ABCG2 transporter plays a key role in the biodistribution of melatonin and its main metabolites.

The ATP-binding cassette G2 (ABCG2) is an efflux transporter expressed in the apical membrane of cells from a large number of tissues, directly affecting bioavailability, tissue accumulation, and secretion into milk of both xenobiotics and endogenous compounds. The aim of this work was to characterize the role of ABCG2 in the systemic distribution and secretion into milk of melatonin and its main metabolites, 6-hydroxymelatonin, and 6-sulfatoxymelatonin. For this purpose, we first showed that these three molecules are transported by this transporter using in vitro transepithelial assays with MDCK-II polarized cells transduced with different species variants of ABCG2. Second, we tested the in vivo effect of murine Abcg2 in the systemic distribution of melatonin and its metabolites using wild-type and Abcg2-/- mice. Our results show that after oral administration of melatonin, the plasma concentration of melatonin metabolites in Abcg2-/-  mice was between 1.5 and 6-fold higher compared to the wild-type mice. We also evaluated in these animals differences in tissue accumulation of melatonin metabolites. The most relevant differences between both types of mice were found for small intestine and kidney (>sixfold increase for 6-sulfatoxymelatonin in Abcg2-/-  mice). Finally, melatonin secretion into milk was also affected by the murine Abcg2 transporter, with a twofold higher milk concentration in wild-type compared with Abcg2-/-  lactating female mice. In addition, melatonin metabolites showed a higher milk-to-plasma ratio in wild-type mice. Overall, our results show that the ABCG2 transporter plays a critical role in the biodistribution of melatonin and its main metabolites, thereby potentially affecting their biological and therapeutic activity.

Longitudinal Associations between Prenatal Exposure to Phthalates and Steroid Hormones in Maternal Hair Samples from the SEPAGES Cohort.

We assessed phthalate-hormone associations in 382 pregnant women of the new-generation SEPAGES cohort (2014-2017, France) using improved exposure and outcome assessments. Metabolites from seven phthalate compounds and the replacement di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) were measured in within-subject pools of repeated urine samples collected at the second and third pregnancy trimesters (≈21 samples/trimester). Metabolites from five steroid hormones were measured in maternal hair samples collected at delivery, reflecting cumulative levels over the previous weeks to months. Adjusted linear regression and Bayesian weighted quantile sum (BWQS) mixture models were performed. Each doubling in third-trimester urinary mono-benzyl phthalate (MBzP) concentrations was associated with an average increase of 13.3% (95% CI: 2.65, 24.9) for ∑cortisol, 10.0% (95% CI: 0.26, 20.7) for ∑cortisone, 17.3% (95% CI: 1.67, 35.4) for 11-dehydrocorticosterone, and 16.2% (95% CI: 2.20, 32.1) for testosterone, together with a suggestive 10.5% (95% CI: -1.57, 24.1) increase in progesterone levels. Each doubling in second-trimester urinary di-isononyl phthalate (DiNP) concentrations was inversely associated with testosterone levels (-11.6%; 95% CI: -21.6, -0.31). For most hormones, a nonsignificant trend toward a positive phthalate mixture effect was observed in the third but not in the second trimester. Our study showed that exposure to some phthalate metabolites, especially MBzP, may affect adrenal and reproductive hormone levels during pregnancy.

Determination of well-being-related markers in nails by liquid chromatography tandem mass spectrometry.

Well-being is a multifactorial positive state that is highly influenced by some endogenous molecules that control happiness and euphoric feelings. These molecules, e.g., neurotransmitters, hormones and their derivatives, play a crucial role in metabolism and may be referred to as "well-being-related markers". The deregulation of well-being-related markers can lead to organism malfunctions and life-threatening states. In this research, we aimed to evaluate the potential of nails for the chronic production of several well-being-related markers. For this purpose, we developed an LCMS /MS-based method for the determination of 10 well-being-related markers, including melatonin, serotonin, cortisol, kynurenine and several precursors and metabolites. The method was optimized regarding different analytical steps: required sample amount, extraction time, number of required extractions, preconcentration, injection volume and MS conditions. Method validation was performed by two different approaches: (i) using surrogate nail matrix and (ii) using authentic nail samples by standard additions. The method was found to be linear in the expected endogenous range and sensitive enough to determine the low endogenous concentration levels in nails. Accuracy and precision were appropriate in both validation approaches. As proof of concept, the method was used (i) to correlate fingernail and toenail levels for all metabolites in 22 volunteers, (ii) to establish the endogenous concentration range of all metabolites in females (n = 50) and males (n = 34) and (iii) to correlate the metabolite levels with age. For some metabolites, the calculated ranges have been reported for the first time. In summary, the present strategy to evaluate well-being-related markers in nails may be a useful tool for the evaluation of the production of these important compounds with high potential for a wide range of clinical purposes.

Formation Mechanism of Inter-Crosslink in DNA by Nitrogen Oxides Pollutants through A Diazonium Intermediate.

Outdoor air pollution is a mixture of multiple atmospheric pollutants, among which nitrogen oxide (NOx) stands out due to its association with several diseases. NOx reactivity can conduct to DNA damage as severe as interstrand crosslinks (ICL) formation, that in turn is able to block DNA replication and transcription. Experimental studies have suggested that the ICL formation due to NOx is realized through a diazonium intermediate (DI). In this work, we have modeled the DI structure, including a DNA double-strand composed of two base pairs GC/CG, being diazotized as one of the guanine nucleotides. The structural stability of DNA with DI lesion was essayed through 500 ns molecular dynamics simulations. It was found that the DNA structure of the oligonucleotide is stable when the DI is present since the loss of a Guanine-Cytosine hydrogen bond is replaced by the presence of two cation-π interactions. Additionally, we have studied the mechanism of formation of a crosslink between the two guanine nucleobases from the modeled DI by carrying out DFT calculations at the M06-L/DNP+ level of theory. Our results show that the mechanism is thermodynamically favored by a strong stabilization of the ICL product, and the process is kinetically viable since its limiting stage is accessible.

Nail Melatonin Content: A Suitable Non-Invasive Marker of Melatonin Production.

Melatonin plays multiple physiological roles in the human body. Evaluation of melatonin production by the determination of urinary 6-sulfatoxymelatonin in 24-h samples has important drawbacks which hinder the successful evaluation of melatonin production in large cohorts. Here, we evaluated the potential of nail analysis for estimating melatonin production. Firstly, mass spectrometry methodology for the determination of melatonin in nails was optimized and successfully validated. The method was found to be linear in the range 6.5-830 fg/mg with intraday and interday accuracy in the range 100-104 %, precision below 15 % and a LOD of 3.5 fg/mg. Secondly, nail melatonin concentrations from 84 volunteers (age 5-96) were determined. The expected correlation between melatonin and age was obtained (correlation coefficient -0.615; p < 0.001). Additionally, we showed that fingernails are preferable to toenails to determine nail melatonin content. Finally, fingernails collected for 180 days after melatonin administration (two volunteers, 1.9 mg/night during 5 days) were analyzed. Nail melatonin concentrations immediately rose after administration and went back to pre-administration values after ≈100 days in both volunteers. Our results suggest that melatonin determination in nails is a suitable non-invasive tool for the estimation of global melatonin production. Due to the easy collection and storage of nails, the long-term information obtained and the multiple functions of melatonin, nail melatonin content might complement dim light melatonin onset, which is commonly measured from plasma/saliva samples, paving the way for melatonin research.

Metabolic Signatures Associated with Severity in Hospitalized COVID-19 Patients.

The clinical evolution of COVID-19 pneumonia is poorly understood. Identifying the metabolic pathways that are altered early with viral infection and their association with disease severity is crucial to understand COVID-19 pathophysiology, and guide clinical decisions. This study aimed at assessing the critical metabolic pathways altered with disease severity in hospitalized COVID-19 patients. Forty-nine hospitalized patients with COVID-19 pneumonia were enrolled in a prospective, observational, single-center study in Barcelona, Spain. Demographic, clinical, and analytical data at admission were registered. Plasma samples were collected within the first 48 h following hospitalization. Patients were stratified based on the severity of their evolution as moderate (N = 13), severe (N = 10), or critical (N = 26). A panel of 221 biomarkers was measured by targeted metabolomics in order to evaluate metabolic changes associated with subsequent disease severity. Our results show that obesity, respiratory rate, blood pressure, and oxygen saturation, as well as some analytical parameters and radiological findings, were all associated with disease severity. Additionally, ceramide metabolism, tryptophan degradation, and reductions in several metabolic reactions involving nicotinamide adenine nucleotide (NAD) at inclusion were significantly associated with respiratory severity and correlated with inflammation. In summary, assessment of the metabolomic profile of COVID-19 patients could assist in disease severity stratification and even in guiding clinical decisions.

Sperm physiology and in vitro fertilising ability rely on basal metabolic activity: insights from the pig model.

Whether basal metabolic activity in sperm has any influence on their fertilising capacity has not been explored. Using the pig as a model, the present study investigated the relationship of energetic metabolism with sperm quality and function (assessed through computer-assisted sperm analysis and flow cytometry), and fertility (in vitro fertilisation (IVF) outcomes). In semen samples from 16 boars, levels of metabolites related to glycolysis, ketogenesis and Krebs cycle were determined through a targeted metabolomics approach using liquid chromatography-tandem mass spectrometry. High-quality sperm are associated to greater levels of glycolysis-derived metabolites, and oocyte fertilisation and embryo development are conditioned by the sperm metabolic status. Interestingly, glycolysis appears to be the preferred catabolic pathway of the sperm giving rise to greater percentages of embryos at day 6. In conclusion, this study shows that the basal metabolic activity of sperm influences their function, even beyond fertilisation.

Loss of dyskerin facilitates the acquisition of metastatic traits by altering the mevalonate pathway.

The initial dissemination of cancer cells from many primary tumors implies intravasation to lymphatic nodes or blood vessels. To investigate the mechanisms involved, we analyzed the expression of small non-coding RNAs in cutaneous squamous cell carcinoma (cSCC), a prevalent tumor that mainly spreads to lymph nodes. We report the reduced expression of small nucleolar RNAs in primary cSCCs that metastasized when compared to non-metastasizing cSCCs, and the progressive loss of DKC1 (dyskerin, which stabilizes the small nucleolar RNAs) along the metastasis. DKC1 depletion in cSCC cells triggered lipid metabolism by altering the mevalonate pathway and the acquisition of metastatic traits. Treatment of DKC1-depleted cells with simvastatin, an inhibitor of the mevalonate pathway, blocked the expression of proteins involved in the epithelial-to-mesenchymal transition. Consistently, the expression of the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 1 was associated with pathological features of high metastatic risk in cSCC patients. Our data underpin the relevance of the mevalonate metabolism in metastatic dissemination and pave the possible incorporation of therapeutic approaches among the antineoplastic drugs used in routine patient care.

Human Metastatic Cholangiocarcinoma Patient-Derived Xenografts and Tumoroids for Preclinical Drug Evaluation.

PURPOSE: Cholangiocarcinoma (CCA) is usually diagnosed at advanced stages, with limited therapeutic options. Preclinical models focused on unresectable metastatic CCA are necessary to develop rational treatments. Pathogenic mutations in IDH1/2, ARID1A/B, BAP1, and BRCA1/2 have been identified in 30%-50% of patients with CCA. Several types of tumor cells harboring these mutations exhibit homologous recombination deficiency (HRD) phenotype with enhanced sensitivity to PARP inhibitors (PARPi). However, PARPi treatment has not yet been tested for effectiveness in patient-derived models of advanced CCA. EXPERIMENTAL DESIGN: We have established a collection of patient-derived xenografts from patients with unresectable metastatic CCA (CCA_PDX). The CCA_PDXs were characterized at both histopathologic and genomic levels. We optimized a protocol to generate CCA tumoroids from CCA_PDXs. We tested the effects of PARPis in both CCA tumoroids and CCA_PDXs. Finally, we used the RAD51 assay to evaluate the HRD status of CCA tissues. RESULTS: This collection of CCA_PDXs recapitulates the histopathologic and molecular features of their original tumors. PARPi treatments inhibited the growth of CCA tumoroids and CCA_PDXs with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1. In line with these findings, only CCA_PDX and CCA patient biopsy samples with mutations of BRCA2 showed RAD51 scores compatible with HRD. CONCLUSIONS: Our results suggest that patients with advanced CCA with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1, are likely to benefit from PARPi therapy. This collection of CCA_PDXs provides new opportunities for evaluating drug response and prioritizing clinical trials.

Untargeted detection of the carbonyl metabolome by chemical derivatization and liquid chromatography-tandem mass spectrometry in precursor ion scan mode: Elucidation of COVID-19 severity biomarkers.

Metabolomics (both targeted and untargeted) has become the gold standard in biomarker discovery. Whereas targeted approaches only provide information for the selected markers, thus hampering the determination of out-of-the-box markers, the common bottleneck of untargeted metabolomics is the identification of detected biomarkers. In this study, we developed a strategy based on derivatization and LC-MS/MS detection in a precursor ion scan for the untargeted determination of a specific part of the metabolome (carbonyl-containing metabolites). The usefulness of this guided metabolomics approach has been demonstrated by elucidating carbonyl-containing biomarkers of COVID-19 severity. First, the LC-MS/MS behavior of 63 model compounds after O-benzylhydroxylamine derivatization was studied. A precursor ion scan of m/z 91 was selected as a suitable approach for the untargeted detection of carbonyl-containing metabolites. The method was able to detect ≈300 potential carbonyl-containing molecules in plasma, including mono-/di-/tricarbonylic compounds with satisfactory intra-day and inter-day repeatability and RSDs commonly <15%. Additionally, the semiquantitative nature of the precursor ion scan method was confirmed by comparison with a fully validated targeted method. The application of the guided metabolomics method to COVID-19 plasma samples revealed the presence of four potential COVID-19 severity biomarkers. Based on their LC-MS/MS behavior, these biomarkers were elucidated as 2-hydroxybutyrate, 2,3-dihydroxybutyrate, 2-oxobutyrate and 2-hydroxy-3-methylbutyrate. Their structures were confirmed by comparison with reference materials. The alterations of these biomarkers with COVID-19 severity were confirmed by a target analysis of a larger set of samples. Our results confirm that guided metabolomics is an alternative approach for the untargeted detection of selected families of metabolites; this approach can accelerate their elucidation and provide new perspectives for the establishment of health/disease biomarkers.

Metabolomics and integrated network analysis reveal roles of endocannabinoids and large neutral amino acid balance in the ayahuasca experience.

There has been a renewed interest in the potential use of psychedelics for the treatment of psychiatric conditions. Nevertheless, little is known about the mechanism of action and molecular pathways influenced by ayahuasca use in humans. Therefore, for the first time, our study aims to investigate the human metabolomics signature after consumption of a psychedelic, ayahuasca, and its connection with both the psychedelic-induced subjective effects and the plasma concentrations of ayahuasca alkaloids. Plasma samples of 23 individuals were collected both before and after ayahuasca consumption. Samples were analysed through targeted metabolomics and further integrated with subjective ratings of the ayahuasca experience (i.e., using the 5-Dimension Altered States of Consciousness Rating Scale [ASC]), and plasma ayahuasca-alkaloids using integrated network analysis. Metabolic pathways enrichment analysis using diffusion algorithms for specific KEGG modules was performed on the metabolic output. Compared to baseline, the consumption of ayahuasca increased N-acyl-ethanolamine endocannabinoids, decreased 2-acyl-glycerol endocannabinoids, and altered several large-neutral amino acids (LNAAs). Integrated network results indicated that most of the LNAAs were inversely associated with 9 out of the 11 subscales of the ASC, except for tryptophan which was positively associated. Several endocannabinoids and hexosylceramides were directly associated with the ayahuasca alkaloids. Enrichment analysis confirmed dysregulation in several pathways involved in neurotransmission such as serotonin and dopamine synthesis. In conclusion, a crosstalk between the circulating LNAAs and the subjective effects is suggested, which is independent of the alkaloid concentrations and provides insights into the specific metabolic fingerprint and mechanism of action underlying ayahuasca experiences.

Targeted metabolomics in formalin-fixed paraffin-embedded tissue specimens: Liquid chromatography-tandem mass spectrometry determination of acidic metabolites in cancer research.

Formalin-fixed paraffin-embedded (FFPE) tissues play an irreplaceable role in cancer research. Although extensive research has been conducted for the detection of DNA, RNA and proteins in FFPE samples, literature dealing with the FFPE determination of small molecules is scarce. In this study, we aimed to explore the potential of targeted metabolomics in FFPE specimens. For that purpose, we developed a LC-MS/MS method for the quantification of acidic metabolites in FFPE samples. The method involves trimming tissue slices from FFPE blocks, deparaffinization, lysis of the tissue, o-benzyl hydroxylamine derivatization and LC-MS/MS detection. Deparaffinization and lysis steps were optimized to maximize the analytes extraction and to minimize the effect of the ubiquitous presence of some metabolites in the paraffin. Two validation approaches were applied: (i) using blank paraffin as matrix and (ii) using actual human FFPE tissue samples by standard additions. The method quantified 40 metabolites with appropriate accuracy (commonly 80-120%) and precision (CV 2-19%) in both validation approaches. LLOQs ranging 0.88-2001 pg mg-1 with low-moderate matrix effects (commonly 85-115%) were obtained. FFPE samples from 15 patients with colorectal cancer were analyzed and metabolites concentrations in tumor vs matched normal FFPE tissues were compared. Results show that tumor tissues have a well-established fingerprint including an increase in ketogenesis, a decrease in lipogenesis and an imbalance in the tricarboxylic acid cycle.

Compressibility of 2M1 muscovite-phlogopite series minerals.

Muscovite (Ms) and phlogopite (Phl) belong to the 2:1 dioctahedral and trioctahedral layer silicates, respectively, and are the end members of Ms-Phl series minerals. This series was studied in the 2M1 polytype and modeled by the substitution of three Mg2+ cations in the Phl octahedral sites by two Al3+ and one vacancy, increasing the substitution up to reach the Ms. The series was computationally examined at DFT level as a function of pressure to 9 GPa. Cell parameters as a function of pressure and composition, and bulk moduli as a function of the composition agrees with the existing experimental results. The mixing Gibbs free energy was calculated as a function of composition. From these data, approximated solvi were calculated at increasing pressure. A gap of solubility is found, decreasing the gap of solubility at high pressure.

The accuracy of self-reported hearing loss in older Latino-American adults.

The purpose of this study was to examine the accuracy of self-reported hearing loss in older Latino-American adults. Fifty-nine individuals, 32 women (mean age 62.2 years) and 27 men (mean age 62.4 years), participated. The questionnaire addressed hearing loss, hearing aid use, and other people's views of their hearing abilities. Based on the question, 'Do you feel you have a hearing loss?', the prevalence of self-reported hearing loss was 57.6%, with 56.3% of women and 59.3% of men reporting a hearing loss. Sensitivity, specificity, and accuracy were calculated using this question and compared to the pure-tone average (PTA) at 500, 1000, 2000, and 4000 Hz in the poorer ear. Overall, sensitivity was 75.7%, specificity was 72.7%, and accuracy was 74.6%. Results of this smaller scale study suggest that a single question, used as a screening measure, is both sensitive and specific for hearing loss in older Latino-American adults and may overcome language and cultural barriers. This question can be easily implemented by physicians or other health-care employees to quickly screen for hearing loss.