Human milk oligosaccharides have been recognized as an important, functional biomolecule in mothers’ milk. Moreover, these oligosaccharides have been recognized as the third most abundant component of human milk, ranging from 10−15 g/L in mature milk and up to and over 20 g/L reported in colostrum. Initially, health benefits of human milk oligosaccharides were assigned via observational studies on the differences between breastfed and bottle fed infants. Later, pools of milk oligosaccharides were isolated and used in functional studies and in recent years more specific studies into structure−function relationships have identified some advanced roles for milk oligosaccharides in the healthy development of infants. In other research, the levels, diversity, and complexity of human milk oligosaccharides have been studied, showing a wide variation in results. This review gives a critical overview of challenges in the analysis of human milk oligosaccharides. In view of the myriad functions that can be assigned, often to specific structures or classes of structures, it is very relevant to assess the levels of these structures in the human milk correctly, as well as in other biological sample materials. Ultimately, the review makes a case for a comparative, inter-laboratory study on quantitative human milk oligosaccharide analysis in all relevant biological samples.

Quercetin has been shown to alleviate mucosal damage and modulate the glutathione (GSH) redox system in the colon of rodents. In the current study, we assessed whether quercetin was able to mitigate small intestinal dysfunction in weaned pigs. Here, 224 weaned piglets were fed a diet containing quercetin at either 0, 100, 300, or 900 mg/kg diet until d14 post-weaning, followed by a common basal diet until d42. Eight animals per treatment were sampled at d5 and d14 post-weaning. In these animals, the small intestinal histomorphology, barrier function, and protein abundance of occludin, caspase-3, and proliferating cell nuclear antigen were assessed. None of these parameters were affected, and neither did quercetin improve performance up to d42 post-weaning. The GSH redox system was evaluated in blood, small intestinal mucosa, and liver. Quercetin did not affect the glutathione peroxidase, glutathione reductase, and glutamate&ndash, cysteine ligase activity in these tissues. In contrast, the hepatic glutathione transferase (GST) activity was significantly increased by quercetin supplementation at d5 post-weaning of 100, 300, and 900 mg/kg. Importantly, d5 was characterized by a more oxidized GSH redox status. To conclude, dietary quercetin had little effect on the small intestine, but did upregulate hepatic GST in the occurrence of redox disturbance.

Liquid chromatography (LC) hyphenated to mass spectrometry is currently the most widely used means of determining metabolic phenotypes via both untargeted and targeted analysis. At present a range of analytical separations, including reversed-phase, hydrophilic interaction and ion-pair LC are employed to maximise metabolome coverage with ultra (high) performance liquid chromatography (UHPLC) increasingly displacing conventional high performance liquid chromatography because of the need for short analysis times and high peak capacity in such applications. However, it is widely recognized that these methodologies do not entirely solve the problems facing researchers trying to perform comprehensive metabolic phenotyping and in addition to these "routine" approaches there are continuing investigations of alternative separation methods including 2-dimensional/multi column approaches. These involve either new stationary phases or multidimensional combinations of the more conventional materials currently used, as well as application of miniaturization or "new" approaches such as supercritical HP and UHP- chromatographic separations. There is also a considerable amount of interest in the combination of chromatographic and ion mobility separations, with the latter providing both an increase in resolution and the potential to provide additional structural information via the determination of molecular collision cross section data. However, key problems remain to be solved including ensuring quality, comparability across different laboratories and the ever present difficulty of identifying unknowns.