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[Display omitted] Nanoparticles (NPs) have broad application prospects in the field of biomedicine due to their excellent physicochemical properties. When entering biological fluids, NPs inevitably encountered proteins and were subsequently surrounded by them, forming the termed protein corona (PC). As PC has been evidenced to have critical roles in deciding the biological fates of NPs, how to precisely characterize PC is vital to promote the clinical translation of nanomedicine by understanding and harnessing NPs' behaviors. During the centrifugation-based separation techniques for the PC preparation, direct elution has been most widely used to strip proteins from NPs due to its simpleness and robustness, but the roles of multifarious eluents have never been systematically declared. Herein, seven eluents composed of three denaturants, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and urea (Urea), were applied to detach PC from gold nanoparticles (AuNPs) and silica nanoparticles (SiNPs), and eluted proteins in PC have been carefully characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and chromatography coupled tandem mass spectrometry (LC-MS/MS). Our results showed that SDS and DTT were the main contributors to the efficient desorption of PC on SiNPs and AuNPs, respectively. The molecular reactions between NPs and proteins were explored and verified by SDS-PAGE analysis of PC formed in the serums pretreated with protein denaturing or alkylating agents. The proteomic fingerprinting analysis indicated the difference of the eluted proteins brought by the seven eluents was the abundance rather than the species. The enrichment of some opsonins and dysopsonins in a special elution reminds us that the possibility of biased judgments on predicting NPs' biological behaviors under different elution conditions. The synergistic effects or antagonistic effects among denaturants for eluting PC were manifested in a nanoparticle-type dependent way by integrating the properties of the eluted proteins. Collectively, this study not only underlines the urgent need of choosing the appropriate eluents for identifying PC robustly and unbiasedly, but also provides an insight into the understanding of molecular interactions during PC formation. [ABSTRACT FROM AUTHOR]

6-nitrodopamine (6-ND) is released from rat isolated atria, where it acts as a potent positive chronotropic agent. The release of 6-ND from rat isolated atria and ventricles is significantly reduced when pre-incubated with l -NAME, and the release was not affected by tetrodotoxin pre-treatment, indicating that in the heart, the origin of 6-ND is not neurogenic. Since l -NAME inhibits all three isoforms of NO synthase, it was investigated the basal release of 6-ND from isolated atria and ventricles from nNOS−/−, iNOS−/− and eNOS−/− mice of either sex. The release of 6-ND was measured by LC-MS/MS. There were no significant differences in the 6-ND basal release from isolated atria and ventricles from male control mice, as compared to female control mice. The 6-ND release from atria obtained from eNOS−/− mice was significantly reduced when compared to atria obtained from control mice. The 6-ND release in nNOS−/− mice was not significantly different compared to control animals whereas the 6-ND release from atria obtained from iNOS−/− mice was significantly higher when compared to control group. Incubation of the isolated atria with l -NAME caused a significant decrease in the basal atrial rate of control, nNOS−/−, and iNOS−/− mice, but not in eNOS−/− mice. The results clearly indicate that eNOS is the isoform responsible for the synthesis of 6-ND in the mice isolated atria and ventricles and supports the concept that 6-ND is the major mechanism by which endogenous NO modulates heart rate. • Mouse isolated atria and ventricles present basal release of 6-nitrodopamine, as detected by LC-MS/MS. • The release of 6-nitrodopamine is decreased in atria and ventricles obtained from eNOS−/− mice. • The release of 6-nitrodopamine is not affected in atria and ventricles obtained from nNOS−/− mice. [ABSTRACT FROM AUTHOR]

Fibrinogen-related proteins (FREPs) have been identified in several animals. They are involved in the body's defense, acting as mediators of phagocytosis. Ficolins and intelectins are some of the most studied Fibrinogen-related Domain (FReD)-containing lectins. In this work, we have isolated a singular FReD-containing lectin, which cannot be classified as ficolin or intelectin. ELL (Echinometra lucunter lectin) was isolated from coelomic plasma by affinity chromatography on xanthan gum. Primary structure was determined by tandem mass spectrometry. Moreover, antimicrobial activity of ELL was evaluated against planktonic cells and biofilm of Escherichia coli , Staphylococcus aureus and S. epidermidis. ELL showed hemagglutinating activity in Ca2+ presence, which was inhibited by glycoprotein mucin and thyroglobulin. Complete amino acid sequence consisted of 229 residues, including a FReD in the N-terminal. Searches for similarity found that ELL was very close to putative proteins from Strongylocentrotus purpuratus. ELL showed moderate similarity with uncharacterized sea stars proteins and protochordate intelectins. ELL was able to inhibit the planktonic growth of the Gram-positive bacteria and significantly reduce the biofilm formation of all bacteria tested. In conclusion, we identified a new type of FReP-containing lectin with some structural and functional conservation towards intelectins. • A Fibrinogen-related protein (FREP) was isolated from sea urchin Echinometra lcuunter. • ELL she is the first member of a new family of FREPs found in echinoderms. • ELL seems to be involved in the defense of the E. lucunter. • ELL was able to inhibit the planktonic growth of the Gram-positive bacteria. [ABSTRACT FROM AUTHOR]

DNA polymorphic markers and self-defined ethnicity groupings are used to group individuals with shared ancient geographic ancestry. Here we studied whether ancestral relationships between individuals could be identified from metabolic screening data reported by the California newborn screening (NBS) program. NBS data includes 41 blood metabolites measured by tandem mass spectrometry from singleton babies in 17 parent-reported ethnicity groupings. Ethnicity-associated differences identified for 71% of NBS metabolites (29 of 41, Cohen's d > 0.5) showed larger differences in blood levels of acylcarnitines than of amino acids (P < 1e-4). A metabolic distance measure, developed to compare ethnic groupings based on metabolic differences, showed low positive correlation with genetic and ancient geographic distances between the groups' ancestral world populations. Several outlier group pairs were identified with larger genetic and smaller metabolic distances (Black versus White) or with smaller genetic and larger metabolic distances (Chinese versus Japanese) indicating the influence of genetic and of environmental factors on metabolism. Using machine learning, comparison of metabolic profiles between all pairs of ethnic groupings distinguished individuals with larger genetic distance (Black versus Chinese, AUC = 0.96), while genetically more similar individuals could not be separated metabolically (Hispanic versus Native American, AUC = 0.51). Additionally, we identified metabolites informative for inferring metabolic ancestry in individuals from genetically similar populations, which included biomarkers for inborn metabolic disorders (C10:1, C12:1, C3, C5OH, Leucine-Isoleucine). This work sheds new light on metabolic differences in healthy newborns in diverse populations, which could have implications for improving genetic disease screening. • Ancestral relationships are identified using newborn metabolic screening data. • Machine learning of metabolic screening data could infer metabolic ancestry. • Knowledge of ethnic variability in metabolic screening markers informs NBS. • Ethnic diversity should be considered when establishing NBS metabolite cutoffs. [ABSTRACT FROM AUTHOR]

Fabry disease (FD) is a rare X-linked lysosomal storage disease caused by mutations in the α-galactosidase A (GLA) gene leading to deficiency of α-galactosidase A (α-gal A). This results in progressive multisystemic glycosphingolipid accumulation, especially globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). Enzyme replacement therapy with two recombinant enzymes, agalsidase-α and –β is approved for two different dosages. However, little is known about which enzyme is more effective in decreasing the metabolite load in male and female patients with the classic form of the disease. In this prospective observational study, 14 consecutive adult Fabry patients (10 males) with a classic GLA -mutation, were switched from agalsidase–α to agalsidase–β at the respective licensed doses. Lyso-Gb3 levels were measured before the switch and for a period of 12 months after the switch in dried blood spots by tandem mass spectrometry. Mean age at start of the switch was 36.7 ± 14 years. Plasma Lyso-Gb3 levels decreased from 27.2 ± 17.9 ng/mL before the switch to 16.8 ± 10.5 ng/mL after the switch (mean reduction of 30.1%; p = 0.004). The decrease was maximal in the subgroup of 7 male patients with no or very low residual enzyme activity (mean reduction of 40.4%). However, two females with high residual enzyme activity also showed a reduction >30% after the switch. In male patients, the reduction of plasma Lyso-Gb3 correlated negatively with the residual α-gal A activity: r = −0.803; p = 0.009. Agalsidase-β at licensed dose is significantly more effective than agalsidase-α to reduce Lyso-Gb3 levels in classic Fabry patients, and should be used as first line therapy in classic males with no residual enzyme activity. [ABSTRACT FROM AUTHOR]

Glyphosate is the most widely used herbicide worldwide, both in domestic and industrial settings. Experimental research in animal models has demonstrated changes in muscle physiology and reduced contractile strength associated with glyphosate exposure, while epidemiological studies have shown associations between glyphosate exposure and adverse health outcomes in critical biological systems affecting muscle function. This study used data from a nationally representative survey of the non-institutionalized U.S. general population (NHANES, n = 2132). Urine glyphosate concentrations were determined by ion chromatography with tandem mass spectrometry. Hand grip strength (HGS) was measured using a Takei Dynamometer, and relative strength estimated as the ratio between HGS in the dominant hand and the appendicular lean mass (ALM) to body mass index (ALMBMI) ratio. Low HGS and low relative HGS were defined as 1 sex-, age- and race-specific SD below the mean. Physical function limitations were identified as significant difficulty or incapacity in various activities. In fully-adjusted models, the Mean Differences (MD) and 95% confidence intervals [95%CI] per doubling increase in glyphosate concentrations were −0.55 [-1.09, −0.01] kg for HGS in the dominant hand, and −0.90 [-1.58. −0.21] kg for HGS/ALMBMI. The Odds Ratios (OR) [95% CI] for low HGS, low relative HGS and functional limitations by glyphosate concentrations were 1.27 [1.03, 1.57] for low HGS; 1.43 [1.05; 1.94] for low relative HGS; 1.33 [1.08, 1.63] for stooping, crouching or kneeling difficulty; 1.17 [0.91, 1.50] for lifting or carrying items weighting up to 10 pounds difficulty; 1.21 [1.01, 1.40] for standing up from armless chair difficulty; and 1.47 [1.05, 2.29] for ascending ten steps without pause difficulty. Glyphosate exposure may be a risk factor for decreased grip strength and increased physical functional limitations. More studies investigating the influence of this and other environmental pollutants on functional aging are needed. [Display omitted] • Population strength decline in recent decades is not explained by changes in lifestyle. • Previous experimental research shows altered muscle physiology with glyphosate exposure. • Higher glyphosate exposure is linked to reduced grip strength in the US population. [ABSTRACT FROM AUTHOR]

Legumes are a good source of bioactive compounds, including peptides with antidiabetic potential. The α-glucosidase inhibitory activity of simulated gastrointestinal digested (GID) soybean, chickpea, green pea, and navy bean was determined after using three different household cooking methods (conventional, pressure, and microwave cooking). Samples were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC), and the fractions responsible for the α-glucosidase inhibitory activity were isolated. Lastly, peptides were identified by mass spectrometry in tandem (MS/MS) and in silico analyses were done to hypothesise potentially bioactive sequences. The results indicated that all legume extracts exert α-glucosidase inhibitory activity after thermal treatment and GID. Peptide profiles obtained by RP-HPLC showed the highest generation of peptides after the intestinal digestion phase, with small changes between thermal treatments. Best α-glucosidase inhibitory activity was observed in fraction 2 of all gastrointestinal digested samples, with values between 40 % and 62 % inhibition. In soybean, green pea, and navy bean, conventional-cooked samples showed the highest activity, while the pressure-cooked treatment resulted in significantly higher activity in chickpea samples. Finally, 48 peptides were identified by MS/MS and 13 were found as potentially bioactive using in silico tools, expanding previous knowledge on antidiabetic peptides derived from legumes. • Legumes exert α-glucosidase inhibitory activity after cooking and digestion. • Thermal treatment did not affect the peptide profile of intestinal-digested legumes. • The activity of legumes was concentrated in a polar fraction from RP-HPLC. • 13 of 48 peptides identified by MS/MS were predicted in silico as bioactives. [ABSTRACT FROM AUTHOR]

In recent years, there has been increasing interest in the use of red beets as a valuable source of bioactive compounds. Although saponins may be responsible for the pharmacological and nutraceutical activities of red beets, information on the saponin composition of various red beet cultivars is very limited, and the saponin profiles remain to be fully elucidated. Here, we profiled triterpene saponins from the flesh and skin of different red beetroot cultivars, whose names of Polish cv. are Bonel, Chrobry and Nochowski. In addition, the total triterpene saponin contents in cultivars Bonel, Chrobry, Nochowski and in their various root parts were determined. Triterpene saponins isolated from the skin and flesh of different red beetroot cultivars were qualitatively profiled using two high-performance liquid chromatography systems (HPLC-ESI-IT MS and UPLC-HESI-Q-Orbitrap MS). The total triterpene saponin contents in different cultivars were analyzed using high-performance liquid chromatography-tandem mass spectrometry. In total, 40 triterpene saponins were tentatively identified, and among them, 5 compounds were potentially new saponins, not previously reported in red beets. These saponins are new to red beets and were reported in B. vulgaris for the first time. The analyzed cultivars were different in terms of their total saponin content. The results of our study indicated that the variation in the composition of saponins depended on the red beet cultivars and part of the roots. Higher concentrations of these compounds were found in the skins than in the flesh of roots. We developed an analytical strategy to discover and identify triterpene saponins from B. vulgaris L. using a combination of techniques to detect the comprehensive fragmentation pathways of the saponins present at low concentrations in plant material. • Five triterpene saponins were identified as potentially new compounds of red beet. • The content and composition of saponins differ between red beet cultivars. • The triterpene saponins content were higher in the skin than in the flesh. • 40 triterpene saponins, including 8 pairs of isomers, were tentatively identified. • All compounds of cv. Chrobry and Bonel were glycosides with different aglycone structures. [ABSTRACT FROM AUTHOR]

[Display omitted] • Seven new compounds with four different structural classes were isolated from Talaromyces scorteus. • The configurations of new compounds were elucidated by crystallography, NOESY, and quantum-chemical analyses. • Scortide B (2) and talascortene I (4) significantly inhibited the pathogenic fungus Curvularia spicifera. Marine natural products play an important role in biopesticides. Seven new secondary metabolites with different structural classes, including two cycloheptapeptides, scortide A (1) and scortide B (2), two 19-nor-diterpenoids, talascortene H (3) and talascortene I (4), two diterpenoid acids, talascortene J (5) and talascortene K (6), and one triterpenoid, talascortene L (7) were isolated and identified from the sea-anemone-derived endozoic fungus Talaromyces scorteus AS-242. Their structures were comprehensively assigned by spectroscopic data analysis, single-crystal X-ray diffraction, tandem mass spectrometry, and electronic circular dichroism (ECD) calculations. The result of the antimicrobial assay demonstrated that compounds 1 − 6 have inhibitory activity against several human, aquatic, and plant pathogens with minimum inhibitory concentration (MIC) values ranging from 1 to 64 μg/mL. Specially, compounds 2 and 4 showed significant activities against the pathogenic fungus Curvularia spicifera with the MIC value of 1 μg/mL, providing an experimental basis of 2 and 4 with the potential as lead compounds to be developed into biopesticides. [ABSTRACT FROM AUTHOR]

Oncoprotein SE translocation (SET) is frequently overexpressed in different types of tumors and correlated with poor prognosis of cancer patients. Targeting SET has been considered a promising strategy for cancer intervention. However, the mechanisms by which SET is regulated under cellular conditions are largely unknown. Here, by performing a tandem affinity purification-mass spectrometry (TAP-MS), we identify that the ubiquitin-specific protease 7 (USP7) forms a stable protein complex with SET in cancer cells. Further analyses reveal that the acidic domain of SET directly binds USP7 while both catalytic domain and ubiquitin-like (UBL) domains of USP7 are required for SET binding. Knockdown of USP7 has no effect on the mRNA level of SET. However, we surprisingly find that USP7 depletion leads to a dramatic elevation of SET protein levels, suggesting that USP7 plays a key role in destabilizing oncoprotein SET, possibly through an indirect mechanism. To our knowledge, our data report the first deubiquitinase (DUB) that physically associates with oncoprotein SET and imply an unexpected regulatory effect of USP7 on SET stability. [ABSTRACT FROM AUTHOR]

In this study, a novel antifreeze peptide was isolated from crayfish shells and its antifreeze mechanism and activity were investigated. Firstly, the crayfish shells peptides (CSPs) were prepared by Enzymatic Hydrolysis Technology (alkaline protease and trypsin) and the CSPs could significantly enhance the survival rate of Saccharomyces cerevisiae from 12.5% to 88.5% after 24h freeze-thaw cycle. Then, a total 1004 peptides were identified from CSPs by liquid chromatography/tandem mass spectrometry (LC-MS/MS), among those peptides, two peptide sequences YWDHPIRDGFAPH (AFP1) and GPPGKPGIPDIVDW (AFP1) were selected as potential antifreeze polypeptides (AFPs) according the physicochemical properties: instability index, hydrophilicity, and confidence level. Furthermore, the antifreeze activity and mechanism of AFP1 and AFP2 were studied through molecular dynamics simulation, revealing that the AFP1 and AFP2 could adsorb to ice surface with 7 and 14 hydrogen bonds, respectively. Moreover, AFP2 has a more stable binding capacity and could interact with 33 water molecules. At last, the results of differential scanning calorimetry (DSC) and cold-stage polarization microscope indicated that the thermal hysteresis (TH) values of AFP2 were 1.62–2.09 °C and could inhibit ice recrystallization. Therefore, this study provides a theoretical basis for developing safe peptide-based cryoprotectants which will improve the quality of frozen foods. [Display omitted] • A novel antifreeze polypeptide (AFP2) was derived from CPS. • Crayfish shell peptides (CPS) showed good antifreeze for S.cerevisiae. • AFP2 bound to ice crystals and water molecules to form 14 and 56 hydrogen bonds. • AFP2 possessed significant thermal hysteresis value of 2.09 °C and inhibiting ice crystal growth. [ABSTRACT FROM AUTHOR]

Florfenicol is a broad-spectrum antibiotic commonly used in livestock and poultry. However, its embryotoxicity and hematotoxicity are widely concerned. In this study, a monoclonal antibody (mAb) with high specificity to florfenicol was generated. A highly sensitive and specific quantum dots lateral flow immunoassay (QDs-LFIA) was established, which can qualitatively or quantitatively detect the residual amount of florfenicol in food. The optimal method parameters were determined by optimizing the activated pH, mAb diluents, mAb dosage, probe amount and standard diluents. The visual limit of detection (vLOD) and the instrumental limit of detection (LOD) for florfenicol in QDs-LFIA were 3.2 and 0.02 ng mL−1, respectively. The detection time for the test strip is 6 min. The cross reactivities (CRs) with common structurally similar compounds were below 1.6%. The average recovery rates for pork, eggs, and milk were 90.53% to 114.9%. The sample pretreatment method is simple, fast, and takes no more than 30 min. A comparison between the QDs-LFIA and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) exhibited a high level of agreement. This approach can meet the needs of specific, rapid, and visual detection of florfenicol in the field. Homology modeling and molecular docking were used to explore the main forces and key amino acids between the antibody and florfenicol, as well as structurally similar compounds. These results contribute to the understanding of the antigen-antibody binding mechanism. [Display omitted] • A mAb-based quantum dots lateral flow immunoassay for florfenicol (FF) detection. • A rapid detection method of FF in food, with high specificity and sensitivity. • Homology modeling and molecular docking conducted to explore the mAb structure. • Key amino acid residues and major binding forces of the antibody were revealed. [ABSTRACT FROM AUTHOR]

A highly sensitive, efficient and rugged analytical method was developed and validated for determination of residues of more than 400 multiclass pesticides in honey using QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach in combination with liquid and gas chromatography coupled to tandem mass spectrometry (LC-MS/MS and GC-MS/MS). Various combinations of extracting solvents and cleanup sorbents were evaluated to optimize the sample preparation step. The method involving acetonitrile as extracting solvent and primary secondary amine (PSA) with magnesium sulphate (MgSO 4) as cleanup sorbent proved to be the most effective. The analytical method was validated at 5, 10, and 20 µg kg−1 in accordance with SANTE/11312/2021 and FDA guidelines and found suitable for simultaneous determination of 206 pesticides on LC-MS/MS and 197 pesticides on GC-MS/MS with acceptable recovery (70–120%), precision (RSD <20.0%), linearity (correlation coefficient ≥ 0.99) at limit of quantification (LOQ) ranging from 5 and 10 µg kg−1. The method was successfully applied to monitor pesticide residues in 80 market honey samples collected from different parts of India. The developed method enabled the quantitative determination of a wide spectrum of pesticide residues in honey in a single chromatographic run. [Display omitted] • Multi-residue method was developed for determination of > 400 pesticides in honey. • Method validation was done using LC-MS/MS and GC-MS/MS. • Extraction and cleanup was done using QuEChERS based method. • ACN in combination with PSA gave best results for recovery. • Method implemented successfully on 80 honey samples collected from Indian markets. [ABSTRACT FROM AUTHOR]

The aim of this survey was to evaluate the residue levels, distribution and exposure risk of the 38 most commonly used pesticides in rapeseed samples collected from the main production areas in China over a two-year period. The sampling area covered 12 provinces, including Guizhou, Shaanxi, Yunnan, Hunan, Jiangxi, Sichuan, Chongqing, Anhui, Henan, Hubei, Zhejiang, and Jiangsu provinces. The pesticide residues were determined using a QuEChERS (Quick Easy Cheap Effective Rugged and Safe) method coupled with gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry. 8.4% of the rapeseed samples contained pesticides with a residue level ranging from 0.001 to 0.634 mg/kg. The detected analytes were imidacloprid, quizalofop-P-ethyl, thiamethoxam, paclobutrazol, prochloraz, tebuconazole, difenoconazole, s-metolachlor, carbofuran, and carbendazim. The concentrations of four analytes, including thiamethoxam, difenoconazole, carbendazim and prochloraz, exceeded the maximum residue level set by the Chinese government for rapeseed, with exceedance rates of 0.1%, 0.1%, 0.1%, and 1.1%, respectively. Based on the index of quality for residues (IqR) values, 91.6% of the total rapeseed samples had an IqR category of Excellent (IqR = 0). Only 1.5% of the tested samples were of inadequate quality. Furthermore, the assessment of chronic and acute exposure, as well as health risks associated with pesticide residues in rapeseed, was conducted for different age groups within the Chinese population, including adults (6–14 years), children (15–49 years), and the elderly (50–74 years). The results of this assessment indicated that pesticide residues in rapeseed cultivated in China are not expected to be of short- or long-term risks to the Chinese customers. • A study of the pesticide residues in rapeseeds in China was conducted. • Levels of pesticide residue showed significant differences between studied areas. • The risk of chronic and acute dietary exposure to pesticides is negligible. [ABSTRACT FROM AUTHOR]

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency has been a target of expanded newborn screening (ENBS) using tandem mass spectrometry in Japan. Since the implementation of ENBS, a number of novel ACADVL variants responsible for VLCAD deficiency have been identified. In this study, genotypic differences in Japanese patients with VLCAD deficiency were investigated before and after ENBS. The ACADVL variants in 61 subjects identified through ENBS (ENBS group) and in 40 patients who subsequently developed clinical symptoms without undergoing ENBS (pre-ENBS group) were compared. Subjects in the ENBS group underwent genetic testing and/or VLCAD enzyme activity measurements. Patients in the pre-ENBS group were stratified into three clinical phenotypes and underwent genetic testing. This study revealed that the variants p.K264E, p.K382Q and c.996dupT were found in both groups, but their frequencies were lower in the ENBS group (5.2%, 3.1% and 4.2%, respectively) than in the pre-ENBS group (16.5%, 12.7% and 10.1%, respectively). In addition, p.C607S, p.T409M, p.M478I, p.G289R, p.C237R, p.T260M, and p.R229* were exclusively identified in the ENBS group. Among these variants, p.C607S exhibited the highest frequency (18.8%). The patients who were heterozygous for p.C607S demonstrated 7–42% of control enzyme activity. p.C607S is suspected to be unique to Japanese individuals. According to a comparison of enzyme activity, patients with the p.C607S variant may exhibit higher enzyme activity than those with the p.A416T, p.A180T, p.R450H, and p.K264E variants, which are responsible for the myopathic form of the disease. The VLCAD deficiency genotypes have changed since the initiation of ENBS in Japan. [ABSTRACT FROM AUTHOR]

To explore the metabolic mechanism of differential plasma interleukin (IL)-6 expression in patients with rheumatoid arthritis (RA). A total of 240 RA patients were enrolled in the non-target metabolomics study cohort and 69 healthy volunteers were included as healthy controls (HCs). Plasma IL-6 levels were detected by electrochemiluminescence assay. Plasma metabolites were detected by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Patients with active RA (n = 20) and remissive RA (n = 20) and 20 HCs were enrolled in the targeted validation cohort. Metabolites identified by non-target metabolomics were quantitatively analyzed by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry. Effects of 1-oleoyl- sn -glycero-3-phosphocholine (OGPC) associated with IL-6 on MH7A cells were assessed. After 24-h or 48-h induction by TNF-α, the supernatants were collected for IL-6 quantification by enzyme-linked immunosorbent assay. Furthermore, Western blot was performed to investigate the relative JAK2 and p -JAK2 expressions. With an increasing IL-6 level, OGPC shown to be related to the glycerophospholipid metabolism pathway by Kyoto Encyclopedia of Genes and Genomes analysis displayed a significant decrease. In the validating RA cohort, the OGPC concentrations in remissive RA group and active RA group decreased compared with HC group. OGPC down-regulated IL-6 secretion and p -JAK2 expression in TNF-α-induced MH7A cells in vitro. In conclusion, glycerophospholipid metabolism is the main metabolic pathway associated with the differential IL-6 expression in RA patients. The down-regulated OGPC is a promoting factor for the increased IL-6 plasma level in RA patients, which further affects the downstream JAK signaling pathway. • Abnormal glycerophospholipid metabolism occurs in patients with rheumatoid arthritis. • OGPC negatively correlated with IL-6 in patients with rheumatoid arthritis. • OGPC had an inhibitory effect on MH7A cells through the IL-6/JAK signaling pathway. [ABSTRACT FROM AUTHOR]

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder that results in reduced activity of guanidinoacetate methyltransferase, an accumulation of guanidinoacetate (GUAC), and a lack of cerebral creatine (CRE). Lack of CRE in the brain can cause intellectual disability, autistic-like behavior, seizures, and movement disorders. Identification at birth and immediate therapy can prevent intellectual disability and seizures. If started early in life, treatment with creatine supplements is highly effective. Because there are reliable biomarkers for GAMT deficiency, GUAC and CRE, and because the disorder is readily treatable with a significant improvement in outcomes, GAMT deficiency is an excellent candidate for newborn screening. Several programs have conducted pilot programs or started screening. An isobaric interferant of the GUAC marker has been reported which may cause false positive results. To reduce the number of false positives, a second-tier HPLC test to separate GUAC from unknown, isobaric interferants may be incorporated into the screening algorithm. New York State began screening for GAMT deficiency in October 2018 using a three-tiered screening approach. Quantification of GUAC and CRE were incorporated into routine screening for amino acids and acylcarnitines. In the first year of screening a total of 263,739 samples were tested for GAMT deficiency. Of these, 3382 required second tier testing. After second tier testing, 210 repeat specimens were requested for borderline results and 10 referrals were made to specialty care centers for confirmatory testing. In the first year of screening there were no confirmed cases of GAMT deficiency detected. To reduce the number of samples needing second tier testing and the number false positives we explored the use of a second MS transition to confirm the identity of the GUAC marker. GUAC and its internal standard are detected as butylated esters after sample preparation and derivatization. The original method used transition of the GUAC molecular ion of m / z 174.1 to a reactant ion of m/z 101.1. To confirm the identity of the GUAC marker we selected a qualifier ion of 174.1 > 73. The alternative product ion results were found to agree more closely with the second tier HPLC-MS/MS results for GUAC. It was found that the alternative transition may be used for quantification of the GUAC marker with acceptable analytical performance (linearity, accuracy, and precision). On March 5, 2020, the method of analysis for GUAC was modified to use the alternative product ion. For a comparable 6-month period, the modified method reduced the number of samples requiring second tier testing by 98%, reduced the number of borderline results requiring a repeat sample by 87.5%, and reduced the number of referrals to specialty care centers by 85%. Using the modified method, the correlation (r-squared) of the first and second tier screening results for GUAC is greater than 0.95. Since the first-tier results correlate well with the second-tier results, the second-tier screening is no longer necessary with the modified method. [ABSTRACT FROM AUTHOR]

• AFM stretching and SMD simulations are combined to study protein folding pathways. • Coiled-coils probes for the identification of the unfolding pathways of proteins are described. • Theoretical background and sample preparation for proteins force spectroscopy are provided. Most proteins in proteomes are large, typically consist of more than one domain and are structurally complex. This often makes studying their mechanical unfolding pathways challenging. Proteins composed of tandem repeat domains are a subgroup of multi-domain proteins that, when stretched, display a saw-tooth pattern in their mechanical unfolding force extension profiles due to their repetitive structure. However, the assignment of force peaks to specific repeats undergoing mechanical unraveling is complicated because all repeats are similar and they interact with their neighbors and form a contiguous tertiary structure. Here, we describe in detail a combination of experimental and computational single-molecule force spectroscopy methods that proved useful for examining the mechanical unfolding and refolding pathways of ankyrin repeat proteins. Specifically, we explain and delineate the use of atomic force microscope-based single molecule force spectroscopy (SMFS) to record the mechanical unfolding behavior of ankyrin repeat proteins and capture their unusually strong refolding propensity that is responsible for generating impressive refolding force peaks. We also describe Coarse Grain Steered Molecular Dynamic (CG-SMD) simulations which complement the experimental observations and provide insights in understanding the unfolding and refolding of these proteins. In addition, we advocate the use of novel coiled-coils-based mechanical polypeptide probes which we developed to demonstrate the vectorial character of folding and refolding of these repeat proteins. The combination of AFM-based SMFS on native and CC-equipped proteins with CG-SMD simulations is powerful not only for ankyrin repeat polypeptides, but also for other repeat proteins and more generally to various multidomain, non-repetitive proteins with complex topologies. [ABSTRACT FROM AUTHOR]

Aflatoxins (AFs) are toxic metabolic products of fungi found in feed and food. Sheep, goats and cattle can convert aflatoxin B1 (AFB1) to aflatoxin M1 (AFM1). AFB1 concentrations in feed contaminated with AFB1 result in high AFM1 levels in milk and dairy foods. In this study, milk samples (relatively 792) collected from the Mediterranean region of Turkey between 2020 and 2022 were evaluated for the presence of AFM1 whereas in feed AFB1, aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) contents were determined using ultra-high-performance liquid chromatography with fluorescence (UHPLC-FLD) and tandem mass spectrometry (LC-MS/MS). The results of the study indicated that of the 550 feed samples analyzed, only 74 (13.45 %) showed AFB1 values above the EU threshold limit of 5 µg/kg. AFs were detected in approximately more than half of the feed samples (234 feed samples), representing 42.54 %. AFB1 contents of 27.27 % of feed samples (150) ranged from 0.12 to 9.25 µg/kg. Out 234 of feed samples, 7.26 % contained AFB2, 22.22 % contained AFG1 and 5.12 % contained AFG2. A total of 113 milk samples taken in 3 days were positive for aflatoxin. The AFM1 levels of 62 samples taken after 24 h and 50 samples taken after 48 h were positive for aflatoxin. In addition, the number of the aflatoxin positive samples was found to be higher at the end of three days in June, July and August 2020 and in August 2021. Strict agricultural practices, proper harvesting and drying techniques, and frequent testing of feed components for the presence of aflatoxin can all help lower aflatoxin levels. [Display omitted] • The effects of feeds and seasons on AFs content of milks from Mediterranean region were determined. • 234 of 550 feed samples were positive for aflatoxin. • High AFB1 content of feeds were corn silage, cottonseed meal, peanut silage and corn. • AFM1 was positive in 113 of the total milk samples. • The AFs content in raw milk and feeds, especially summer time should be monitored regularly. [ABSTRACT FROM AUTHOR]

The regulation of solar radiation is a pivotal agricultural practice in enhancing the quality of grapes and subsequently, wine. In regions like Yantai, where solar radiation levels are typically low, the accumulation of wine phenolic compounds is negatively impacted. In light of this, our study sought to assess the efficacy of silver reflective mulch (RM) treatment on grape yield, maturity, and phenolic composition. Specifically, we applied RM treatment post-veraison to a Cabernet Gernischt (Vitis vinifera L.) vineyard in the Yantai wine region of China. Our findings indicate a consistent improvement in grape maturity with RM treatment, alongside enhanced accumulation of total phenols (TP), total tannins (TT), and total anthocyanins (TAC) in grape skin. Consequently, this led to elevated levels of phenolic compounds in wines and heightened color intensity. Moreover, tandem mass spectrometry analysis unveiled higher concentrations of key compounds such as petunidin-3-glucoside, malvidin-3-glucoside, procyanidin B1, quercetin-3-glucoside, cis -resveratrol, and piceatannol in the resultant wines. This study underscores the potential of RM treatment to bolster wine phenolics, particularly in regions characterized by a Temperate Monsoon Climate. ● Reflective mulch (RM) treatment was conducted at post-veraison in the vineyard of Yantai wine region of China. ● RM treatment hastened grape ripening and enhanced wine phenolic compounds including TP, TT and TAC. ● RM treatment consistently increased total flavan-3-ols in the two vintage. ● RM treatment significantly improved wine color intensity. [ABSTRACT FROM AUTHOR]

Due to methodological advances in the archaeological sciences, an increasing number of archaeological specimens undergo destructive sampling. However, the preservation of cultural heritage is a primary concern. This leads to a dilemma between accessing sample material and obtaining sufficient information for a meaningful analytical outcome. Ideally, sampling a specimen would preserve the object for further macro, micro, and molecular analyses. For palaeoproteomics, a number of minimally invasive sampling approaches have been proposed, representing different benefits and limitations. There have been studies comparing a selection of these protocols, however, these have focused on specimens from a homogenous preservation environment using Zooarchaeology by Mass Spectrometry (ZooMS). Here we expand on earlier work by extending the comparison to specimens from two highly different preservation environments through both ZooMS and liquid-chromatography tandem mass spectrometry (LC-MS/MS). We compare five sampling approaches and seven extraction protocols in total, on 10 Bos sp. humeri from the Early Neolithic site of La Draga, Spain, utilising MALDI-ToF MS and LC-MS/MS to generate proteomic output, while assessing protocol invasiveness using microscopy and 3D imaging. Five humeri originate from Sector A, which is mostly related to dry, terrestrial preservation conditions, while the other five humeri stem from Sector B, which is characterised by its phreatic/aquatic preservation conditions. We show that there is a significant difference in protein recovery and taxonomic specificity between the sampling techniques applied, as well as between burial conditions. Additionally, various surface modifications were observed depending on the specific sampling technique applied. It is therefore essential to assess protein preservation for each sedimentological context within an archaeological site before performing extensive sampling, as protein preservation can be highly inter- and intra-site-specific. • Preservation influences the proteomic outcome of different sampling methods. • Minimally invasive sampling extracts protein adequately if preservation is sufficient. • The HCl protocol produces the best proteomic results regardless of preservation state. • Minimally invasive sampling methods complicate use-wear analysis to various degrees. [ABSTRACT FROM AUTHOR]

Organic food consumption in children has been shown to reduce the body burden of chemical pesticides. However, there is little evidence of human health benefits associated with the consumption of organic foods. The objectives were to i) determine the effectiveness of an organic food intervention treatment in reducing the magnitude of an inflammation biomarker (C-reactive protein, CRP) in children (10–12 years) and ii) assess the association between the urinary biomarkers of exposure to pesticides and CRP. This work was part of the ORGANIKO cluster-randomized cross-over trial entailing a 40-day organic food treatment in healthy children. Urinary biomarkers of exposure to pesticides and inflammation (CRP) were measured using tandem mass spectrometry and ELISA immunoassay, respectively. Linear mixed-effect regression models of CRP were used to account for the effect and duration of organic food treatment. Multiple comparisons were handled using Benjamini-Hochberg correction. Results supported an anti-inflammatory effect of organic food treatment in children, albeit with mixed results, depending on the creatinine adjustment method; biomarker levels were divided by urinary creatinine (method a1), or urinary creatinine was used as a fixed effect variable (a2). In the a1 method, a time-dependent reduction for creatinine-adjusted CRP (β = −0.019; 95% CI: −0.031, −0.006; q = 0.045) was observed during the organic food intervention period. A statistically significant association (β = 0.104; 95% CI: 0.035, 0.173; q = 0.045) was found between the biomarker of pyrethroids exposure (3-PBA) and CRP inflammatory biomarker, but not for 6-CN. In the a2 method, similar trend of time-dependent reduction for creatinine-adjusted CRP (β = −0.008; 95% CI: −0.021, 0.004; p = 0.197) was observed during the organic food intervention period, but did not reach statistical significance (q > 0.05); the associations of pyrethroid and neonicotinoid biomarkers with CRP were not statistically significant (q > 0.05). More studies are warranted to sufficiently understand the potential anti-inflammatory response of an organic food treatment. • The anti-inflammatory action of a 40-day organic food treatment in healthy children was examined. • A time-dependent CRP reduction effect of the organic food treatment was observed for one of the two creatinine adjustment methods used. • More longitudinal studies are warranted to replicate observed trends for children's health. [ABSTRACT FROM AUTHOR]

Blueberry production and consumption are gradually increasing in China; however, the potential risks of pesticide residues are essentially unknown. In this study, a modified QuEChERS method was used for the analysis of twenty pesticides and their nine metabolites in blueberry by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) and gas chromatography with tandem mass spectrometry (GC-MS/MS). This method achieved high linearity (R 2 > 0.99), accuracy (average recovery, 70–125%), precision (relative standard deviation, RSD ≤12%), and sensitivity (limit of quantification, LOQ, 0.01 mg/kg). The dissipation of most pesticides fitted the first-order model, and the half-lives were in the range of 2.21–35.65 d. The residual levels of the analytes on the first day after application were below maximum residue limits (MRLs) on fruits, and ten pesticides with MRLs in China were categorized as low-risk pesticides according to the risk ranking. This study provides a reference for the rational application of pesticides for blueberry cultivation. • Methods for the determination of 29 analytes were well optimized and validated. • The half-lives ranged from 2.21 to 35.65 d during blueberry cultivation. • Pesticides with MRLs in China were identified to be low-risk based on risk ranking. [ABSTRACT FROM AUTHOR]

In this study, four plant matrices, including soybean, chickpea, pea, and lens, underwent fermentation by Bacillus subtilis var. natto. Then, the process influence on the content of four pesticides (clomazone, azoxystrobin, fluazifop-p-butyl, and quizalofop-p-ethyl) was evaluated, depending on the fermentation length. Fermentation was conducted for 312 h in controlled conditions of temperature and relative humidity (37 °C, 75%). The pesticide residue content in fermented seeds was assessed with the QuEChERS multiresidue method, combined with gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The studies demonstrated that Bacillus subtilis can biodegrade pesticides in legume seeds, with those microorganisms having a greater potential to degrade herbicides, especially fluazifop-p-butyl, and quizalofop-p-ethyl, than azoxystrobin and clomazone. The highest pesticide degradation occurred during first hours of fermentation. The quizalofop-p-ethyl and fluazifop-p-butyl levels reached < 0.02 mg/kg in nearly all studied seeds already after 48 h. At the end of fermentation (after 312 h), the azoxystrobin and clomazone levels ranged from 0.0203 mg/kg (soybean) to 0.0475 mg/kg (lens) and from 0.001 mg/kg (chickpeas) to 0.0753 mg/kg (lens), respectively. In all studied plant matrices, levels of pesticide residues were below the established MRLs, with clomazone being the sole exception. [Display omitted] • Fermentation with Bacillus subtilis can effectively reduce the pesticide residue content. • The greatest % degradation enhancement with respect to control occurred during the first hours of the process. • Azoxystrobin, clomazone, fluazifop-p-butyl and quizalofop-p-ethyl were determined in this study. [ABSTRACT FROM AUTHOR]

An orange-red fraction was obtained from fully sun-dried Ume (Prunus mume Sieb. et Zucc.) using a layer extraction method with acetone, methanol, and chloride as organic solvents. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis revealed a series of (-)-epicatechin (EC) dimers and trimers. The polymers were compared with published m/z negative mode and their fragments were characterized as procyanidin B2–2 (A-type dimer), procyanidin C1–2 (A-type trimer), and procyanidin C1–4 (dehydrotriepicatechin B1). High-performance liquid chromatography coupled with diode array detection (HPLC-DAD) analysis showed that the dehydrotriepicatechin B1 peak had an absorption maximum of 425 nm in the visible light range (400–600 nm). Similar results were obtained when salted Ume were subjected to simulated sunlight drying conditions using a low-temperature artificial drying system. Testing of blue light in an (-)-epicatechin (EC) model system showed the strongest catalytic effect. This study suggests that during sun-drying of salted Ume, (-)-epicatechin (EC) concentrates on the epidermis and undergoes photooxidative polymerization after sufficient sun exposure, further producing double bonds that cause the surface of sun-dried Ume to appear orange-red. This research has led to an innovative drying system that can replace sun-drying and avoid external factors such as insects, bacteria, meteorology, and food nutrition improvement that affect the quality of dried products. • Ume (Prunus mume) that has been dried in the sun develops a valuable orange-red hue. • Acetone extracts contain trimer compounds, detected by tandem mass spectrometry. • The visible spectrum absorption peak for HPLC-DAD is at 425 nm. • Blue light enhances photooxidation of trimer compounds in (-)-epicatechin model system. • The full spectrum artificial sun light effectively mimics traditional sun-drying. [ABSTRACT FROM AUTHOR]

Subretinal fluid (SRF) accumulates between photoreceptor outer segments and retinal pigment epithelium during rhegmatogenous retinal detachment. Biomolecular components such as lipids originate from cells surrounding the SRF. Knowledge of the composition of these molecules in SRF potentially provides mechanistic insight into the physiologic transfer of lipids between retinal tissue compartments. Using mass spectrometry and tandem mass spectrometry analysis on an electrospray ionization quadrupole-time-of-flight mass spectrometer, we identified a total of 115 lipid molecular species of 11 subclasses and 9 classes in two samples from two patients with rhegmatogenous retinal detachment. These included 47 glycerophosphocholines, 6 glycerophosphoethanolamines, 1 glycerophosphoinositol, 18 sphingomyelins, 9 cholesteryl esters, free cholesterol, 3 ceramides, 22 triacylglycerols and 8 free fatty acids. Glycerophosphocholines were of the highest intensity. By minimizing the formation of different adduct forms or clustering ions of different adducts, we determined the relative intensity of lipid molecular species within the same subclasses. The profiles were compared with those of retinal cells available in the published literature. The glycerophosphocholine profile of SRF was similar to that of cone outer segments, suggesting that outer segment degradation products are constitutively released into the interphotoreceptor matrix, appearing in SRF during detachment. This hypothesis was supported by the retinal distributions of corresponding lipid synthases' mRNA expression obtained from an online resource based on publicly available single-cell sequencing data. In contrast, based on lipid profiles and relevant gene expression in this study, the sources of free cholesterol and cholesteryl esters in SRF appeared more ambiguous, possibly reflecting that outer retina takes up plasma lipoproteins. Further studies to identify and quantify lipids in SRF will help better understand etiology of diseases relevant to outer retina. • Subretinal fluid is rich in glycerophosphocholine lipids. • The glycerophosphocholine lipid profile resembles that of cone outer segments. • Retinal cells express mRNA for synthases of subretinal fluid lipids. • Cone outer segments are a major source of subretinal fluid lipids. [ABSTRACT FROM AUTHOR]

Aromatic l-amino acid decarboxylase deficiency (AADCD) is a rare, autosomal-recessive neurometabolic disorder caused by variants in dopa decarboxylase (DDC) gene, resulting in a severe combined deficiency of serotonin, dopamine, norepinephrine, and epinephrine. Birth prevalence of AADCD varies by population. In pilot studies, 3- O -methyldopa (3-OMD) was shown to be a reliable biomarker for AADCD in high-throughput newborn screening (NBS) allowing an early diagnosis and access to gene therapy. To evaluate the usefulness of this method for routine NBS, 3-OMD screening results from the largest three German NBS centers were analyzed. A prospective, multicenter (n = 3) NBS pilot study evaluated screening for AADCD by quantifying 3-OMD in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). In total, 766,660 neonates were screened from January 2021 until June 2023 with 766,647 with unremarkable AADCD NBS (766,443 by 1st-tier analysis and 204 by 2nd-tier analysis) and 13 with positive NBS result recalled for confirmatory diagnostics (recall-rate about 1:59,000). Molecular genetic analysis confirmed AADCD (c.79C > T p.[Arg27Cys] in Exon 2 und c.215 A > C p.[His72Pro] in Exon 3) in one infant. Another individual was highly suspected with AADCD but died before confirmation (overall positive predictive value 0.15). False-positive results were caused by maternal L-Dopa use (n = 2) and prematurity (30th and 36th week of gestation, n = 2). However, in 63% (n = 7) the underlying etiology for false positive results remained unexplained. Estimated birth prevalence (95% confidence interval) was 1:766,660 (95% CI 1:775,194; 1:769,231) to 1:383,330 (95% CI 1:384,615; 1:383,142). The identified child remained asymptomatic until last follow up at the age of 9 months. The proposed screening strategy with 3-OMD detection in DBS is feasible and effective to identify individuals with AADCD. The estimated birth prevalence supports earlier estimations and confirms AADCD as a very rare disorder. Pre-symptomatic identification by NBS allows a disease severity adapted drug support to diminish clinical complications until individuals are old enough for the application of the gene therapy. [ABSTRACT FROM AUTHOR]

[Display omitted] Protein trafficking is a fundamental process with profound implications for both intracellular and intercellular functions. Proximity labeling (PL) technology has emerged as a powerful tool for capturing precise snapshots of subcellular proteomes by directing promiscuous enzymes to specific cellular locations. These enzymes generate reactive species that tag endogenous proteins, enabling their identification through mass spectrometry-based proteomics. In this comprehensive review, we delve into recent advancements in PL-based methodologies, placing particular emphasis on the label-and-fractionation approach and TransitID, for mapping proteome trafficking. These methodologies not only facilitate the exploration of dynamic intracellular protein trafficking between organelles but also illuminate the intricate web of intercellular and inter-organ protein communications. [ABSTRACT FROM AUTHOR]

[Display omitted] • BX17 secretes metabolites that antagonize the phytopathogenic fungus Fusarium graminearum. • BX17 proteome includes the metabolic machinery involved in the biosynthesis of bioactive metabolites. • BX17 strain synthesizes bioactive molecules probably involved in their ecology. • Antifungal metabolite is probably synthesized by Shikimate pathway in the BX17 strain. Soil-isolated Amycolatopsis sp. BX17 releases antifungal extracellular metabolites, capable of antagonizing the phytopathogenic fungus Fusarium graminearum and protecting maize plants from fungus colonization. A metabolomic analysis was performed to identify the extracellular metabolites produced by this strain. Also, a shot-gun proteomic analysis of axenic BX17 strain cultures was conducted using tandem mass spectrometry, and identified 1821 proteins of which 1039 were classified according to their biological function by KEGG. Results showed that central carbon metabolism, genetic information processing, environmental information processing, and amino acid metabolism were the most enriched pathways. Furthermore, an essential set of proteins was found related to the metabolism of terpenoids and polyketides. Overall, 20 compounds were found, and echinosporin identified in the global profile by MSE analysis. This evidence suggests that Amycolatopsis BX17 synthesize this antifungal molecule by converting of shikimate to chorismate. Our results advance our understanding of the mechanisms of bioactive compounds production in Amycolatopsis , and the metabolic machinery involved. [ABSTRACT FROM AUTHOR]

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells. [Display omitted] • Techniques for quantitating acetyl-coenzyme A (acetyl-CoA) were compared. • Commercial ELISA was unable to measure acetyl-CoA in standards and samples tested. • Fluorometric enzymatic assay varied from LC-MS depending on matrix and extraction. • High-throughput sample processing for LC-MS was achieved via automation. • Multiplexed analysis of acyl-coenzyme A metabolites was demonstrated in AML cells. [ABSTRACT FROM AUTHOR]

Polybrominated diphenyl ethers (PBDEs) are a group of widely used chemicals and humans are exposed to them in their daily life. PBDEs exposure during pregnancy may have adverse effects on pregnant women and their fetuses. Nevertheless, limited information is available on the levels and determinants of PBDEs exposure in Chinese pregnant women. The internal exposure levels of eight PBDEs (BDE-28, 47, 99, 100, 153, 154, 183, and 209) in placental samples of 1280 pregnant women from Zunyi birth cohort were analyzed using gas chromatography tandem mass spectrometry. All PBDEs concentrations were lipid adjusted (ng/g lw). Determinants of exposure were assessed by multivariable logistic regression model. Eight PBDE homologues were quantifiable in more than 70% of the samples. The highest median concentrations were found for BDE-209 (2.78 ng/g lw), followed by BDE-153 (1.00 ng/g lw) and BDE-183 (0.93 ng/g lw). The level of ΣPBDEs ranged from 0.90 to 308.78 ng/g lw, with a median concentration of 10.02 ng/g lw. Multivariate logistic regression analysis showed that maternal age older than 30 years old (OR: 1.59; 95% CI: 1.14, 2.23), pre-pregnancy obesity (1.51; 1.08, 2.10), home renovation within 2 years (1.43; 1.08, 1.91), spending more time outdoors during pregnancy (0.70; 0.55, 0.89), high consumption of fish/seafood (1.46; 1.13, 1.90) and eggs (1.44; 1.04, 2.00), male infant sex (1.69; 1.18, 2.42) were associated with PBDEs exposure. The study population is generally exposed to PBDEs, of which BDE-209 is the dominant congener, indicating extensive application of products containing deca-BDE mixtures. Maternal age, pre-pregnancy BMI, home decoration, average outdoor time during pregnancy, fish, seafood, eggs consumption, and fetal sex were exposure-determinning factors. This study contributes to the knowledge on region-specific PBDEs contamination in pregnant women and related risk factors. [Display omitted] • PBDEs remain ubiquitously present in pregnant women in Zunyi, Southwest China. • BDE-209 is the predominant homologue in human placenta. • PBDEs exposure significantly related with maternal age, BMI and fetal sex. • Increased exposure found in women with home decoration and less time outdoors. • Fish, seafood and eggs consumption significantly related with increased exposure. [ABSTRACT FROM AUTHOR]

Sex steroids are pervasive in mammals and evolutionarily conserved, but differences in the nuances of endocrine profiles characterize distinct species. Two sex steroids, testosterone and progesterone, feature prominently in the life history of mammalian taxa, but neither one has been analyzed from wild giraffes. Our study was designed to address this gap in knowledge by examining how these sex steroids are related to biological features and giraffe life history. We conducted the research at Rooipoort Nature Reserve a 44,000 ha private nature reserve in South Africa on a population of South African giraffes (Giraffa camelopardalis giraffa). Eleven adult giraffe cows and seven adult bulls were immobilized and various biological samples and morphological measurements were obtained. We analyzed both testosterone and progesterone using Ultra High Performance Liquid Chromotography – Tandem Mass Spectrometry (UHPLC-MS/MS). We found that non-pregnant females had lower progesterone concentrations than pregnant females and that those in the last trimester of pregnancy showed a slight drop in progesterone, as well as an increase in testosterone. Among males, chronological age was correlated with testicle size, testosterone concentrations, and ossicone volume. We propose that the progesterone decline functions partly to accelerate resumption of ovulation because giraffes become pregnant while lactating, and that the testosterone elevation provides an endocrine mileu for female defense of neonatal calves, given that lion predation is a major threat to calf survival. We suggest that male reproductive strategies are mediated by the age-related impacts of testosterone on growth in both body mass and ossicone volume as a consequence of sexual selection. We conclude by noting that the robust and solid ossicones of male giraffes function in mate competition as wedges that are used to topple opponents by raising their legs and placing them in an off-balanced position that can seriously injure them when falling to the ground. [ABSTRACT FROM AUTHOR]

The present study utilized a combined quick, easy, cheap, effective, rugged, safe (QuEChERS) method with ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) to detect and quantify pesticide residues in both whole banana fruit and pulp samples collected from five provinces in southern China between June 2018 and December 2021. The analysis identified the occurrence of 29 widely used insecticides, fungicides, and herbicides on bananas, including organophosphates, organochlorines, pyrethroids, triazoles, etc. The limits of quantification for the pesticides analyzed in the banana matrix ranged from 1.0 to 5.0 μg/kg. Among the 501 samples collected, the detection rates of pesticides in whole fruit and pulp samples were 65.1 % and 40.9 %, respectively. Specifically, 20 kinds of pesticides were detected in whole fruit samples, while 16 were detected in pulp samples. Furthermore, imidacloprid was detected across various regions and years. The evaluation of risk of hazard quotient (HQ) to the identified pesticides in whole fruit and pulp samples revealed values ranging from 0.0016-0.0313 and 0.0002–0.0131, respectively. Overall, the results suggest that the detected pesticide residues in bananas are unlikely to pose an unacceptable health risk to consumers of different age groups in China. [Display omitted] • QuEChERS combined UPLC-MS/MS and GC-MS/MS method was successfully validated. • Total 501 banana samples from five provinces of southern China were monitored. • The detection rates of pesticides in whole fruit and pulp samples were 65.1 % and 40.9 %, respectively. • Imidacloprid was detected in both whole fruit and pulp samples across different regions and years. • Chronic dietary risks were extremely low for different genders and ages of China. [ABSTRACT FROM AUTHOR]

Previously we developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay using dried blood spots for all subtypes of mucopolysaccharidoses (MPS) except MPS-IIID. Here we show that the MPS-IIID enzyme N -acetylglucosamine-6-sulfatase (GNS) is inhibited in dried blood spot (DBS) extracts, but activity can be recovered if the extract is diluted to reduce the concentrations of endogenous inhibitors. The new GNS assay displays acceptable characteristics including linearity in product formation with incubation time and amount of enzyme, low variability, and ability to distinguish MPS-IIID-affected from healthy patients using DBS. The assay can be added to the LC-MS/MS multiplex panel for all MPS subtypes requiring ∼2 min per newborn for the LC-MS/MS run. [ABSTRACT FROM AUTHOR]

Inborn errors of metabolism (IEMs) frequently result in progressive and irreversible clinical consequences if not be diagnosed or treated timely. The tandem mass spectrometry (MS/MS)-based newborn screening (NBS) facilitates early diagnosis and treatment of IEMs. The aim of this study was to determine the characteristics of IEMs and the successful deployment and application of MS/MS screening over a 19-year time period in Shanghai, China, to inform national NBS policy. The amino acids and acylcarnitines in dried blood spots from 1,176,073 newborns were assessed for IEMs by MS/MS. The diagnosis of IEMs was made through a comprehensive consideration of clinical features, biochemical performance and genetic testing results. The levels of MS/MS testing parameters were compared between various IEM subtypes and genotypes. A total of 392 newborns were diagnosed with IEMs from January 2003 to June 2022. There were 196 newborns with amino acid disorders (50.00%, 1: 5910), 115 newborns with organic acid disorders (29.59%, 1: 10,139), and 81 newborns with fatty acid oxidation disorders (20.41%; 1:14,701). Phenylalanine hydroxylase deficiency, methylmalonic acidemia and primary carnitine deficiency were the three most common disorders. Some hotspot variations in eight IEM genes (PAH, SLC22A5, MMACHC, MMUT, MAT1A, MCCC2, ACADM, ACAD8), 35 novel variants and some genotype-biochemical phenotype associations were identified. A total of 28 types of IEMs were identified, with an overall incidence of 1: 3000 in Shanghai, China. Our study offered clinical guidance for the implementation of MS/MS-based NBS and genetic counseling for IEMs in this city. [ABSTRACT FROM AUTHOR]

Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS. An IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genome-editing strategy targeting IVD exons 2–3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity. c.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p.Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants. We have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs. [ABSTRACT FROM AUTHOR]

Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited metabolic disorder that impairs the synthesis of creatine (CRE). Lack of CRE in the brain can cause intellectual disability, autistic-like behavior, seizures, and movement disorders. Identification at birth and immediate therapy can prevent intellectual disability and seizures. Here we report the first two cases of GAMT deficiency identified at birth by newborn screening (NBS) in Utah and New York. NBS dried blood spots were analyzed by tandem mass spectrometry (MS/MS) using either derivatized or non-derivatized assays to detect guanidinoacetate (GUAC) and CRE. For any positive samples, a second-tier test using a more selective method, ultra-performance liquid chromatography (UPLC) combined with MS/MS, was performed to separate GUAC from potential isobaric interferences. NBS for GAMT deficiency began in Utah on June 1, 2015 using a derivatized method for the detection of GUAC and CRE. In May 2019, the laboratory and method transitioned to a non-derivatized method. GAMT screening was added to the New York State NBS panel on October 1, 2018 using a derivatized method. In New York, a total of 537,408 babies were screened, 23 infants were referred and one newborn was identified with GAMT deficiency. In Utah, a total of 273,902 infants were screened (195,425 with the derivatized method, 78,477 with the non-derivatized method), three infants referred and one was identified with GAMT deficiency. Mean levels of GUAC and CRE were similar between methods (Utah derivatized: GUAC = 1.20 ± 0.43 μmol/L, CRE = 238 ± 96 μmol/L; Utah non-derivatized: GUAC = 1.23 ± 0.61 μmol/L, CRE = 344 ± 150 μmol/L, New York derivatized: GUAC = 1.34 ± 0.57 μmol/L, CRE = 569 ± 155 μmol/L). With either Utah method, similar concentrations of GUAC are observed in first (collected around 1 day of age) and the second NBS specimens (routinely collected at 7–16 days of age), while CRE concentrations decreased in the second NBS specimens. Both infants identified with GAMT deficiency started therapy by 2 weeks of age and are growing and developing normally at 7 (Utah) and 4 (New York) months of age. Newborn screening allows for the prospective identification of GAMT deficiency utilizing elevated GUAC concentration as a marker. First-tier screening may be incorporated into existing methods for amino acids and acylcarnitines without the need for new equipment or staff. Newborn screening performed by either derivatized or non-derivatized methods and coupled with second-tier testing, has a very low false positive rate and can prospectively identify affected children. Summary Cerebral creatine deficiency syndromes caused by defects in creatine synthesis can result in intellectual disability, and are preventable if therapy is initiated early in life. This manuscript reports the identification of two infants with GAMT deficiency (one of the cerebral creatine deficiency syndromes) by newborn screening and demonstrates NBS feasibility using a variety of methods. [ABSTRACT FROM AUTHOR]

Clinical and animal studies have suggested a possible beneficial effect of sodium-glucose cotransporter 2 (SGLT2) inhibitors on nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH). Although SGLT2 inhibitors have been shown to reduce hepatic fat deposition in association with loss of body weight, the mechanism of this action has remained unknown. We here show that the SGLT2 inhibitor canagliflozin ameliorated fatty liver and hyperglycemia without affecting body weight or epididymal fat weight in obese diabetic KKAy mice. Lipidomics analysis based on liquid chromatography and tandem mass spectrometry revealed that canagliflozin treatment increased the amounts of prostaglandin E 2 (PGE 2) and resolvin E3 in the liver of these mice. We also found that PGE 2 attenuated fat deposition in mouse primary hepatocytes exposed to palmitic acid. Our results thus suggest that PGE 2 may play an important role in the amelioration of hepatic fat deposition by canagliflozin, with elucidation of its mechanism of action potentially providing a basis for the development of new therapeutics for NAFLD-NASH. • Canagliflozin attenuates hepatic fat deposition in obese diabetic KKAy mice. • Canagliflozin increases the amounts of PGE 2 and resolvin E3 in the KKAy mouse liver. • PGE 2 inhibits deposition of lipid droplets in mouse primary hepatocytes. • PGE 2 may mediate a beneficial effect of SGLT2 inhibitors on NAFLD. [ABSTRACT FROM AUTHOR]

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare inherited autosomal recessive disorder of biogenic amine metabolism. Diagnosis requires analysis of neurotransmitter metabolites in cerebrospinal fluid, AADC enzyme activity analysis, or molecular analysis of the DDC gene. 3- O -methyldopa (3-OMD) is a key screening biomarker for AADC deficiency. We describe a rapid method for 3-OMD determination in dried blood spots (DBS) using flow-injection analysis tandem mass spectrometry with NeoBase™ 2 reagents and 13C 6 -tyrosine as an internal standard, which are routinely used in high-throughput newborn screening. We assessed variability using quality control samples over a range of 3-OMD concentrations. Within-day and between-day precision determined with quality control samples demonstrated coefficients of variation <15%. 3-OMD concentrations in 1000 healthy newborns revealed a mean of 1.33 μmol/L (SD ± 0.56, range 0.61–3.05 μmol/L), 100 non-AADC control subjects (age 7 days – 1 year) showed a mean of 1.19 μmol/L (SD ± 0.35–2.00 μmol/L), and 81 patients receiving oral L-Dopa had a mean 3-OMD concentration of 14.90 μmol/L (SD ± 14.18, range 0.4–80.3 μmol/L). A patient with confirmed AADC was retrospectively analyzed and correctly identified (3-OMD 10.51 μmol/L). In April 2020, we started a pilot project for identifying AADC deficiency in DBSs routinely submitted to the expanded newborn screening program. 3-OMD concentrations were measured in 21,867 samples; no patients with AADC deficiency were identified. One newborn had a high 3-OMD concentration due to maternal L-Dopa treatment. We demonstrated a rapid new method to identify AADC deficiency using reagents and equipment already widely used in newborn screening programs. Although our study is limited, introduction of our method in expanded neonatal screening is feasible and could facilitate deployment of screening, allowing for early diagnosis that is important for effective treatment. [ABSTRACT FROM AUTHOR]

Shotgun proteomics is a powerful method for comprehensively identifying and quantifying tryptic peptides, but it is difficult to analyze proteolytic events. One-dimensional gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) enables the separation of proteolytic fragments using SDS-PAGE followed by identification using LC-MS/MS. GeLC-MS/MS is thus an excellent method for identifying fragmentation. However, the lower reproducibility of gel extraction and nano flow LC-MS/MS can produce inaccurate results in comparative analyses of protein quantification among samples. In this study, a novel GeLC-MS/MS method coupled with stable isotope dimethyl labeling was developed. In the method, a mixture of light- and heavy-labeled samples is loaded onto an SDS-PAGE gel, and proteins with different isotopes in one extracted band are quantitatively analyzed by one-shot injection. This procedure enables accurate determination of the abundance ratio of peptides between two samples, even in cases of low peptide abundance, and it is not affected by the reproducibility of the gel extraction or LC-MS procedures. Therefore, our new GeLC-MS/MS method coupled with stable isotope dimethyl labeling provides high accuracy and comprehensive peptide comparisons, enabling the detection of proteolysis events caused by disease or physiological processes. • A GeLC-MS/MS method coupled with dimethyl labeling was established. • High-quality comparative analysis was achieved by high yield labeling of proteins. • Differences in protein abundance and proteolysis between samples were elucidated. [ABSTRACT FROM AUTHOR]

Cryopreserved oocytes are inevitably exposed to cold stress, which negatively affects the cellular aspects of the oocytes. Lipidomic analysis of the oocytes reveals quantitative changes in lipid classes under conditions of cold stress, leading to potential freezing-vulnerability. We had previously shown that specific phospholipids are significantly downregulated in vitrified-warmed mouse oocytes compared to those in fresh oocytes. In this study, we examined whether supplementation of polyethylene glycol 8000 (PEG 8000) during vitrification influences the lipidome of the oocytes. We used liquid chromatography with tandem mass spectrometry (LC-MS/MS) to study the alteration in the lipidome in three groups of mouse oocytes: fresh, vitrified-warmed, and vitrified with PEG 8000-warmed during vitrification. In these groups, we targeted to analyze 21 lipid classes. We profiled 132 lipid species in the oocytes and statistical analyses revealed lipid classes that were up- or downregulated in these groups. Overall, our data revealed that several classes of lipids were affected during vitrification, and that oocytes vitrified with PEG 8000 to some extent alleviated the levels of changes in phospholipid and sphingolipid contents during vitrification. These results suggest that phospholipids and sphingolipids are influenced by PEG 8000 during vitrification and that PEG 8000 can be considered as a potential candidate for preserving membrane integrity during oocyte cryopreservation. [ABSTRACT FROM AUTHOR]

Cerebrospinal fluid (CSF) contains glycosphingolipids, including lactosylceramide (LacCer, Galβ(1,4)Glcβ-ceramide). LacCer and its structural isomer, galabiosylceramide (Gb 2 , Galα(1,4)Galβ-ceramide), are classified as ceramide dihexosides (CDH). Gb 2 is degraded by α-galactosidase A (GLA) in lysosomes, and genetic GLA deficiency causes Fabry disease, an X-linked lysosomal storage disorder. In patients with Fabry disease, Gb 2 accumulates in organs throughout the body. While Gb 2 has been reported to be in the liver, kidney, and urine of healthy individuals, its presence in CSF has not been reported, either in patients with Fabry disease or healthy controls. Here, we isolated CDH fractions from CSF of patients with idiopathic normal pressure hydrocephalus. Purified CDH fractions showed positive reaction with Shiga toxin, which specifically binds to the Galα(1,4)Galβ structure. The isolated CDH fractions were analyzed by hydrophilic interaction chromatography (HILIC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS). HILIC-ESI-MS/MS separated LacCer and Gb 2 and revealed the presence of Gb 2 and LacCer in the fractions. We also found Gb 2 in CSF from neurologically normal control subjects. This is the first report to show Gb 2 exists in human CSF. • Ceramide dihexoside was isolated from human CSF. • Ceramide dihexoside isolated from human CSF reacted with Shiga toxin. • LC/MS revealed the presence of LacCer and Gb 2 in the isolated ceramide dihexoside. • The presence of Gb 2 in human CSF was demonstrated for the first time. [ABSTRACT FROM AUTHOR]

Organophosphorus pesticides (OPPs) and organophosphate esters (OPEs) are organophosphorus compounds created as substitutes for persistent environmental pollutants, namely organochlorines pesticides and brominated flame retardants, respectively. However, there is evidence that organophosphorus compounds are also widespread across the environment and have adverse effects on biota. In humans, OPPs and OPEs were reported to be carcinogenic, neurotoxic, hepatotoxic, nephrotoxic, amongst others. As lipophilic compounds, these accumulate in fat tissues as adipose tissue. Yet biomonitoring studies and analytical methodologies to assess these compounds in the human body are scarce, particularly in adipose tissue. In this study, the presence of six OPPs and seven OPEs was determined in samples of subcutaneous adipose tissue (scAT) and visceral adipose tissue (vAT) from 188 adult obese women. OPPs and OPEs were quantified by gas chromatography (GC) flame photometric detection and confirmed in GC tandem mass spectrometry. The detection frequencies ranged between 0.5-1.6% and 48–53%, respectively for OPPs and OPEs. Organophosphorus pollutants were present in both adipose tissues and median concentrations were 0.008 ± 0.020 μg/g scAT and 0.009 ± 0.020 μg/g vAT. A total of 32 Spearman's correlations were found between organophosphorus pollutants concentrations in adipose tissue and several biochemical parameters (18 positive and 14 negative). Our results show that anthropometric and hormonal parameters, cholesterol, glycaemia, macrominerals, urea and sedimentation velocity might be influenced by the presence of these compounds. The presence of organophosphorus pollutants in the environmental and their possible effect on female metabolic processes is concerning. Particularly because presently OPEs usage is not controlled or limited by any regulation. More studies are needed to fully understand these pollutants behaviour and hazard effects on human health, biota, and the environment so control regulations can be drawn to prevent and lessen their effects. The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license (https://creativecommons.org/licenses/by/3.0/). [Display omitted] • Organophosphorus compounds were analysed in adipose tissue of a female obese cohort. • From 13 compounds, two OPPs and five OPEs were found in samples of scAT and vAT. • The detection frequency of ΣOPP + OPE is higher in vAT (53.2%) than in scAT (48.4%). • OPPs and OPEs levels were linked to patient's clinical and biochemical data. • Organophosphorus compounds might alter metabolic processes. [ABSTRACT FROM AUTHOR]

Due to adverse health effects, di-(2-ethylhexyl) phthalate (DEHP), a plasticizer used to soften plastic medical devices (PMDs), was restricted, and gradually replaced by alternative plasticizers (APs). Up to this date, urine was the sole matrix studied for plasticizer exposure in neonates hospitalized in the neonatal intensive care unit (NICU), a population highly vulnerable to toxic effects of plasticizers. The primary aim of this study was to assess simultaneous measurement of phthalate and AP metabolites in neonatal scalp hair. In addition, we aimed to use this matrix to investigate exposure of premature neonates to plasticizers during their stay in the NICU. Hair samples in this study were collected from premature neonates and their mothers included in a prospective birth cohort study in a tertiary NICU at the Antwerp University Hospital (UZA), Belgium. Samples from premature neonates (n = 45) and their mothers (n = 107) as well as from control neonates (n = 24) and mothers (n = 29) were analyzed using liquid-chromatography coupled to tandem mass spectrometry. This is the first study reporting metabolites of phthalate and alternative plasticizers in neonatal hair samples as biomarkers for exposure to these plasticizers. Results showed that hair sampled from premature neonates after a NICU stay contained significantly higher metabolite concentrations of both phthalates (DEHP, DiBP, and DnBP; 9.0–2500, 9.3–2200, and 24.7–5300 ng/g), and alternative plasticizers (DEHA, DEHT, and TOTM; 38.8–3400, 127.5–5700, and 10.8–8700 ng/g) – when compared to healthy control neonates. Besides, DEHP and DEHT metabolite concentrations were significantly higher than in hair sampled from adult populations. In addition, prolonged NICU exposure to non-invasive respiratory support devices and gastric tubes was correlated with increased concentrations in hair samples, indicating accumulation of plasticizers in this alternative matrix. In conclusion, our data indicate that preterm neonates are still highly exposed to phthalate and alternative plasticizers during NICU stay, despite the EU Medical Devices Regulation. • This is the first study on phthalate and alternative plasticizers in neonatal hair. • DEHP, DEHA and TOTM metabolite concentrations were higher in NICU than control neonates. • Respiratory support and a nasogastric tube correlate with increased concentrations. • Preterm neonates are still highly exposed to phthalate and alternative plasticizers. [ABSTRACT FROM AUTHOR]

Recombinant monoclonal antibodies (mAbs) have been spurring the rapid growth of commercial biotherapeutics. During production their charge heterogeneity must be assessed as a critical quality attribute to ensure safety, efficacy, and potency. Although imaged capillary isoelectric focusing (icIEF) is a powerful tool for this process, it could be improved further with tandem high-resolution mass spectrometry (HRMS). In this work, a nano-electrospray ionization (nano-ESI) apparatus was constructed to directly couple icIEF to HRMS. The system was evaluated with the standard NISTmAb, as well as more complex mAb, bi-specific antibody, and fusion protein samples. NISTmAb concentrations as low as 0.25 mg/ml demonstrated excellent sensitivity. There were good repeatabilities at 1 mg/ml with 7.58% and 8.01% RSDs for intention time and MS intensity, respectively, and the HRMS signal showed a strong linearity (R = 0.9983) across different concentrations. Meanwhile, the fingerprinting of the complex samples illustrated the versatility and potential of icIEF-HRMS. icIEF-HRMS developed can provide a comprehensive understanding of the underlying structural modifications that impact protein charge heterogeneity. Compared to the traditional ESI, nano-ESI can significantly improve sensitivity while maintaining a reasonable repeatability and throughput. Furthermore, the interface is much easier to connect, and is compatible with many commercial HRMS instruments. [Display omitted] • ICIEF/MS online characterizations with Nano ESI. • Rapid peak pI characterization and corresponding mass identification. • A robust nano emitter to optimize sensitivity and robustness. • Extensive research based on previous published work, contributing to the existing knowledge in the field. [ABSTRACT FROM AUTHOR]

Newborn screening (NBS) for the full set of mucopolysaccharidoses (MPSs) is now possible by either measuring all of the relevant enzymatic activities in dried blood spots (DBS) using tandem mass spectrometry followed by measurement of accumulated glycosaminoglycans (GAGs) or the vice-versa approach. In this study we considered multiple factors in detail including reagent costs, time per analysis, false positive rates, instrumentation requirements, and multiplexing capability. Both NBS approaches are found to provide acceptable solutions for comprehensive MPS NBS, but the enzyme-first approach allows for better multiplexing to include numerous additional diseases that are appropriate for NBS expansion. By using a two-tier NBS approach, the false positive and false negatives rates are expected to acceptably low and close to zero. [ABSTRACT FROM AUTHOR]

Viral hemorrhagic septicemia causes considerable economic losses for Korea's olive flounder (Paralichthys olivaceus) aquaculture farms; therefore, effective antiviral agents for controlling viral hemorrhagic septicemia virus (VHSV) infection are imperative. The present study implemented a Box–Behnken design and cytopathic reduction assay to derive an optimized extract of Sanguisorba officinalis L. roots (OE-SOR) with maximum antiviral activity against VHSV. OE-SOR prepared under optimized extraction conditions (55% ethanol concentration at 50 °C for 5 h) exhibited potent antiviral activity against VHSV, with a 50% effective 0.21 μg/mL concentration and a 340 selective index. OE-SOR also showed direct virucidal activity in the plaque reduction assay. Administering OE-SOR to olive flounder exhibited substantial efficacies against VHSV infection. Fish receiving 100 mg/kg body weight/day of OE-SOR as a preventive (40.0%; p < 0.05) or therapeutic (44.4%; p < 0.05) exhibited a higher relative survival than the untreated VHSV-infected control group (mortalities of 100% and 90%, respectively). In addition, fish fed with OE-SOR (100 mg/kg body weight/day) for two weeks conveyed a significantly higher inflammatory cytokine expression (nuclear factor kappa-light-chain-enhancer of activated B cells [ NF-κB ], interleukin-1 beta [ IL-1β ], and tumor necrosis factor-alpha [ TNF-α ]) than the control group one to two days post-administration. Moreover, no hematological or histological changes were observed in olive flounder treated with OE-SOR over four weeks. Liquid chromatography-quadrupole-time of flight tandem mass spectrometry and -triple quadrupole tandem mass spectrometry analyses identified ziyuglycoside I as a prominent OE-SOR constituent and marker compound (content: 14.5%). This study verifies that OE-SOR is an effective alternative for controlling viral hemorrhagic septicemia in olive flounder farms as it exhibits efficient in vivo anti-VHSV activity and increases innate immune responses. • BBD optimized the anti-VHSV S. officinalis root extract (OE-SOR). • OE-SOR attested exceptional preventive and therapeutic efficacy against VHSV. • Inflammatory cytokines were highly expressed after OE-SOR administration. • OE-SOR-treated olive flounder exhibited no signs of toxicity after four weeks. [ABSTRACT FROM AUTHOR]

The aim of the current study was to validate a screening and confirmatory method for the determination of nitrofuran residues: 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), semicarbazide (SEM), 3,5-dinitrosalicylic acid hydrazine (DNSAH), nifurpirinol (NPIR), nifuroxazide (metabolite 4-HBH), and nitrovin (metabolite AMG) in muscle, milk, eggs, honey, and casings, in accordance with the legislation 2002/657/EC and 2021/808/EC. Sample preparation included hydrolysis and derivatization, followed by ethyl acetate extraction and measurement by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Validation parameters specified in this method are specificity/selectivity, trueness, precision, decision limit (CCα), and detection capability (CCβ). In the screening method, the CCβ value ranged from 0.275 to 0.582 µg kg-1, while in the confirmatory method, the CCα value ranged from 0.264 to 0.407 µg kg-1. The recovery values ranged from 93.5% to 127.5%. A total of 497 routine samples from the National Residue Monitoring Control Plan (NRCP) and border inspection were analysed from 2021 to 2022. SEM was detected in a total of 11 samples. Only four samples had a concentration above the Reference Point for Action (RPA = 0.5 µg kg-1). The validated method is suitable for monitoring nitrofuran residues at levels below the recently established RPA. • A sensitive UHPLC-MS/MS method for the analysis of eight nitrofurans was validated. • New analytes were introduced: nifursol, nifuroxazide, nitrovin, and nifurpirinol. • Method validation was performed on muscle, milk, eggs, honey, and casings. • The validated method was used for the analysis of 497 routine samples. • This method is applicable for monitoring nitrofuran residues in various matrices. [ABSTRACT FROM AUTHOR]

A rapid and simple approach based on ion-pair liquid chromatography for the estimation of all B vitamins in a single run of 35 min using UV detector is developed. The estimation is based on the separation of all B vitamins on the C18 column using gradient elution comprising mobile phase A (65% methanol) and B (sodium dihydrogen phosphate, pH 3.05 containing 5 mM hexane sulphonic acid as an ion-pair reagent). Vitamins B1, B2, B3, B6, B9, and B12 were detected at 255 nm, while vitamins B5 and B7 were best detected at 200 and 210 nm, respectively. The standardized protocol was validated in buffer and milk permeate. The correlation coefficient for all B vitamins determined using a linear regression equation in the range of 1–30 µg mL−1 was > 0.99. The relative standard deviation for intra-day and inter-day repeatability of vitamins (10 µg mL−1) was less than 6%, and recoveries were in the 75–120% range. The applicability of the developed method has also been demonstrated for the quantification of B vitamins (B2, B3, B5, B6) in fortified commercial energy drinks. [Display omitted] [ABSTRACT FROM AUTHOR]

A sensitive and selective method was developed using hydrophilic interaction chromatography combined with tandem mass spectrometry to detect 18 aminoglycosides in bovine milk. Aminoglycosides were extracted from milk using a mixture of trichloroacetic acid, ammonium acetate, ethylenediaminetetraacetic acid, and sodium chloride, and then isolated using an Oasis HLB solid-phase extraction cartridge. We evaluated the performance of chromatographic columns with different stationary phases and optimized the mobile phases to achieve satisfactory separation of the analytes. The limits of detection and quantification were 0.05–2 µg/kg and 0.1–5 µg/kg, respectively. The recoveries of the method ranged from 78.9% to 101.1%, with intra- and inter-day relative standard deviations of less than 8.1% and 9.0%, respectively. This method was applied to 30 milk samples collected from markets, and neomycin (16.8 µg/kg) was detected in one fresh bovine milk sample. The proposed method provides a powerful tool for the surveillance of aminoglycosides in bovine milk. [Display omitted] • A UHPLC-MS/MS method was developed for analysis of 18 aminoglycosides. • Satisfactory separation of 18 AGs was achieved on zwitterionic HILIC column. • Optimized sample pretreatment provided excellent accuracy and precision. • High sensitivity of the method facilitates the detection of trace AGs in milk. [ABSTRACT FROM AUTHOR]