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• We applied liquid chromatography-tandem mass spectrometry to the analysis of historical iron gall ink recipes. • We characterised ageing markers of iron gall inks by HPLC-ESI-Q-ToF. • We highlighted different degradation trends depending on historical recipes and ageing conditions. • We proved that it is possible to use 15 µg as minimum amount of sample for the HPLC-ESI-Q-ToF analysis of iron gall inks. Several manuscripts and drawings of our historical and artistic heritage have been produced with iron gall inks. To obtain an iron gall ink, ancient treatises cite the addition of ferrous sulphate and gum Arabic to a decoction of oak galls as a basic procedure. Owing to the development of synthetic chemistry, iron gall ink recipes were improved with new materials and procedures in the late 19th and early 20th century. Notably, many conservation issues arise from the interaction between iron gall inks and the paper support of manuscripts and drawings. To date, most of the research on the topic are focused on paper preservation by non-destructive analytical methods, which provide only limited information on degradation process trends and minor components, representative of iron gall ink's recipes. In the present work, three historical recipes of iron gall inks (alizarine ink, Reid ink, modern gall ink) , dated to 19th-20th century and differing for the preparation method and additives, were characterized. The molecular markers of iron gall inks and of gallic acid degradation were detected by an optimized protocol based on high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC HRMS). Furthermore, by performing ageing tests on reference materials in different indoor conditions (natural light and stored in the dark), two degradation mechanisms were observed: hydrolysis of poly-galloyl glucose species and auto-oxidation of gallic acid. Thus, different chemical profiles and ageing trends were revealed depending on the starting recipe. The procedure Limit of Detection (LOD) was estimated, improving the approaches reported so far in the literature. Finally, the strategy was successfully applied for the characterisation of the ink employed in a very degraded 16th century manuscript, granting access to the complete molecular profile of an iron gall ink with just 15 µg of sample. [Display omitted] [ABSTRACT FROM AUTHOR]

Several generic formulations of rivaroxaban were recently marketed to be used interchangeably with their branded equivalent. However, there have been no previously published studies that directly compared the in vitro anticoagulant effect of branded vs. generic rivaroxaban. The aim of this in vitro study was to compare the effects of three raw rivaroxaban materials, obtained from the branded (Xarelto®) and two generic (Rivarolto® and Rivaroxaban Sandoz®) rivaroxaban formulations on an array of coagulation assays. A pool of normal plasma was spiked with several concentrations of the three rivaroxaban (range 50–750 ng/ml). The concentrations were assessed with a rivaroxaban calibrated anti-Xa assay and confirmed by ultra-high-performance liquid chromatography-mass spectrometry coupled with tandem mass spectrometry (UHPLC-MS/MS). The following assays were performed: Prothrombin time (PT), activated Partial Thromboplastin time (aPTT), Diluted Russell's Viper Venom Test (dRVVT), Thrombin time (TT), Clauss Fibrinogen, Factor VII, VIII and IX assays, and thromboelastography. The results obtained by the three rivaroxaban at similar concentrations were comparable. Increasing concentrations of the three rivaroxaban showed a strong positive correlation with the PT, aPTT and dRVVT assays (r > 0.95, p < 0.01 for all), and a strong negative correlation with the Factors assays (r < −0.95, p < 0.01 for all). TT and Clauss Fibrinogen were not affected by rivaroxaban. No significant difference was identified in the mean assays' results obtained by the three rivaroxaban. This study showed that the branded and generic rivaroxaban exert an identical in vitro anticoagulant effect across a wide range of concentrations. [Display omitted] • No published study compared the in vitro anticoagulant effect of branded vs generic rivaroxaban. • Normal plasma was spiked with three raw rivaroxaban materials (concentrations 50–750 ng/ml). • Branded and generic rivaroxaban exerted identical in vitro effect on several coagulation assays. [ABSTRACT FROM AUTHOR]

• Natural and synthetic dyes in Central Asian ikats were identified by HPLC-DAD-MS/MS. • Cotton weft threads were often dyed with red synthetic dyes. • Yellow from larkspur kept being used well after synthetic dyes were available. • A local species of scale insect (cochineal) might have been used to obtain red. • The introduction of synthetic dyes in ikat dyeing was a gradual and experimental process. Central Asian ikat textiles are characterised by their bold and large abstract patterns, made up of vibrant colours with a characteristic "blurred edge" effect, which makes them some of the most recognisable fabrics worldwide. Eleven ikats from the collection of the National Museum of Asian Art, Smithsonian Institution (Washington, DC, USA) and six ikats from the collection of the British Museum (London, UK) were investigated by high pressure liquid chromatography coupled to diode array detector and high-resolution tandem mass spectrometry (HPLC-DAD-HRMS/MS) to identify the dyes and expand on recent research focused on the integration of synthetic dyes in ikat dyeing during the second half of the 19th century. The results confirmed that the palette of natural dyes includes cochineal (probably mostly from a local species native to central Asia) for red, larkspur (Delphinium semibarbatum) for yellow, and indigo for blue as the main dye sources. In some of the textiles, madder (Rubia tinctorum) for red and pagoda tree (Sophora japonica) for yellow were also detected as minor components mixed with cochineal and larkspur, respectively. Synthetic dyes, such as scarlet reds (C.I. 16150 and 16155), rose bengale (C.I. 45435), eosin (C.I. 45380), erythrosine (C.I. 45430), fuchsin (C.I. 42510/42520), methyl violet (C.I. 42535/42555), malachite green (C.I. 42000) and brilliant green (C.I. 42040) were detected in eight textiles. In three textiles, natural dyes were identified in the ikat-dyed silk warp threads and synthetic dyes in the red cotton weft threads, highlighting interesting mixtures of materials and textile-making practices. Natural yellow dyes were used in all textiles, even those for which all the other colours are synthetic. These results provide significant new information about the changes in ikat production that occurred during the 19th century in Central Asia. [ABSTRACT FROM AUTHOR]

To determine the pharmacokinetic profile of hydromorphone 0.2 mg kg–1 administered by the intravenous (IV) and subcutaneous (SC) route in ferrets. Randomized, crossover study. A group of eight adult ferrets weighting (mean ± standard deviation) 1.02 ± 0.22 kg. Hydromorphone hydrochloride 0.2 mg kg–1 was administered IV or SC with a washout period of 7 days. Blood samples were collected from a jugular catheter before administration of hydromorphone and at 5, 10, 15, 20, 30, 45, 60, 90, 120, 240, 360, 480 and 720 minutes after hydromorphone administration. Plasma hydromorphone concentrations were determined by liquid chromatography/tandem mass spectrometry. Data were analyzed using a non-linear mixed effects model. The hydromorphone effective half-life was (t 1/2) 45 min–1. Systemic clearance (Cl s) and the volume of distribution (Vd ss) following IV administration were 84.8 mL kg–1 min–1 and 5.59 L kg–1, respectively. The maximum observed plasma concentration was 59.53 ± 14.02 ng mL–1 within 10 minutes following SC administration. The SC bioavailability was 102.0%. Administration of IV and SC hydromorphone (0.2 mg kg–1) was characterized by a high clearance, short terminal half-life and large volume of distribution. Hydromorphone plasma concentrations remained greater than 2 ng mL–1 for 2 hours in most ferrets, a threshold reported to provide antinociceptive effects in other species. Hydromorphone was well absorbed following SC injection, providing an alternative administration route for clinical use in ferrets. [ABSTRACT FROM AUTHOR]

Cholemic nephropathy (CN) is a severe complication of cholestatic liver diseases for which there is no specific treatment. We revisited its pathophysiology with the aim of identifying novel therapeutic strategies. Cholestasis was induced by bile duct ligation (BDL) in mice. Bile flux in kidneys and livers was visualized by intravital imaging, supported by MALDI mass spectrometry imaging and liquid chromatography-tandem mass spectrometry. The effect of AS0369, a systemically bioavailable apical sodium-dependent bile acid transporter (ASBT) inhibitor, was evaluated by intravital imaging, RNA-sequencing, histological, blood, and urine analyses. Translational relevance was assessed in kidney biopsies from patients with CN, mice with a humanized bile acid (BA) spectrum, and via analysis of serum BAs and KIM-1 (kidney injury molecule 1) in patients with liver disease and hyperbilirubinemia. Proximal tubular epithelial cells (TECs) reabsorbed and enriched BAs, leading to oxidative stress and death of proximal TECs, casts in distal tubules and collecting ducts, peritubular capillary leakiness, and glomerular cysts. Renal ASBT inhibition by AS0369 blocked BA uptake into TECs and prevented kidney injury up to 6 weeks after BDL. Similar results were obtained in mice with humanized BA composition. In patients with advanced liver disease, serum BAs were the main determinant of KIM-1 levels. ASBT expression in TECs was preserved in biopsies from patients with CN, further highlighting the translational potential of targeting ASBT to treat CN. BA enrichment in proximal TECs followed by oxidative stress and cell death is a key early event in CN. Inhibiting renal ASBT and consequently BA enrichment in TECs prevents CN and systemically decreases BA concentrations. Cholemic nephropathy (CN) is a severe complication of cholestasis and an unmet clinical need. We demonstrate that CN is triggered by the renal accumulation of bile acids (BAs) that are considerably increased in the systemic blood. Specifically, the proximal tubular epithelial cells of the kidney take up BAs via the apical sodium-dependent bile acid transporter (ASBT). We developed a therapeutic compound that blocks ASBT in the kidneys, prevents BA overload in tubular epithelial cells, and almost completely abolished all disease hallmarks in a CN mouse model. Renal ASBT inhibition represents a potential therapeutic strategy for patients with CN. [Display omitted] • Bile acid enrichment in proximal tubular epithelial cells triggers cholemic nephropathy. • Blocking renal ASBT prevents bile acid enrichment and death of proximal tubular epithelial cells, thereby preventing cholemic nephropathy. • Renal ASBT inhibition enhances urinary bile acid excretion and lowers the systemic bile acid pool. • Renal ASBT expression is preserved in patients with cholemic nephropathy. [ABSTRACT FROM AUTHOR]

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications. [ABSTRACT FROM AUTHOR]

The cases were a pair of siblings with a carnitine palmitoyltransferase (CPT2) deficiency detected by tandem mass spectrometry. Their C16 and C18:1 levels were both within the normal range, while C0 was low, and the (C16+C18:1)/C2 ratio was high. Following genetic testing, a novel CPT2 gene mutation was identified in both patients. The male patient had a normal growth rate during 5 years of follow-up after treatment. By contrast, the female patient did not take l -carnitine supplements and died after an infectious disease-associated illness when she was 1 year old. These data emphasize the need to raise awareness about CPT2 deficiency so as to correctly diagnose and accurately manage the disease. [ABSTRACT FROM AUTHOR]

Lectins presents the ability to interact with glycans and trigger varied responses, including the inhibition of the development of various pathogens. Structural studies of these proteins are essential to better understand their functions. In marine sponges, so far only a few lectins have their primary structures completely determined. Thus, the objective of this work was to structurally characterize and evaluate antibacterial potential, in association with different antibiotics, of the lectin isolated from the marine sponge Aplysina lactuta (ALL). ALL is a homotetramer of 60 kDa formed by four 15 kDa-subunits. The lectin showed affinity only for the glycoproteins fetuin, asialofetuin, mucin type III, and bovine submaxillary mucin type I. The complete amino acid sequences of two isoforms of ALL, named ALL-a and ALL-b, were determined by a combination of Edman degradation and overlapped peptides sequenced by tandem mass spectrometry. ALL-a and ALL-b have 144 amino acids with molecular masses of 15,736 Da and 15,985 Da, respectively. Both structures contain conserved residues typical of the galectin family. ALL is a protein with antibacterial potential, when in association with ampicillin and oxacillin the lectin potentiates its antibiotic effect, included Methicillin-resistant Staphylococcus strains. Thus, ALL shows to be a molecule with potential for the development of new antibacterial drugs. • ALL conserves structural motifs and carbohydrate-binding residues of galectins. • The predicted 3-D structure of ALL is a typical galectin β-sandwich. • ALL in association with ampicillin potentiates the effect of the antibiotic. • ALL in association with tetracycline confers an additive effect to the antibiotic. [ABSTRACT FROM AUTHOR]

Kabuki syndrome (KS) and spinocerebellar ataxia (SCA) are both rare conditions with neurodevelopmental abnormalities. Approaching a patient with complex phenotypes and differentiating the role of mutations may be beneficial but challenging in predicting the disease prognosis. A boy presented with progressive ataxia, developmental regression, and myoclonus since 4 years of age. Additional features included growth hormone deficiency, excessive body hair, dysmorphic facies, hypoparathyroidism, and bilateral sensorineural hearing impairment. Brain magnetic resonance imaging depicted T2-weighted hyperintensities over bilateral globus pallidus, thalamus, subcortical white matter, and brainstem. The results of tandem mass spectrometry, mitochondrial deletion, and mitochondrial DNA sequencing were inconclusive. Whole-exome sequencing (WES) on genomic DNA obtained from peripheral blood cells revealed a known pathogenic variant at KMT2D gene (c.5993A > G, p.Tyr1998Cys) related to KS and two compound heterozygous, likely pathogenic variants at VPS13D gene (c.908G > A, p.Arg303Gln and c.8561T > G, p.Leu2854Arg) related to autosomal recessive SCA type 4 (SCAR4). SCAR4 is mainly adult-onset, but a few pediatric cases have recently been reported with progressive gait instability and developmental delay. The VPS13D gene has been suggested to play a role in mitochondrial size, autophagy, and clearance, thus explaining the clinical and imaging phenotypes. Our case showed a rare co-existence of KS and SCAR4, highlighting the utility of WES in atypical cases that a single-gene disease cannot fully explain. [ABSTRACT FROM AUTHOR]

The terrestrial crustacean Porcellio scaber is an established test organism in environmental research. We analysed the haemolymph proteome of P. scaber using a classical proteomic approach based on one-dimensional gel electrophoresis and tandem mass spectrometry. Using a publicly available protein database and our P. scaber transcriptome data, we have identified 76 proteins involved in cytoskeleton formation, protein degradation, vesicular transport, genetic information processing, detoxification, carbohydrate and lipid metabolism reflecting haemocyte metabolic activity, active intracellular transport, and intercellular communication. Compared with the data reported for other crustaceans, 28 of these P. scaber proteins have been linked to its immunity, among them hemocyanin, α-2-macroglobulin, phenoloxidase 3, superoxide dismutase, glutathione S-transferase, haemolymph clottable protein, and histones H4 and H2B. Our results thus provide a firm base for studying the innate immune response of P. scaber at the level of the haemolymph proteome. This knowledge is of particular importance in ecotoxicity studies with various environmental stressors where understanding physiological changes is important to reveal possible modes of action. • This study presents proteome profile in the haemolymph of P. scaber under basal condition. • In total, 76 proteins were identified in the haemolymph without pre-treatment. • Many proteins are involved in intracellular trafficking and intercellular communication. • 28 proteins were found associated with immunity. • The most abundant protein was hemocyanin. [ABSTRACT FROM AUTHOR]

Identity testing is a critical part in the development of a therapeutic synthetic oligonucleotide. Tandem Mass Spectrometry (MS/MS) is commonly used for the analysis of oligonucleotides to obtain structural and sequence information, however there are challenges resulting from chemical modifications introduced to improve their pharmacokinetics and stability. For these structurally complex oligonucleotides, Nuclear Magnetic Resonance (NMR) Spectroscopy has found limited use for characterisation and identity testing, as only partial NMR resonance assignment for oligonucleotides is achieved without isotopic labelling methodologies. Regardless of the choice of method used for oligonucleotide analysis, the specificity is of critical importance. In this work, in-source dissociation mass spectrometry and proton (1H) and carbon (13C) NMR at high temperature were used to analyse danvatirsen, a 16 nucleotide phosphorothioate antisense oligonucleotide, and its closely related switch sequences. Both approaches have shown specificity to distinguish danvatirsen from these similar sequences. [ABSTRACT FROM AUTHOR]

An excessive blood level of homocysteine (HcySH) is associated with numerous cardiovascular and neurodegenerative disease conditions. It has been suggested that direct S-homocysteinylation, of proteins by HcySH, or N-homosteinylation by homocysteine thiolactone (HTL) could play a causative role in these maladies. In contrast, ascorbic acid (AA) plays a significant role in oxidative stress prevention. AA is oxidized to dehydroascorbic acid (DHA) and if not rapidly reduced back to AA may degrade to reactive carbonyl products. In the present work, DHA is shown to react with HTL to produce a spiro bicyclic ring containing a six-membered thiazinane-carboxylic acid moiety. This reaction product is likely formed by initial imine condensation and subsequent hemiaminal product followed by HTL ring opening and intramolecular nucleophilic attack of the resulting thiol anion to form the spiro product. The reaction product was determined to have an accurate mass of 291.0414 and a molecular composition C 10 H 13 NO 7 S containing five double bond equivalents. We structurally characterized the reaction product using a combination of accurate mass tandem mass spectrometry, 1D and 2D-nuclear magnetic resonance. We also demonstrated that formation of the reaction product prevented peptide and protein N-homocysteinylation by HTL using a model peptide and α-lactalbumin. Furthermore, the reaction product is formed in Jurkat cells when exposed to HTL and DHA. [Display omitted] • Dehydroascorbate reacts with homocysteine thiolactone. • The reaction prevents protein N-homocysteinylation by homocysteine thiolactone. • The stability of the reaction product decreases at raising pH. • The reaction takes place intracellularly in Jurkat cells. [ABSTRACT FROM AUTHOR]

To develop a population pharmacokinetic (PK) model with data from the largest polymyxin B-treated patient population studied to date to optimize its dosing in hospitalized patients. Hospitalized patients receiving intravenous polymyxin B for ≥48 hours were enrolled. Blood samples were collected at steady state and drug concentrations were analysed by liquid chromotography tandem mass spectrometry (LC-MS/MS). Population PK analysis and Monte Carlo simulations were performed to determine the probability of target attainment (PTA). One hundred and forty-two patients received intravenous polymyxin B (1.33–6 mg/kg/day), providing 681 plasma samples. Twenty-four patients were on renal replacement therapy, including 13 on continuous veno-venous hemodiafiltration (CVVHDF). A 2-compartment model adequately described the PK with body weight as a covariate on the volume of distribution that affected C max , but it did not impact clearance or exposure. Creatinine clearance was a statistically significant covariate on clearance, although clinically relevant variations of dose-normalized drug exposure were not observed across a wide creatinine clearance range. The model described higher clearance in CVVHDF patients than in non-CVVHDF patients. Maintenance doses of ≥2.5 mg/kg/day or ≥150 mg/day had a PTA ≥90% (for non-pulmonary infections target) at a steady state for minimum inhibitory concentrations ≤2 mg/L. The PTA at a steady state for CVVHDF patients was lower. Fixed loading and maintenance doses of polymyxin B seemed to be more appropriate than weight-based dosing regimens in patients weighing 45–90 kg. Higher doses may be needed in patients on CVVHDF. Substantial variability in polymyxin B clearance and volume of distribution was found, suggesting that therapeutic drug monitoring may be indicated. [ABSTRACT FROM AUTHOR]

• Massive iron overload, infertility and fatigue in 35-year-old man homozygous for a novel hepcidin mutation. The bioactive peptide hormone hepcidin-25 regulates iron levels by inhibiting iron transport to plasma via ferroportin. Hepcidin-25 is synthesized in the liver where the 84 amino acids pro-hepcidin is cleaved into the bioactive hepcidin-25. A patient admitted to the hospital presented with infertility and fatigue. Genomic DNA was purified from whole blood using the Maxwell 16 system (Promega). MLPA analysis was performed to detect large genomic rearrangements using the SALSA MLPA kit # P347, Hemochromatosis (MRC Holland, Holland). Plasma hepcidin measurements were performed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). A novel HAMP mutation (homozygous one base deletion in c.215delG, p.Cys72Serfs*?) was detected. The deletion in nucleotide 215 causes a frameshift altering the predicted protein sequence from cysteine13 in mature peptide. Whether this leads to nonsense mediated decay of the mRNA or synthesis of an aberrant peptide in unknown, but bioactive hepcidin-25 was undetectable in plasma. The patient had massive iron overload with ferritin up to 8360 µg/L. He was anaemic with a Hb at 7.0 mmol/L (11.3 g/dL) and suffered from hypogonadotropic hypogonadism with a total testosterone of 1.2 nmol/l. Continued treatment with venesection and gonadotropins led to reduced fatigue, reduction in iron overload, a normalized Hb and improvement of semen quality. A novel hepcidin mutation was detected in a patient with massive iron overload, fatigue and hypogonadotropic hypogonadism. [ABSTRACT FROM AUTHOR]

To characterize the pharmacokinetics of buprenorphine and norbuprenorphine in isoflurane-anesthetized cats. Prospective experimental study. A group of six healthy adult male neutered cats. Cats were anesthetized with isoflurane in oxygen. Catheters were placed in a jugular vein for blood sampling and in a medial saphenous vein for buprenorphine and lactated Ringer's solution administration. Buprenorphine hydrochloride (40 μg kg–1 over 5 minutes) was administered intravenously. Blood samples were collected before buprenorphine administration and at various times up to 12 hours after administration. Plasma buprenorphine and norbuprenorphine concentrations were measured using liquid chromatography/tandem mass spectrometry. Compartment models were fitted to the time-concentration data using nonlinear mixed effect (population) modeling. A five-compartment model (three compartments for buprenorphine and two compartments for norbuprenorphine) best fitted the data. Typical value (% interindividual variability) for the three buprenorphine volumes of distribution, and the metabolic clearance to norbuprenorphine, the remaining metabolic clearance and the two distribution clearances were 157 (33), 759 (34) and 1432 (43) mL kg–1, and 5.3 (33), 16.4 (11), 58.7 (27) and 6.0 (not estimated) mL minute–1 kg–1, respectively. Typical values (% interindividual variability) for the two norbuprenorphine volumes of distribution, and the norbuprenorphine metabolic and distribution clearances were 1437 (30) and 8428 (not estimated) mL kg–1 and 48.4 (68) and 235.9 (not estimated) mL minute–1 kg–1, respectively. The pharmacokinetics of buprenorphine in isoflurane-anesthetized cats were characterized by a medium clearance. [ABSTRACT FROM AUTHOR]

Mammalian brain tubulins undergo a reversible posttranslational modification--polyglutamylation--which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the β-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for a-isoform than β-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of a1- and β2-tubulins and a recombinant tubulin. Particularly, in the recombinant a1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct role in vivo. [ABSTRACT FROM AUTHOR]

• A highly accurate and precise candidate Reference Measurement Procedure (cRMP) was developed for CDC Clinical Standardization Programs (CDC CSP) free thyroxine (FT4) measurements. • The FT4 cRMP was based on equilibrium dialysis (ED) and isotope dilution liquid chromatography – tandem mass spectrometry (ID-LC-MS/MS) procedures. • The accuracy and precision of the cRMP were validated via an interlaboratory comparison study with excellent agreement between four independent laboratories. • The low limit of detection of cRMP provided sufficient sensitivity to determine FT4 for patients with hypothyroidism. Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays. [ABSTRACT FROM AUTHOR]

[Display omitted] • M. anisopliae phenylethyl alcohol exerts an avoidance effect to locusts. • M. anisopliae infection elevates the expression of LmOBP11 in locusts. • Treatment with phenylethyl alcohol upregulates LmOBP11 in locusts. • Locust LmOBP11 is involved in the detection of fungal phenylethyl alcohol. • LmOBP11 negatively regulates the locust immune system to benefit fungal infection. Odorant-binding proteins (OBPs) are a class of small molecular weight soluble proteins that exist as expanded gene families in all insects, acting as ligand carriers mediating olfaction and other physiological processes. During fungal infection, a subset of insect OBPs were shown to be differentially expressed. We tested whether the altered expression of insect OBP s during pathogenic infection plays a role in behavioral or immune interactions between insect hosts and their pathogens. A wide range of techniques including RNAi-directed knockdown, heterologous protein expression, electrophysiological/behavioral analyses, transcriptomics, gut microbiome analyses, coupled with tandem mass spectrometry ion monitoring, were used to characterize the function of a locust OBP in host behavioral and immune responses. The entomopathogenic fungus Metarhizium anisopliae produces the volatile compound phenylethyl alcohol (PEA) that causes behavioral avoidance in locusts. This is mediated by the locust odorant binding protein 11 (LmOBP11). Expression of LmOBP11 is induced by M. anisopliae infection and PEA treatment. LmOBP11 participates in insect detection of the fungal-produced PEA and avoidance of PEA-contaminated food, but the upregulation of LmOBP11 upon M. anisopliae infection negatively affects the insect immune responses to ultimately benefit successful mycosis by the pathogen. RNAi knockdown of LmOBP11 increases the production of antimicrobial peptides and enhances locust resistance to M. anisopliae infection, while reducing host antennal electrophysiological responses to PEA and locust avoidance of PEA treated food. Also, transcriptomic and gut microbiome analyses reveal microbiome dysbiosis and changes in host genes involved in behavior and immunity. These results are consistent with the elevated expression of LmOBP11 leading to enhanced volatile detection and suppression of immune responses. These findings suggest a crosstalk between olfaction and immunity, indicating manipulation of host OBPs as a novel target exploited by fungal pathogens to alter immune activation and thus promote the successful infection of the host. [ABSTRACT FROM AUTHOR]

• Sixteen CPT intermediates from nothapodytes nimmoniana were identified and confirmed. • Isomers of CPT post-strictosidine intermediates were relatively quantified in root, bark, fruit and leaf. • Roots accumulated most of the CPT intermediates. • Both carboxylic acid and methyl ester forms of CPT intermediates are present in N. nimmoniana. Nothapodytes nimmoniana is a medicinally important plant producing camptothecin (CPT) which is a well-known anti-cancer drug. Its analogues viz. topotecan and irinotecan are currently used to treat variety of cancers like lung, colon, ovarian cancers. The CPT is a monoterpene indole alkaloid class of compound and synthesised by the combination of terpene and indole pathway. Secologanin from terpene pathway and tryptamine from indole pathway condense together to form strictosidine which is a precursor of CPT. In this study, four different tissues viz. root, leaf, bark and fruit of N. nimmoniana were subjected to LC-QTOF-MS analysis. Total sixteen intermediate compounds in the CPT biosynthetic pathway with their relative accumulation were detected and confirmed by tandem mass spectrometry analysis. Two or more than two isomers of post-strictosidine pathway intermediates like deoxypumiloside, pumiloside and strictosamide, strictosamide epoxide were detected in roots and bark. Accumulation pattern of intermediate metabolites in roots suggests active biosynthesis of CPT in roots. Moreover, unlike Camptotheca acuminata (another CPT producing plant), presence of both carboxylic acid derivatives and methyl esters of iridoid pathway intermediates such as loganic acid/ loganin, secologanic acid/ secologanin and strictosidinic acid/ strictosidine suggests existence of two or more interwoven pathways leading to CPT biosynthesis in N. nimmoniana. [ABSTRACT FROM AUTHOR]

In this paper, a near-field thermophotovoltaic (TPV) system with a dual TPV cell design is considered and studied numerically. The fluctuational electrodynamics accompanied by the dyadic Green's function are utilized for calculating the radiation heat flux. The generated photocurrent and the conversion efficiency are obtained by solving the photon-coupled charge transport equations. It was discovered that the addition of the second TPV cell to the system, increases the generated photocurrent of the system due to the absorption of the low-energy photons and generation of the electron-hole pairs. By increasing the gap size between the emitter and Si TPV cell from 50 nm to 1 μm, the generated photocurrent decreases because of the reduction of the near-field thermal radiation effects. Finally, the tandem nano-gap TPV system shows higher conversion efficiency compared with the single Si TPV cell for different values of the vacuum gaps, and the gap size of d 1 = 200 nm is the optimum gap size due to the improvement of the conversion efficiency of the system. [ABSTRACT FROM AUTHOR]

Exocrine meibomian glands (MGs) play a central role in the ocular physiology and biochemistry by producing in situ and, mostly, de novo a secretion (meibum), which is composed of a complex mixture of homologous lipids of various classes, in a metabolic pathway termed meibogenesis. Recent in vivo experiments with a number of mouse models demonstrated that inactivation of any of the major genes of meibogenesis led to alterations in the lipid composition of meibum and severe ocular and MG abnormalities that replicated various human ocular pathologies. However, the role of dietary lipids in meibogenesis, and in the onset and/or alleviation of these diseases, remains controversial. To uncover the role of dietary lipids, the metabolic transformations of a dietary lipid tracer--stable isotope-labeled glyceryl tri(oleate-1,2,3,7,8-13C5) (13C15-TO)--were investigated using liquid chromatography-high-resolution mass spectrometry. We demonstrated that major metabolic transformations of the tracer occurred in the stomach and small intestines where 13C15-TO underwent immediate and extensive transesterification into 13C5- and 13C10-substituted triacylglycerols of various lengths, giving a mixture of 13Clabeled compounds that remain virtually unchanged in the mouse plasma, liver, and white adipose tissue but were almost undetectable in the feces. Importantly, the tracer and its metabolites were virtually undetectable in MGs, even after 4 weeks of daily supplementation. Notably, unbiased principal component analysis of the data revealed no measurable changes in the overall chemical composition of meibum after the treatment, which implies no direct effect of dietary triacylglycerols on meibogenesis, and left their systemic effects as the most likely mechanism. [ABSTRACT FROM AUTHOR]

There is an increased need for the sensitive and accurate measurement of estradiol levels in patients with estradiol-related endocrine disorders. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a reliable and accurate method for measuring steroid hormone levels. Here, we aimed to establish an LC-MS/MS-based method to quantify estradiol levels without sample derivatization and evaluated its analytical performance. Sciex Triple Quad 6500 + LC-MS/MS was used for estradiol analysis. We evaluated its analytical performance, including linearity, precision, the lower limit of detection and quantification (LoD and LoQ, respectively), accuracy, and carryover. The estradiol results determined by LC-MS/MS were compared with those obtained using a chemiluminescent microparticle immunoassay. The LC-MS/MS output was linear for serum estradiol concentrations in the range of 0.2–10311.6 pmol/L. The intra-laboratory precision (coefficient of variation) was 3.0–10.1 %. The LoD and LoQ were 2.8 and 7.5 pmol/L, respectively. The overall accuracy was within 15 % of bias, and the carryover was within the acceptable range (<1.0 %). The results of the estradiol analysis determined by LC-MS/MS were comparable to those obtained by the chemiluminescent microparticle immunoassay (r2 = 0.9843), although there was a negative bias of − 17.82 (95 % confidence interval, −27.21 to − 8.44). A highly sensitive, derivatization-free LC-MS/MS method was successfully developed in this study. This may be beneficial for estradiol measurements in patients with estradiol-related endocrine disorders. [ABSTRACT FROM AUTHOR]

• First established reference intervals for estradiol using a UHPLC-MS/MS-based method in South Korea. • Serum samples from 634 pediatric patients, including neonates, infants, children, and adolescents. • The highly analytical sensitive UHPLC-MS/MS method was useful for estradiol determination in children. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) is a reliable and accurate method for measuring steroid hormone levels. There is an increasing need for sensitive and precise methods to measure estradiol in pediatric patients. Here, we established reference intervals for estradiol in healthy children using a UHPLC–MS/MS-based method for the first time in South Korea. Serum estradiol was measured using a Sciex Triple QuadTM 6500 + UHPLC–MS/MS (Sciex, Framingham, MA, USA). Reference intervals for estradiol were established according to the CLSI document EP28-A3c:2008. The reference intervals were validated using serum samples from 634 pediatric patients, including neonates, children, and adolescents. Among them, 389 specimens were used in analysis of the specimen acceptance time. Statistical analysis was performed using MedCalc (MedCalc, Ostend, Belgium) and Analyse–it (Analyse–it Software Ltd., Leeds, United Kingdom) software. Reference intervals for boys (n = 297) were <16.6, <7.3, <19.0, <30.5, 7.6–96.5, and 10.6–134.4 pmol/L among those aged <1, 1–5, 6–9, 10–11, 12–14, and 15–17 years, respectively. Reference intervals for girls (n = 337) were <114.7, <24.2, <34.8, 8.0–177.0, 10.4–480.5, and 9.1–486.7 pmol/L among those aged <1, 1–5, 6–9, 10–11, 12–14, and 15–17 years, respectively. Overall, there was no effect of specimen acceptance time on estradiol measurements in boys or girls, except for that in the group aged 10–11 years. The reference intervals for healthy children were validated using a UHPLC–MS/MS-based method. The highly analytical sensitive UHPLC–MS/MS method may be useful for estradiol determination in pediatric patients. [ABSTRACT FROM AUTHOR]

Uterine secretions provide a suitable environment for sperm selective migration during a couple of days preceding ovulation and for early embryo development before implantation. Our goal was to identify and quantify proteins in the bovine uterine fluid during the periovulatory period of the estrous cycle. Genital tracts with normal morphology were collected from adult cyclic Bos taurus females in a local slaughterhouse and classified into pre-ovulatory or post-ovulatory stages of cycle (around days 19–21 and 0–5 of cycle, respectively; n = 8 cows per stage) based on ovarian morphology. Proteins from uterine fluid collected from the utero-tubal junction to the base of each horn (four pools of two cows per condition) were analyzed by nanoLiquid Chromatography coupled with tandem Mass Spectrometry (nanoLC-MS/MS). A total of 1214 proteins were identified, of which 91% were shared between all conditions. Overall, 57% of proteins were predicted to be secreted and 17% were previously reported in uterine extracellular vesicles. Paired comparisons between uterine horns ipsilateral and contralateral to ovulation evidenced 12 differentially abundant proteins, including five at pre-ovulatory stage. Furthermore, 35 proteins differed in abundance between pre- and post-ovulatory stages, including 21 in the ipsilateral side of ovulation. Functional analysis of identified proteins demonstrated roles in binding, metabolism, cellular detoxification and the immune response. This study provides a valuable database of uterine proteins for functional studies on sperm physiology and early embryo development. • This is the first proteomic profiling of the peri-ovulatory uterine fluid in cattle. • Albumin, complement C3, myosin 9 and mucin 5B were among the most abundant proteins. • 35 differentially abundant proteins between peri-ovulatory stages were identified. • 12 differentially abundant proteins between uterine horns were identified. • Proteins were mostly involved in metabolism, detoxification and immune system. [ABSTRACT FROM AUTHOR]

In several neurodegenerative disorders, the neuronal proteins tau and α-synuclein adopt aggregation-prone conformations capable of replicating within and between cells. To better understand how these conformational changes drive neuropathology, we compared the interactomes of tau and α-synuclein in the presence or the absence of recombinant fibril seeds. Human embryonic stem cells with an inducible neurogenin-2 transgene were differentiated into glutamatergic neurons expressing (1) WT 0N4R tau, (2) mutant (P301L) 0N4R tau, (3) WT α-synuclein, or (4) mutant (A53T) α-synuclein, each genetically fused to a promiscuous biotin ligase (BioID2). Neurons expressing unfused BioID2 served as controls. After treatment with fibrils or PBS, interacting proteins were labeled with biotin in situ and quantified using mass spectrometry via tandem mass tag labeling. By comparing interactions in mutant versus WT neurons and in fibril-versus PBS-treated neurons, we observed changes in protein interactions that are likely relevant to disease progression. We identified 45 shared interactors, suggesting that tau and α-synuclein function within some of the same pathways. Potential loci of shared interactions include microtubules, Wnt signaling complexes, and RNA granules. Following fibril treatment, physiological interactions decreased, whereas other interactions, including those between tau and 14-3-3 η, increased. We confirmed that 14-3-3 proteins, which are known to colocalize with protein aggregates during neurodegeneration, can promote or inhibit tau aggregation in vitro depending on the specific combination of 14-3-3 isoform and tau sequence. [ABSTRACT FROM AUTHOR]

• Differences and similarities between ice and aqueous photochemistry were revealed. • Photodegradation kinetics are clearly influenced by the matrix phases, ice and water. • Different photodegradable potential orders between in the two phases were discovered. • Unidentical effects of key constituents in the two phases account for the differences. • Ice photolysis shows fewer products / simpler pathways compared to water photolysis. The photochemical behavior of organic pollutants in ice is poorly studied in comparison to aqueous photochemistry. Here we report a detailed comparison of ice and aqueous photodegradation of two representative OH-PAHs, 2-hydroxyfluorene (2-OHFL) and 9-hydroxyfluorene (9-OHFL), which are newly recognized contaminants in the wider environment including colder regions. Interestingly, their photodegradation kinetics were clearly influenced by whether they reside in ice or water. Under the same simulated solar irradiation (λ > 290 nm), OHFLs photodegraded faster in ice than in equivalent aqueous solutions and this was attributed to the specific concentration effect caused by freezing. Furthermore, the presence of dissolved constituents in ice also influenced photodegradation with 2-OHFL phototransforming the fastest in 'seawater' ice (k = (11.4 ± 1.0) × 10−2 min−1) followed by 'pure-water' ice ((8.7 ± 0.4) × 10−2 min−1) and 'freshwater' ice ((8.0 ± 0.7) × 10−2 min−1). The presence of dissolved constituents (specifically Cl−, NO 3 −, Fe(III) and humic acid) influences the phototransformation kinetics, either enhancing or suppressing phototransformation, but this is based on the quantity of the constituent present in the matrixes, the specific OHFL isomer and the matrix type (e.g., ice or aqueous solution). Careful derivation of key photointermediates was undertaken in both ice and water samples using tandem mass spectrometry. Ice phototransformation exhibited fewer by-products and 'simpler' pathways giving rise to a range of hydroxylated fluorenes and hydroxylated fluorenones in ice. These results are of importance when considering the fate of PAHs and OH-PAHs in cold regions and their persistence in sunlit ice. [Display omitted] [ABSTRACT FROM AUTHOR]

• Interpretation of data from studies that have explored a possible association between depression and testosterone is limited by methodological heterogeneity. • Studies that have measured testosterone by liquid/gas chromatography with tandem mass spectrometry are lacking. • The available data does not support an association between testosterone and depression in postmenopausal women. The contribution of testosterone to depression in older women is uncertain. This review was conducted to investigate the association between endogenous testosterone blood concentrations and depression in postmenopausal women. We searched Ovid MEDLINE, EMBASE, PsycINFO, and Web of Science databases for observational studies with at least 100 community-dwelling participants. The results were categorised by study design, and the reporting of total, bioavailable and free testosterone findings is narrative. The search strategy retrieved 28 articles for full-text review, of which eight met the criteria for inclusion; these described 6 cross-sectional and 2 longitudinal studies. Testosterone was measured by immunoassay in all of the included studies. No association was seen between total testosterone or free testosterone and depression in either the cross-sectional or the longitudinal studies. A significant association between bioavailable testosterone and incident depressive symptoms was limited to women at least 21 years postmenopause in one study. Most of the cross-sectional studies were not representative of national populations and lacked random selection. This systematic review does not support an association between testosterone and depression in postmenopausal women. However, as the included studies had substantial methodological limitations, studies of community-based samples, employing validated instruments for depression and precise measurement of blood testosterone, are needed to address this knowledge gap. [ABSTRACT FROM AUTHOR]

• CTX newborn screening is possible using only metabolite ratios. • Screening of 20,076 deidentified newborn blood spots showed no CTX screen positives. • CTX newborn screening is possible using either FIA-MS/MS or UPLC-MS/MS. Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1 - leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t -CDCA) and tauro-trihydroxycholestanoic acid (t -THCA). To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/ t -CDCA and t -THCA/GlcA-tetrol ratios. Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/ t -CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/ t -CDCA and t -THCA/GlcA-tetrol ratios also did not yield any screen positives. Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS. [ABSTRACT FROM AUTHOR]

Neonicotinoid pesticides (NNs) have been reported to have neurobehavioral effects on offspring after fetal and lactational exposure. In this study, clothianidin (CLO), an NN, was administered orally as a single dose (6.5 mg/kg: 1/10 of the no-observed-adverse-effect level in the current Pesticide Evaluation Report) to 10-day post-partum ICR mice, and CLO and its metabolites desmethyl-CLO (dm-CLO) were quantified using liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) after collecting maternal breast milk and blood samples over time (1, 3, 6, 9, 12, and 24 h after administration). CLO and dm-CLO were detected in the breast milk at 1 h after the administration, and their concentrations were significantly higher than those in blood at all time points. The concentrations of CLO and dm-CLO in the breast milk were at their highest levels at 1 and 3 h, respectively, and then decreased over time to become almost undetectable at 24 h after the administration. These results show that CLO is metabolized in the mother's body and is rapidly transferred to and concentrated in the breast milk. Since CLO concentrations in breast milk are higher than those in the blood, there is concern about the effects of CLO during lactation. • CLO is metabolized maternally in mice and rapidly transferred in breast milk. • CLO and metabolites are more highly concentrated in breast milk than maternal blood. • Most of CLO and its metabolites were eliminated from the mother's body within 24 h. • Infants are sensitive to chemicals, so adverse CLO effects in infants are a concern. [ABSTRACT FROM AUTHOR]

A convenient and nonradioactive method for quantifying in vitro NR biosynthesis is presented that is based upon the quantitation of substrate depletion by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). NR oligomers could be in vitro biosynthesized with the enzyme source from Hevea brasiliensis (Hevea) or Taraxacum kok-saghyz (TKS) by exogenous monomers (IPP) and initiators (FPP). The IPP incorporation rate and FPP consumption rate were 62.24% and 51.14% respectively when the washed rubber particles (WRP) of Hevea was the enzyme source. The IPP incorporation rate and the FPP consumption rate were 74.49% and 95.90% respectively when the sediment bottom fraction (BF) of Hevea was the enzyme source. The in vitro NR biosynthesis can be divided into two stages:(1) the initiation reaction of FPP, which occurs more in BF, and (2) the growth reaction of IPP, which occurs more in WRP. In addition, the IPP incorporation and FPP consumption rates were 59.39% and 34.15% respectively when the BF of TKS was selected as an enzyme source. • The NR oligomers were biosynthesized in vitro by exogenous IPP and FPP. • The BF and WRP of Hevea latex and the BF of TKS latex were transferase sources. • The contents of IPP and FPP were determined by UHPLC-MS/MS. • The chain initiation reaction of FPP occurred more in the BF than in the WRP. • The chain growth reaction of IPP occurred more in the WRP than in the BF. [ABSTRACT FROM AUTHOR]

• Interferents of methylmalonic acid can lead to invalid diagnoses in clinical assays. • Samples from patients with propionic and methylmalonic acidurias were analyzed. • Six isobaric interferents were detected in serum, urine and dried blood spots. • QTOF HR–MS, MS/MS, NMR, HPLC and UPLC techniques were used for identification. • The results facilitate diagnostic and quantitative reliability in newborn screening. Determination of methylmalonic acid (MMA) from dried blood spots (DBS) is commonly performed in clinical diagnostics and newborn screening for propionic acidemia (PA) and methylmalonic acidemia. Isobaric compounds of MMA having the same mass can affect diagnostic reliability and quantitative results, which represents a previously unrecognized pitfall in clinical assays for MMA. We set out to identify interfering substances of MMA in DBS, serum and urine samples from confirmed patients with PA and methylmalonic acidemia. Techniques included quadrupole time–of–flight high-resolution mass spectrometry (QTOF HR–MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC) and tandem mass spectrometry (MS/MS). The five isobaric metabolites detected in DBS, serum and urine from PA and methylmalonic acidemia patients were confirmed as 2–methyl-3-hydroxybutyrate, 3–hydroxyisovalerate, 2–hydroxyisovalerate, 3–hydroxyvalerate and succinate using a series of experiments. An additional unknown substance with low abundance remained unidentified. The presented results facilitate the diagnostic and quantitative reliability of the MMA determination in clinical assays. Isobaric species should be investigated in assays for MMA to eliminate possible interference in a wide range of conditions including PA, methylmalonic acidemia, a vitamin B 12 deficiency, ketosis and lactic acidosis. [ABSTRACT FROM AUTHOR]

• An HPLC-DMS/MS/MS method was developed to assay steroid hormones in human serum. • A novel DMSPE method was developed to purify and to enrich the analytes in serum. • In situ derivatization was conducted on the adsorbent after DMSPE procedures. Androgens play important roles in polycystic ovarian syndrome (PCOS). However, measures of androgens based on mass spectrometry (MS) remain complex due to endogenous inferences of isomers or compounds with similar structures. Lack of sensitivity can also affect the accurate quantification of androgens, especially for very low level of 11-oxygenated androgens. We developed a fast and sensitive high-performance liquid chromatography-differential mobility spectrometry tandem mass spectrometry (HPLC-DMS/MS/MS) method for the simultaneous determination of seven androgens and 17-hydroxyprogesterone. Dispersive magnetic solid phase extraction (DMSPE) was conducted with core–shell structured nanoparticles of magnetic graphene oxide (Fe 3 O 4 @GO). In situ derivatization was performed using Girard's Reagent P. Linear ranges of the eight analytes were set in terms of clinical use. Intra- and inter-run precisions were < 16.7 % and 12.9 % for all the analytes and relative error was − 14.7–13.3 % and − 9.3–11.0 %, respectively. Extraction recoveries were 54.0–92.7 % for different analytes. The method was validated and was applied to assay 432 clinical samples. The developed method is green, fast, sensitive and accurate for the determination of endogenous androgens. It can be readily implemented in medical laboratories to provide superior analytical performance over the traditional electrochemiluminescence immunoassay method. [ABSTRACT FROM AUTHOR]

Elevated concentrations of triglyceride-rich lipoproteins (TGRL) in the fasting and postprandial states are risk factors for cardiovascular events, especially in type 2 diabetes (T2D). T2D modifies the lipid composition of plasma and lipoproteins and some sphingolipids (SP) have been validated as potent predictive biomarkers of cardiovascular disease occurrence. The main objectives of the present study were to characterize the plasma SP profile in fasting T2D patients and to determine whether SP are modified in postprandial TGRL from these patients compared to fasting TGRL. In a randomized parallel-group study, 30 T2D women ingested a breakfast including 20g lipids from either hazelnut cocoa palm oil-rich spread (Palm Nut) or Butter. Plasma was collected and TGRL were isolated by ultracentrifugation at fasting and 4h after the meal. Fasting samples of 6 control subjects from another cohort were analyzed for comparison. SP were analyzed by tandem mass spectrometry. Plasma from fasting T2D patients had higher ceramide (Cer) and ganglioside GM3 concentrations, and lower concentrations of sphingosylphosphorylcholine vs healthy subjects. In postprandial TGRL from T2D patients compared to those in the fasting state, Cer concentrations and especially C16:0, C24:1 and C24:0 molecular species, increased after the Palm Nut or Butter breakfast. A positive correlation was observed in the Palm Nut group between changes (Δ4h-fasting) of summed C16:0+C22:0+C24:1+C24:0 Cer concentrations in TGRL, and changes in plasma TG, TGRL-TG and TGRL-C16:0 concentrations. Altogether in T2D, the altered profile of plasma SP and the increased Cer concentrations in postprandial TGRL could contribute to the increased atherogenicity of TGRL. [Display omitted] • Elevated postprandial triglyceride-rich lipoproteins (TGRL) and triglycerides are risk factors for cardiovascular disease. • Some plasma sphingolipids are bioactive lipid mediators in type 2 diabetes (T2D). • Higher concentrations of ceramides (Cer) and ganglioside GM3 in fasting T2D women. • A fat-rich meal increased Cer concentrations in postprandial TGRL from T2D women compared to fasting ones. • A fat-rich meal particularly increased C16:0, C24:0 and C24:1 Cer in postprandial T2D TGRL. [ABSTRACT FROM AUTHOR]

Three new very long chain polyunsaturated fatty acids (VLC PUFA) belonging to the omega-3 family have been identified in meibum samples collected by Schirmer strips. These VLC PUFA, namely FA (32:3), FA (34:3) and FA (36:3), were detected in O-acyl-ω-hydroxy fatty acids using a molecular network approach, and as free fatty acids. Identification was supported by retention time prediction model, exact mass determination and isotopic patterns. Double bond location was determined using cross metathesis reaction associated to tandem mass spectrometry. In meibum, synthesis of these VLC PUFA is likely to be mediated by elongation of very long chain fatty acids 4 enzyme. The biological role of these newly VLC PUFA and their occurrence in other tissues and biological fluids remains to be elucidated. [ABSTRACT FROM AUTHOR]

• The incidence of MADD was approximately 1:26,670. • Half of the MADD patients would not be detected under conventional screening protocols. • A novel MALDI-TOF MS assay targeting 23 ETFDH variants in the Chinese population was developed. • Incorporating second-tier genetic screening into the current NBS could improve the performance of MADD NBS. Newborn screening (NBS) for multiple acyl-CoA dehydrogenase deficiency (MADD) has poor sensitivity. This study aimed to evaluate the feasibility of incorporating second-tier genetic screening for MADD. A total of 453,390 newborns were screened for inherited metabolic disorders using tandem mass spectrometry from January 2017 to May 2022. A matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assay was developed to identify 23 common ETFDH variants and used for second-tier screening of MADD. Overall, 185 newborns with suspected MADD received second-tier genetic screening. Thirty-three (17.8 %) newborns with positive results, of which 7 were homozygotes, 5 were compound heterozygotes, 21 were heterozygotes. Further genetic analysis revealed that 6 of the 21 newborns had a second ETFDH variant. Therefore, 18 patients were finally diagnosed with MADD, with a positive predictive value of 9.73 %. The detection rate and diagnostic rate of MALDI-TOF MS assay were 83.33 % and 66.67 %, respectively. Thus the incidence of MADD in our population was estimated at 1:25,188. Nine different ETFDH variants were identified in MADD patients. The most common ETFDH variant being c.250G > A with an allelic frequency of 47.22 %, followed by c.524G > A (13.89 %) and c.998A > G (13.89 %). All patients had elevation of multiple acylcarnitines at NBS. However, seven patients had normal acylcarnitine levels and two patients showed mild elevation of only two acylcarnitines during the recall review. We have established a high throughput MALDI-TOF MS assay for MADD screening. Half of the MADD patients would not be detected under conventional screening protocols. Incorporating second-tier genetic screening into the current NBS could improve the performance of MADD NBS. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is a complex disease with metabolic and functional changes that can alter an individual's proteome. An LC-MS/MS analytical method, in an HDMSE system, was used to identify differentially expressed proteins in the high abundance protein-depleted serum of T1D patients and healthy controls. Samples were processed in Progenesis QI for Proteomics software. A functional enrichment of the proteins was performed with Gene Ontology and ToppGene, and the interactions were visualized by STRING 11.5. As a result, 139 proteins were identified, 14 of which were downregulated in the serum of patients with T1D compared to controls. Most of the differentially expressed proteins were shown to be involved with the immune system, inflammation, and growth hormone stimulus response, and were associated with the progression of T1D. Differential protein expression data showed for the first-time changes in CPN2 expression levels in the serum of patients with T1D. Our findings indicate that these proteins are targets of interest for future investigations and for validation of protein biomarkers in T1D. • UPLC-HDMSE was used to identify possible biomarkers in adults with T1D. • 139 serum proteins from adults with T1D were identified in the proteomic analysis. • Fourteen differentially expressed proteins were described and all downregulation. • Identified proteins were involved with the immune and inflammatory system. • The first evidence of changes in CPN2 expression in T1D was identified. [ABSTRACT FROM AUTHOR]

• Lactic acid bacteria were isolated from fermented black cherry fruit, which had never been investigated before and they are afforded generally regarded as safe status and have positive influence on gastrointestinal tract of human and other consumer animals. • The LAB strains exhibited strong survival properties and antimicrobial activities, highly desirable features for probiotic application against foodborne pathogens. • Salmonella paratyphi a sessile cells were effectively reduced after treatment with the CFS of lactiplantibacillus plantarum MYSAS1 and enterococcus faecium MYSAS4 all the in-vitro assays. Salmonella spp is one of the leading causes of bacterial foodborne illnesses worldwide resulting in acute gastroenteritis. The current study aimed to explore the inhibitory activity of two newly isolated probiotic candidates Lactiplantibacillus plantarum MYSAS1 and Enterococcus faecium MYSAS4 from fermented black cherry fruit, against Salmonella enterica subsp. enterica serovar Paratyphi A. Results demonstrated effective inhibitory activity of selected Lactic acid bacteria (LAB) strains against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella Paratyphi A. In addition, the cell-free supernatant (CFS) of both strains showed potent antimicrobial activity on growth of S. Paratyphi A were demonstrated by agar well diffusion, broth microdilution assay, in vitro time-kill assay, and co-culture experiments. Besides, Salmonella sessile cells were effectively reduced by 70% after treatment with the CFS of L. plantarum MYSAS1 and E. faecium MYSAS4, as determined by crystal violet and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetra bromide (MTT) biofilm assays. The antimicrobial substances of CFS were thermostable but sensitive to pH neutralization, suggesting that the observed antisalmonella activity is likely due to organic acids. Liquid Chromatography- Tandem Mass Spectrometry (LC-MS/MS) analysis of CFS revealed that the antimicrobial activity is likely due to the presence of 11 different organic acids. The isolated strains exhibited important probiotic characteristics, such as sensitivity to most of the antibiotics tested (except vancomycin and kanamycin), anti-hemolytic activity, tolerance to acidic pH and 0.3% bile, and survival ability at different temperatures. L. plantarum MYSAS1 and E. faecium MYSAS4 also exhibited auto-aggregation and cell surface hydrophobicity of 45 and 80%, respectively. Our findings demonstrated important probiotic features isolated from black cherry fruits while inhibiting multiple pathogens, suggesting possible applications of these potential probiotic strains as alternatives for antibiotics and chemical preservatives in the veterinary and food sectors. [ABSTRACT FROM AUTHOR]

• Cerebral creatine deficiency syndromes (CCDS) are metabolic disorders affecting creatine synthesis or transport. • Creatine and guanidinoacetate (GAA) are two biomarkers used to diagnose CCDS. • Different analytical methods were developed to measure the level of creatine and GAA in different body fluids. • In the current study a new UHPLC-MS/MS was developed. • Four novel intronic variants were discovered. Cerebral creatine deficiency syndromes (CCDS) are disorders affecting creatine synthesis or transport. Several methods have been developed to measure creatine and guanidinoacetate (GAA) in different body fluids including methods based on gas chromatography-mass spectrometry (GC–MS) and High-pressure liquid chromatography mass spectrometry (HPLC-MS). The diagnosis of CCDS is then confirmed by sequencing of creatine biosynthesis genes guanidinoacetate methyltransferase (GAMT) and Arginine: glycine amidinotransferase (GATM) and creatine transporter gene solute carrier family 6 member 8 (SLC6A8) or by functional enzymatic assay. The aim of the current study was to find the most reliable and accurate screening method for CCDS by comparing methods using Nuclear Magnetic Resonance spectroscopy (NMR), GC–MS and HPLC-MS. Additionally, this study was performed to estimate the prevalence of CCDS in a cohort of Egyptian patients and potentially to discover novel variants. The study was conducted on 150 subjects with clinical signs and symptoms consistent with CCDS. Metabolic profiling of urine samples was performed using three techniques: 1) GC–MS 2) Ultra high-pressure (or performance) liquid chromatography – Tandem Mass Spectrometry (UHPLC- MS/MS) and 3) NMR. The linearity of peak areas for creatine and GAA by UHPLC-MS/MS and NMR covered and exceeded the ranges normally found in urine. The limit of quantification and the inter-day precision results for creatine and GAA were more robust by UHPLC-MS/MS than NMR. Ten cases were identified as being positive for CCDS by our analytical approaches and underwent next generation sequencing (NGS) for GAMT, GATM and SLC6A8 genes. NGS was performed and confirmed one patient with one likely Pathogenic variant in GAMT gene: (NC_000019.10:g.1401317C > G, NP_000147.1:p.Ala54Pro). Additionally, we describe four novel intronic variants in the GATM gene: c.1043-357del and c.1043-357_1043-356insT, and were predicted to activate cryptic acceptor site with potential alteration of splicing, c.979-227G > A was found to significantly alter the Exon Splice Enhancer (ESE) xon Splice Silencer (ESS) motifs ratio and c.1042 + 262del which was found to have no implications on splicing. Both UHPLC-MS/MS and NMR spectroscopy are comparable to GC–MS in screening for CCDS. Nonetheless, the UHPLC-MS/MS method had better performance than NMR spectroscopy. Additionally, Sequencing of the full length of GATM, GAMT, and SLC6A8 genes is needed to identify intronic variants that could cause CCDS via affecting splice sites. [ABSTRACT FROM AUTHOR]

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography - tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1–2 and 10–12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1–2, 10–12, and 22–24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study. [ABSTRACT FROM AUTHOR]

• The incidence of HPA in Quanzhou, China was estimated to be 1 in 10,365 newborns. • The incidences of PAHD and BH 4 D in Quanzhou were 1:12,350 and 1:64,496, respectively. • The incidence of BH 4 D was as high as 15.1% in the southern Chinese population. • The most common PAH variants were c.158G > A (22.3 %) and c.721C > T (14.9 %). • The most common PTS variants were c.155A > G (50.0 %) and c.259C > T (33.3 %). Hyperphenylalaninemia (HPA) is the most common congenital amino acid metabolism-related defect, but its incidence differs substantially between northern and southern China. We aimed to elucidate the incidence, proportion, and genetic features of HPA in a southern Chinese population. We analyzed the HPA screening results for 580,460 newborns from 2014 to 2021. Of the 296 newborns who tested HPA positive, 56 were diagnosed with HPA, including 47 with phenylalanine hydroxylase deficiency and nine with tetrahydrobiopterin deficiency (BH 4 D). HPA incidence was estimated to be 1:10,365 newborns. All patients had elevated Phe and Phe/Tyr levels. Thirty-three PAH variants and five PTS variants were detected in HPA patients; 80.6 % PAH variants and 100 % PTS variants were classified as pathogenic or likely pathogenic. In silico tools predicted the remaining variants to be damaging. PAH variants clustered in exons 3, 5, 7, 11, and 12 and PTS variants clustered in exons 2 and 5. The most common PAH variants were c.158G > A (p.R53H, 22.3 %) and c.721C > T (p.R241C, 14.9 %). The most common PTS variants were c.155A > G (p.N52S, 50.0 %) and c.259C > T (p.P87S, 33.3 %). Newborn screening is an effective method for early detection of HPA, but differential diagnosis of BH 4 D is necessary. [ABSTRACT FROM AUTHOR]

The zinc finger ubiquitin ligase RNF6 has been proposed as a potential therapeutic target in several cancers, but understanding its molecular mechanism of degradation has been elusive. In the present study, we find that RNF6 is degraded via auto-ubiquitination in a manner dependent on its Really Interesting New Gene (RING) domain. We determine that when the RING domain is deleted (ΔRING) or the core cysteine residues in the zinc finger are mutated (C632S/C635S), the WT protein, but not the ΔRING or mutant RNF6 protein, undergoes polyubiquitination. We also identify USP7 as a deubiquitinase of RNF6 by tandem mass spectrometry. We show that USP7 interacts with RNF6 and abolishes its K48-linked polyubiquitination, thereby preventing its degradation. In contrast, we found a USP7-specific inhibitor promotes RNF6 polyubiquitination, degradation, and cell death. Furthermore, we demonstrate the anti-leukemic drug Nilotinib and anti-myeloma drug Panobinostat (LBH589) induce RNF6 K48- linked polyubiquitination and degradation in both multiple myeloma (MM) and leukemia cells. In agreement with our hypothesis on the mode of RNF6 degradation, we show these drugs promote RNF6 auto-ubiquitination in an in vitro ubiquitination system without other E3 ligases. Consistently, reexpression of RNF6 ablates drug-induced MM and leukemia cell apoptosis. Therefore, our results reveal that RNF6 is a RING E3 ligase that undergoes auto-ubiquitination, which could be abolished by USP7 and induced by anti-cancer drugs. We propose that chemical induction of RNF6 autoubiquitination and degradation could be a novel strategy for the treatment of hematological malignancies including MM and leukemia. [ABSTRACT FROM AUTHOR]

Polymyxin B (PMB) and sodium deoxycholate sulfate (SDCS) ratios were optimized. The self-assembly of PMB-SDCS was characterized using dynamic light scattering. Five different mole ratios of SDCS to PMB (5:1, 10:1, 15:1, 30:1, and 45:1) were prepared after optimization. FTIR and 1H-NMR were employed to characterize PMB formulations. The chemical stability of PMB was quantified with tandem mass spectrometry. Both PMB and SDCS formed micelles at 14 and 8 μg/ml, respectively. At the critical micelle concentration (CMC), the hydrodynamic diameter of 213 nm was obtained. PMB had a positive charge (+6 mV) while SDCS had a negative charge (‒33 mV). Increasing in SDCS content decreased the charges from ‒6 to ‒25 mV. FTIR revealed H-bonding between PMB and SDCS. The NMR spectra confirmed that chemical shifts of PMB and SDCS did not change. The hydrodynamic size of PMB-SDCS was from 193 to 318 d.nm. Our results suggest that the lower mole ratios of SDCS (< 15:1) were able to stabilize PMB and released PMB within 30 min. Moreover, 5:1 mole ratio maintained the antimicrobial activity against Pseudomonas aeruginosa (MBC = 2 μg/ml). PMB-SDCS micelles of particular mole ratio is able to provide physical and chemical stability of PMB. [ABSTRACT FROM AUTHOR]

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a highly selective and sensitive method for the quantification of kinase inhibitors, yet not widely available in clinical routine for therapeutic drug monitoring (TDM). To provide a more accessible alternative, a high-performance liquid chromatography method with ultraviolet/diode array detection (HPLC-UV/DAD) to quantify cabozantinib, dabrafenib, nilotinib and osimertinib, was developed and validated. Results were compared to LC-MS/MS. After liquid–liquid-extraction and reconstitution of the residue in 20 mM potassium dihydrogen phosphate (KH2PO4) (pH4.6), acetonitrile and methanol (50:25:25,v/v/v), chromatographic separation was achieved in 20.0 min using a Luna® C18(2)-HST column (100 × 2 mm, 2.5 μm), protected by a C18 guard column (4 × 2 mm) (column temperature: 30 °C, autosampler: 10 °C). Mobile phase A and B consisted of 20 mM KH2PO4 (pH4.9) and acetonitrile (9:1,v/v) and acetonitrile:20 mM KH2PO4 (pH4.9) (7:3,v/v), respectively. Gradient elution was performed at 200 µL/min. Analytes were quantified at 250, 280 and 330 nm, using sorafenib as internal standard. Calibration curves were linear (35–2,000 ng/mL). Method validation assays met requirements by U.S. Food and Drug Administration and European Medicines Agency. Compared to the more sensitive and specific LC-MS/MS, HPLC-UV/DAD showed a good correlation and a strong positive association (Kendall's tau 0.811¬–0.963, p < 0.05). Bland-Altman-plots revealed 100% (cabozantinib), 98.6% (dabrafenib), 98.6% (nilotinib) and 96.2% (osimertinib) of relative differences inside the limits of agreement. Regulatory agency criteria for sample reanalysis and cross validation were met (±20%-criterion:100% (cabozantinib), 94.3% (dabrafenib), 92% (nilotinib) and 84.6% (osimertinib). The developed HPLC-UV/DAD method is "fit-for-TDM" in clinical routine and serves as a genuine alternative to LC-MS/MS. [ABSTRACT FROM AUTHOR]

[Display omitted] • A strategy comprising three UHPLC-MS/MS methods was developed for measuring 43 mycotoxins. • Method validation was evaluated for all 43 mycotoxins in 12 complex food matrices. • The methods were applied for 72 dietary samples collected from the sixth China total diet study. • The most detected mycotoxins were DON, SMC, FB1, ZEN, BEA, ENNB1, and ENNB. • The 43 mycotoxins were accurately investigated in a total diet study for the first time. Mycotoxins are toxic metabolites produced by fungi that commonly contaminate foods. As recommended by the World Health Organization, total diet study (TDS) is the most efficient and effective way to estimate the dietary intakes of certain chemical substances for general populations. It requires sensitive and reliable analytical methods applicable to a wide range of complex food matrices and ready-to-eat dishes. A novel strategy with high selectivity and sensitivity, incorporating three methods based on ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), was designed for measuring 43 mycotoxins in dietary samples in a China TDS. The 43 mycotoxins were divided into 3 groups for analysis to achieve better performance. For each group, an UHPLC-MS/MS method was developed to determine the target compounds after clean-up by solid phase extraction. A total of 21 isotope internal standards were employed for accurate quantitation. Method validation in terms of linearity, selectivity, sensitivity, accuracy, and precision was performed for all the 43 mycotoxins in 12 complex food matrices. The limits of detection (LODs) and limits of quantitation (LOQs) were 0.002–1 ng mL−1 and 0.006–3 ng mL−1, respectively. The method recoveries of the 43 mycotoxins spiked in 12 food categories were in the range of 60.3%–175.9% after internal standard correction, with relative standard deviations (RSDs) below 13.9%. For practical application, this method was utilized for 72 dietary samples collected from 6 provinces in the 6th China TDS. More than 80% of the samples were found contaminated by mycotoxins. DON, SMC, FB 1 , ZEN, BEA, ENNB 1 , and ENNB were most detected. The proposed methods with high sensitivity, accuracy, and robustness provide powerful tools for multi-mycotoxin monitoring and dietary exposure assessment, allowing 43 mycotoxins, including some emerging mycotoxins, to be accurately investigated in a total diet study for the first time. [ABSTRACT FROM AUTHOR]

Water disinfection is an essential process that provides safe water by inactivating pathogens that cause waterborne diseases. However, disinfectants react with organic matter naturally present in water, leading to the formation of disinfection by-products (DBPs). Multi-analyte methods based on mass spectrometry (MS) are preferred to quantify multiple DBP classes at once however, most require extensive sample pre-treatment and significant resources. In this study, two analytical methods were developed for the quantification of 32 regulated and unregulated DBPs. A purge and trap (P&T) coupled with gas chromatography mass spectrometry (GC-MS) method was optimized that automated sample pre-treatment and analyzed volatile and semi-volatile compounds, including trihalomethanes (THMs), iodinated trihalomethanes (I-THMs), haloacetonitriles (HANs), haloketones (HKTs) and halonitromethanes (HNMs). LOQs were between 0.02-0.4 µg/L for most DBPs except for 8 analytes that were in the low µg/L range. A second method with liquid chromatography (LC) tandem mass spectrometry (MS/MS) was developed for the quantification of 10 haloacetic acids (HAAs) with a simple clean-up and direct injection. The LC-MS/MS direct injection method has the lowest detection limits reported (0.2-0.5 µg/L). Both methods have a simple sample pre-treatment, which make it possible for routine analysis. Hyperchlorination and uniform formation conditions (UFC) formation potential tests with chlorine were evaluated with water samples containing high and low TOC. Hyperchlorination formation potential test maximized THMs and HAAs while UFC maximized HANs. Ascorbic acid was found to be an appropriate quencher for both analytical methods. Disinfected drinking water from four water utilities in Alberta, Canada were also evaluated. [Display omitted] [ABSTRACT FROM AUTHOR]

Brachial-ankle pulse wave velocity (baPWV) is a noninvasive index of vascular aging. However, the metabolic profile underlying vascular aging has not yet been fully elucidated. The current study aimed to identify circulating markers of vascular aging as assessed by baPWV and to elucidate its mechanism from a metabolomic perspective in older adults. A total of 60 and 61 Chinese male participants aged ≥80 years were recruited to the metabolome and validation cohorts, respectively. The baPWV of participants was measured using an automatic waveform analyzer. Plasma metabolic profile was investigated using ultra-performance liquid chromatography coupled with triple quadrupole linear ion trap tandem mass spectrometry. Orthogonal partial least squares (OPLS) regression modeling established the association between metabolic profile and baPWV to determine important metabolites predictive of vascular aging. Additionally, an enzyme-linked immunosorbent assay was employed to validate the metabolites in plasma and culture media of vascular smooth muscle cells in vitro. OPLS modeling identified 14 and 22 metabolites inversely and positively associated with baPWV, respectively. These 36 biomarkers were significantly enriched in seven metabolite sets, especially in cysteine and methionine metabolism (p <0.05). Notably, among metabolites involved in cysteine and methionine metabolism, S-adenosylmethionine (SAM) level was inversely related to baPWV, with a significant correlation coefficient in the OPLS model (p <0.05). Furthermore, the relationship between SAM and vascular aging was reconfirmed in an independent cohort and at the cellular level in vitro. SAM was independently associated with baPWV after adjustments for clinical covariates (β = −0.448, p <0.001) in the validation cohort. In summary , plasma metabolomics identified an inverse correlation between SAM and baPWV in older males. SAM has the potential to be a novel biomarker and therapeutic target for vascular aging. • High-throughput targeted metabolomics of vascular aging. • OPLS regression analysis associate plasma metabolic profile to baPWV. • S-Adenosylmethionine strongly associated with vascular aging. • Cysteine and methionine metabolism is crucial in vascular aging. [ABSTRACT FROM AUTHOR]

The use of new psychoactive substances derived from ketamine is rarely reported in France. A chronic GHB, 3-MMC, and methoxetamine consumer presented a loss of consciousness in a chemsex context and was referred to the intensive care unit with a rapid and favorable outcome. To investigate the chemicals responsible for the intoxication, a comprehensive analysis was conducted on the ten plasma samples collected over a 29.5-hour period, urine obtained upon admission, a 2-cm hair strand sample, and a seized crystal. These analyses were performed using liquid chromatography hyphenated to high resolution tandem mass spectrometry operating in targeted and untargeted modes. Additionally, analyses using gas chromatography coupled to mass spectrometry and nuclear magnetic resonance were conducted to probe the composition of the seized crystal. The molecular network-based approach was employed for data processing in non-targeted analyses. It allowed to confirm a multidrug exposure encompassing GHB, methyl-(aminopropyl)benzofuran (MAPB), (aminopropyl)benzofuran (APB), methylmethcathinone, chloromethcathinone, and a new psychoactive substance belonging to the arylcyclohexylamine family namely deschloro-N-ethyl-ketamine (O-PCE). Molecular network analysis facilitated the annotation of 27 O-PCE metabolites, including phase II compounds not previously reported. Plasma kinetics of O-PCE allowed the estimation of the elimination half-life of ∼5 hours. Kinetics of O-PCE metabolites was additionally characterized, possibly useful as surrogate biomarkers of consumption. We also observed marked alterations in lipid metabolism related to poly consumption of drugs. In conclusion, this case report provides a comprehensive analysis of exposure to O-PCE in a multidrug user including kinetic and metabolism data in human. • First reported plasma kinetics of O-PCE and its main metabolites in humans. • First identification of plasma O-PCE metabolites in a poisoned patient using a molecular network approach. • Marked alteration in lipid metabolism after O-PCE use. • Multidrug user profile confirmed using hair analysis showing regular synthetic cathinone and recent O-PCE use. [ABSTRACT FROM AUTHOR]

The treatment of waterborne micropollutants, such as diclofenac, presents a significant challenge to wastewater treatment plants due to their incomplete removal by conventional methods. Ozonation is an effective technique for the degradation of micropollutants. However, incomplete oxidation can lead to the formation of ecotoxic by-products that require a subsequent post-treatment step. In this study, we analyze the susceptibility of micropollutant ozonation products to enzymatic digestion with laccase from Trametes versicolor to evaluate the potential of enzymatic treatment as a post-ozonation step. The omnipresent micropollutant diclofenac is used as an example, and the enzymatic degradation kinetics of all 14 detected ozonation products are analyzed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) and tandem mass spectrometry (MS2). The analysis shows that most of the ozonation products are responsive to chemo-enzymatic treatment but show considerable variation in enzymatic degradation kinetics and efficiencies. Mechanistic investigation of representative transformation products reveals that the hydroxylated aromatic nature of the ozonation products matches the substrate spectrum, facilitating their rapid recognition as substrates by laccase. However, after initiation by laccase, the subsequent chemical pathway of the enzymatically formed radicals determines the global degradability observed in the enzymatic process. Substrates capable of forming stable molecular oxidation products inhibit complete detoxification by oligomerization. This emphasizes that it is not the enzymatic uptake of the substrates but the channelling of the reaction of the substrate radicals towards the oligomerization of the substrate radicals that is the key step in the further development of an enzymatic treatment step for wastewater applications. [Display omitted] • Isolated enzymes effectively convert ozonation by-products of diclofenac. • Enzyme substrate spectrum and ozonation product chemistry match. • Different degradation efficiencies result from post-laccase reaction, not enzyme selectivity. • Channelling the post-reaction to oligomerization is the pathway to complete degradation. [ABSTRACT FROM AUTHOR]

Avian leukosis virus subgroup J (ALV-J) is a retrovirus that can cause immunosuppression and tumors in chicken. However, relative pathogenesis is still not clear. At present, metabolomics has shown great potential in the screening of tumor metabolic markers, prognostic evaluation, and drug target design. In this study, we utilize an untargeted metabolomics approach based on ultrahigh-performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) to analyze the metabolic changes in chicken embryo fibroblast (CEF) cells infected by ALV-J. We found that ALV-J infection significantly altered a wealth of metabolites compared with control group. Additionally, most of the differentially expressed metabolites belonged to lipid metabolism, purine nucleotide metabolism and amino acid metabolism. Among them, the proportion of lipid metabolites account for the highest proportion (around 31%). Results suggest that these changes may be conductive to the formation of virion, thereby promoting the replication of ALV-J. These data provided metabolic evidence and potential biomarkers for the cellular metabolic changes induced by ALV-J, and provided important insight for further understanding the replication needs and pathogenesis of ALV-J. [ABSTRACT FROM AUTHOR]

17-β-estradiol, involved in mesothelioma pathogenesis, and its precursors were explored as potential biomarkers for the early diagnosis of mesothelioma. Using enzyme-linked immunosorbent assay(ELISA) for 17-β-estradiol and ultra-high performance liquid chromatography/tandem mass spectrometry(UHPLC-MS/MS) for 19 17-β-estradiol precursors, a comprehensive analysis of 20steroid hormones was conducted in the serum of mesothelioma patients(n=67), asbestos-exposed healthy subjects(n=39), and non-asbestos-exposed healthy subjects(n=35). Bioinformatics analysis explored three potential serum biomarkers: 17-β-estradiol, DHEA-S, and androstenedione. The results revealed significant differences in 17-β-estradiol levels between mesothelioma patients and both non-asbestos-exposed and asbestos-exposed healthy subjects. No significant variations in serum 17-β-estradiol levels were observed among mesothelioma patients at different stages, suggesting its potential as an early diagnostic marker. 17-β-estradiol levels were similar in mesothelioma patients with environmental and occupational asbestos exposure, while males with occupational asbestos exposure exhibited significantly higher levels of 17-β-estradiol compared to females. Significant reduction in androstenedione and an increase in DHEA-S were observed in asbestos-exposed individuals compared to non-asbestos-exposed individuals. The analysis of DHEA-S-androstenedione-17-β-estradiol signature score showed an increase in asbestos-exposed individuals and mesothelioma patients compared to non-asbestos-exposed individuals, and this score effectively distinguished between the groups. The Cancer Genome Atlas data was utilized to analyze the expression of 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 genes. The findings indicated that mesothelioma patients with elevated gene values for 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 have a worse or better prognosis on overall survival, respectively. In conclusion, this study suggests 17-β-estradiol, DHEA-S, and androstenedione as biomarkers for mesothelioma risk and early diagnosis of mesothelioma in asbestos-exposed individuals, aiding timely intervention and improved care. [Display omitted] • E2 increased in healthy asbestos-exposed subjects and MPM patients. • DHEA-S-androstenedione-E2 signature score increased in asbestos-exposed subjects. • The signature differentiates subjects exposed to asbestos from those not exposed. • DHEA-S-androstenedione-E2 as biomarkers of MPM risk and early diagnosis of MPM. [ABSTRACT FROM AUTHOR]