Search Results (686)

Newborn screening in England is a national program with laboratories adhering to common screening algorithms. Until recently, screening for inherited metabolic disorders was provided by ten laboratories using laboratory-developed tests (LDTs) and three using commercial assays: harmonization of results proved challenging. Introduction of hereditary tyrosinemia type 1 screening meant LDTs required modification to include the measurement of succinylacetone, and subsequent re-validation. This provided an opportunity to implement a single commercial reagent kit in all laboratories. It was anticipated that this would improve analytical performance and harmonization. This study aimed to determine whether these goals were achieved. Verification across the 13 laboratories revealed that the commercial kit reduced inter-laboratory variation for all analytes demonstrating improved harmonization. However, this was achieved by applying instrument-specific correction factors to all analytes, the magnitude of which were significant, indicating a lack of standardization. Performance of succinylacetone was limited by instrument-dependent background interference from the methionine stable isotope label, underscoring the need to establish evidence-based screening cut-off values (COV) rather than adopting published thresholds. This study emphasizes the need for traceable reference materials to improve laboratory quality and the value of screening outcome data. Full article

Monoclonal antibodies (mAbs) have been the subject of extensive study in recent years due to their recognition as highly promising therapeutic molecules offering high specificity and a low risk of side effects. Monitoring the structure of these molecules is crucial for developing new therapeutics, characterizing interactions with antigens or receptors, and explaining potential changes in activity between antibody production batches. However, commonly used biophysical approaches provide only low-spatial-resolution information, and conventional structural biology techniques such as crystallography and cryo-electron microscopy (cryo-EM) are difficult to apply to these highly dynamic proteins. Solution nuclear magnetic resonance (NMR) spectroscopy is the method of choice for structural studies of flexible proteins at atomic resolution; however, it has traditionally been limited to low-molecular-weight biological systems. In this review, we present recent advances in NMR spectroscopy and advanced isotopic labeling methods that have enabled the atomic-resolution study of both the crystallizable (Fc) and antigen-binding (Fab) fragments of antibodies. We show how NMR is becoming a powerful tool for investigating full-length mAbs at an atomic level, opening up new possibilities for the characterization and in-depth quality control of therapeutic antibodies in solution. Full article

Background: Creatine deficiency syndromes (CDS) are rare neurometabolic disorders caused by defects in creatine biosynthesis (AGAT and GAMT deficiencies) or creatine transport (SLC6A8 deficiency). Early biochemical recognition is crucial for timely treatment of AGAT and GAMT deficiencies and for improving neurodevelopmental outcomes. In Morocco, expanding the liquid chromatography-tandem mass spectrometry (LC-MS/MS) biomarker panel for inherited metabolic disorders is a priority to strengthen diagnostic capacity and reduce diagnostic delay. Methods: We developed and validated a rapid LC-MS/MS method for the simultaneous quantification of creatine (Cr), guanidinoacetate (GAA), and creatinine (Crn) in plasma and urine using isotopically labelled internal standards and a standardized sample preparation procedure. Analytical performance, including linearity, precision, accuracy, sensitivity, matrix effects, carryover, inter-sample contamination, stability, and measurement uncertainty, was assessed in accordance with ISO 15189:2022 requirements. Results: The assay showed excellent linearity across the analytical range (r² > 0.99), with robust intra- and inter-day precision (CV < 10%). Limits of detection (LOD) were 0.05 µmol/L for Cr and 0.03 µmol/L for GAA in urine, and 0.05 µmol/L for Cr and GAA in plasma. The total run time was 1.1 min per sample, supporting high-throughput implementation. Method performance was further supported by satisfactory results in ERNDIM external quality assessment schemes. Preliminary internal reference ranges and expanded measurement uncertainty were calculated from the available anonymized dataset. Conclusions: This rapid LC-MS/MS method enables the measurement of key CDS biomarkers and contributes to expanding the LC-MS/MS biomarker panel for inherited metabolic disorders in Morocco. Full article

This review summarizes recent applications of nuclear magnetic resonance (NMR) in olefin polymerization catalysis. Due to its capability for quantitative characterization of molecular structures and in situ study, NMR is employed to study the structure of catalysts, and to trace catalyst/cocatalyst interactions, the evolution of active species, monomer insertion, and chain-end formation. This review emphasizes the activation mechanisms of molecular catalysts, ion-pair structures, and the measurement of kinetics. It also discusses the potential applications of in situ multinuclear NMR and isotope labeling technologies in olefin polymerization catalysis studies. Full article

Tetrastigma hemsleyanum Diels et Gilg is a high-value edible and medicinal homologous plant, routinely grown under conventional field or greenhouse production systems across Asia. However, mislabeling of conventional products as the rarer (and more expensive) wild version may occur for financial gain. In this study, stable isotopes (δ¹³C, δ¹⁵N, δ²H, and δ¹⁸O) and metal contents (Cr, Cu, Ni, As, Cd, Pb) were used to characterize plant tissues (tuber root, stem, leaf) and corresponding soils originating from simulated-wild-cultivated (WC) and greenhouse-cultivated (GC) pot trials using the same soil. Carbon and nitrogen isotopes served as key indicators for distinguishing GC and WC products. Specifically, δ¹³C values of GC plant tissues were 1.4 to 2.4‰ more positive than those of WC plant tissues (p < 0.05), and δ¹⁵N values in GC tissues were 2.7 to 4.6‰ more positive than δ¹⁵N in WC tissues (p < 0.01). Lower δ¹⁵N values observed in WC products indicate slower nitrogen turnover compared with GC products. Soil metal concentrations had significant differences between the two cultivation systems, but only limited effects on metal bioconcentration factors (BCFs) and translocation factors (TFs) in T. hemsleyanum tissues. Pb and Cd concentrations in root tissues had large differences between cultivation systems, and carbon dynamics in GC plants were more negatively affected by Pb levels in soils. These findings provide the first investigation of T. hemsleyanum grown under different cultivation practices and establish a scientific basis for distinguishing other wild or simulated-wild labeled food and medicinal plant products from conventionally grown products in future studies. Full article

Ultrashort-chain (USC) per- and polyfluoroalkyl substances (PFAS) are highly polar, mobile, and persistent emerging pollutants. While the environmental distribution of USC species is well-documented, their presence in widely consumed beverages remains under-characterized due to the analytical difficulty of capturing such highly polar species. This study established a robust workflow for the simultaneous determination of C1 to C14 perfluoroalkyl carboxylic and sulfonic acids, alongside other PFAS classes, in diverse beverage matrices including teas and fruit juices. Chromatographic separation was achieved using a mixed-mode inert-coated alkyl-phase LC column to enhance USC retention while maintaining performance for longer-chain analytes. A high-throughput, minimal-handling sample preparation was optimized to mitigate matrix effects and contamination. Method performance was evaluated using fortified beverage samples across 2–500 ng/L, with calibration ranges of 1–2000 ng/L and incorporation of 13 isotopically labeled internal standards. Results demonstrated acceptable accuracy (recoveries within 30% of nominal values) and optimal precision (%RSD < 12%). Application to commercial samples revealed frequent PFAS occurrence, specifically highlighting the prevalence of previously overlooked USC species in the human diet. These results demonstrate that ready-to-drink beverages are a significant pathway for human exposure, necessitating the inclusion of USC compounds in future food safety monitoring and risk assessments. Full article

8-Hydroxy-dihydroergotamine is the major human metabolite of the anti-migraine drug dihydroergotamine and is required, along with a stable isotope-labelled derivative, to aid metabolic studies. An efficient, scalable synthesis of the unlabelled compound is described via the coupling of dihydrolysergic acid to the tricyclic amino compound (2R,5S,8R,10aS,10bS)-2-amino-5-benzyl-10b-hydroxy-8-methoxy-2-methyltetrahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazine-3,6(2H,5H)-dione. The tricycle was obtained by a convergent synthesis combining precursors from suitably protected L-glutamic acid and L-phenylalanine, and 2-bromo-2-methylmalonic acid. For the labelled molecule, the tricyclic precursor contained a pentadeutero benzyl group derived from [2,3,4,5,6-²H₅]L-phenylalanine. Considerable experimentation was required to achieve optimal activation of dihydrolysergic acid for efficient amide formation with the tricycle’s amino function affording 8-methoxy-dihydroergotamine. The stereochemical integrity of an intermediate in this synthesis, ethyl (2R,5S,8R,10aS)-5-benzyl-10b-hydroxy-8-methoxy-2-methyl-3,6-dioxooctahydro-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazine-2-carboxylate, was validated by crystal structure analysis. Acid-catalysed hydrolysis of 8-methoxy-dihydroergotamine gave 8-hydroxy-dihydroergotamine. Pentadeuterated 8-hydroxy-dihydroergotamine was obtained in an analogous manner from [2,3,4,5,6-²H₅]L-phenylalanine. Both 8-hydroxy-dihydroergotamine and its ²H₅-derivative were obtained as an equilibrating mixture of C-8 epimers (diastereomers), with the major isomer having (R)-configuration according to ¹H NMR analysis. The syntheses described enable the routine synthesis of 50–100 mg quantities of each target molecule. Full article

Cross-reactions between peroxy radicals (RO₂) derived from different volatile organic compound (VOC) precursors have been proposed as an important pathway during atmospheric oxidation. However, direct molecular evidence has been limited. In this study, α-pinene and fully deuterated toluene (d8-toluene) were oxidized separately and as a mixture in a potential aerosol mass (PAM) flow reactor, and the resulting secondary organic aerosol (SOA) was characterized by a high-resolution mass spectrometer (ESI FT-ICR-MS). A constrained chemical mass balance (CMB) model attributed 82.9% of the mixed-SOA signal to single-precursor sources (66.5% α-pinene, 16.4% d8-toluene), leaving a 17.1% signal-based residual fraction unexplained by linear mixing. Among 2450 residual molecular formulas exclusive to the mixed-SOA, 1858 were identified as cross-reaction candidates, with carbon, oxygen, and double bond equivalents (DBE) distributions consistent with RO₂-RO₂ cross-reactions between toluene- and α-pinene-derived fragments. We also identified representative monomer-dimer pairs, where one monomer corresponded to a known α-pinene oxidation product, while the other matched a primary oxidation product of d8-toluene oxidation based on the Master Chemical Mechanism (MCM), providing strong molecular-level evidence for RO₂-RO₂ cross-reactions. Our findings demonstrate that the mixed VOCs generate unique SOA products that extend beyond simple additive chemistry, with implications for SOA yield parameterizations and chemical transport models. Full article

Nitrate (NO₃⁻) serves as a critical nitrogen source and signaling molecule essential for its growth and quality formation. Although substantial genetic variation in nitrogen use efficiency (NUE) has been documented among tea cultivars, a systematic characterization of nitrate (NO₃⁻) absorption kinetics and the associated genome-wide transcriptional regulatory networks across varying nitrate concentrations remains lacking. This study employed ¹⁵N isotope labeling and transcriptome sequencing to systematically analyze the absorption characteristics and molecular response mechanisms of the cultivars ‘Longjing 43’ and ‘Zhongming 6 hao’ under varying NO₃⁻ concentrations. Results revealed significant differentiation in absorption strategies: ‘Zhongming 6 hao’ exhibited a significantly higher absorption rate at low concentrations, whereas ‘Longjing 43’ demonstrated enhanced performance at high concentrations. Transcriptome analysis indicated that both cultivars shared coordinated regulation of ‘photosynthesis’ and ‘nitrogen metabolism’ pathways. Furthermore, 14 nitrogen metabolism genes and 64 differentially expressed transcription factors (including MYB, NAC, and LBD families) were identified. Specifically, the CsNiR gene (encoding nitrite reductase) was functionally validated; silencing of CsNiR significantly reduced nitrite reductase activity, confirming its positive regulatory role. This study provided a theoretical framework and key candidate genes for breeding nitrogen-use-efficient varieties, which is essential for sustainable tea production. Full article

Alterations in the ratio of oxidized and reduced nicotinamide adenine dinucleotide (NAD⁺/NADH) reflect the intracellular redox state and have been implicated in a broad spectrum of pathological conditions, including neurogenetic disorders, heart failure, and liver diseases. In the present study, we demonstrate the selective detection of probe-derived NAD⁺ and NADH signals in mouse liver extracts by means of a triple-resonance nuclear magnetic resonance (NMR) spectroscopy technique. We prepared ¹³C/¹⁵N-enriched nicotinamide riboside (¹³C/¹⁵N-NR), which undergoes enzymatic conversion to NAD⁺ and NADH in the liver, and detected these labeled metabolites by ¹H–{¹³C–¹⁵N} triple-resonance NMR measurements. This study provides a methodological proof-of-concept for the selective detection of NAD-related signals derived from a stable-isotope labeled probe in mouse liver extracts. Full article

Accurate determination of cobalamin vitamers in foods remains analytically challenging because conventional cyanidation-based methods convert native cobalamins into cyanocobalamin (CNCbl) and may distort their original distribution. In this study, a cyanide-free UHPLC-MS/MS workflow was developed for the analysis of major cobalamin vitamers in foods, with particular emphasis on preserving native forms during sample preparation. Light, temperature, and cleanup procedures were systematically evaluated. Methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) showed pronounced light sensitivity, whereas red-light handling better preserved vitamer integrity during pre-analytical operations. A tandem cleanup procedure combining immunoaffinity and Oasis HLB solid-phase extraction improved extract cleanliness in complex food matrices. The workflow showed good chromatographic separation and excellent linearity (R² > 0.999). The validated limits of detection were 0.5 μg/kg for CNCbl, 1.0 μg/kg for AdoCbl, and 0.75 μg/kg for MeCbl. Application to food samples showed no detectable target cobalamins in the tested plant-derived foods, whereas animal liver and oyster samples showed comparatively high levels of the target cobalamin vitamers, with AdoCbl predominating in liver. The proposed workflow may serve as a practical cyanide-free option for exploratory or comparative native-vitamer analysis of CNCbl, AdoCbl, and MeCbl in foods within the current validation scope, particularly when full sets of matched isotope-labeled standards are not readily available. Full article

Background: Untargeted proteomics enables quantitative host cell protein (HCP) determination in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. Methods: A prospective total-error (TE) validation of label-free ddaPASEF proteomics was performed. A stable isotope-labeled whole-proteome standard was spiked into NISTmAb at seven levels (20–80 ng) and analyzed in four independent assays (198 injections), supporting one-way random-effects ANOVA with Welch–Satterthwaite adjustment. Peptide-level identification error was evaluated by dual entrapment. Results: Empirical false-discovery proportions were below 1% at q = 0.01. Weighted least-squares regression (R² = 0.993) confirmed stable proportional compression with 81–85% recovery. Repeatability dominated the variance structure (median CV 2.7%); intermediate precision SD ranged from 0.69% to 3.81%. Both 95% β-expectation and 95/95 content tolerance intervals were contained within ±30% at all levels, defining a validated range of 20–80 ng. Abundance-stratified TE profiling revealed concentration-dependent calibration heterogeneity, with stratum-specific intervals within ±35% defining an abundance-aware LLOQ of 3.6 ppm (P95 = 3.87 ppm). Robustness under independent search software (FragPipe v24.0, CCC = 0.998) and cross-platform acquisition (Astral, CCC = 0.980) remained within ±30% limits. Conclusions: This constitutes the first prospective ICH Q2(R2)-aligned validation of untargeted proteomics for HCP quantification, with a transferable statistical framework for high-dimensional analytical methods. Full article

Targeted delivery systems offer a promising approach for selectively modulating cellular processes; yet the intracellular consequences of targeted nutrient delivery to trophoblast cells remain poorly defined. Here, we investigated a previously validated placenta-targeting peptide conjugated to liposomes encapsulating stable isotope-labelled L-arginine and L-lysine to examine cellular uptake and downstream molecular responses in a trophoblast-like cell model. Peptide-dependent uptake of fluorescently labelled liposomes was confirmed in BeWo cells, demonstrating selective internalisation compared with non-targeted controls. Encapsulation of isotope-labelled amino acids enabled direct quantification of intracellular delivery and incorporation into the cellular proteome using stable isotope labelling by amino acids in cell culture (SILAC). Quantitative proteomic analysis revealed coordinated changes in proteins associated with translation, metabolism, and nitric oxide synthase regulation following targeted liposomal uptake. Notably, V-type proton ATPase subunit G1 (ATP6V1G1) and large neutral amino acid transporter small subunit 1 (SLC7A5) showed increased incorporation of labelled amino acids and were independently validated by Western blotting. Together, these findings establish a proof-of-concept platform for targeted intracellular amino acid delivery to trophoblast-like cells and define the resulting proteomic responses. This work provides mechanistic insight into intracellular amino acid utilisation and a framework for future studies in placental cell biology. Full article

The origin of life is commonly discussed within two competing conceptual frameworks: the metabolism-first and information-first hypotheses. While each emphasizes a different defining property of early life, modern biochemistry reveals a fundamental interdependence between metabolic processes and genetic information transfer, leading to a persistent chicken-and-egg problem. In this study, we investigate a prebiotically plausible reaction system that enables the concurrent formation of molecular precursors associated with both frameworks. Under simulated Hadean hydrothermal conditions, acetylene, ammonia, cyanide, and carbon monoxide were reacted in aqueous solution in the presence of transition metal sulfides. Using gas chromatography–mass spectrometry combined with stable isotope labeling, we demonstrate the simultaneous formation of the nucleobase uracil and the amino acids alanine and aspartic acid. Isotopic incorporation patterns allow reconstruction of the underlying reaction pathways and confirm the contribution of all starting materials to product formation. While amino acids are produced continuously over the observed period in significantly higher yields than uracil, uracil formation exhibits a pronounced time-dependent maximum after three days. Variations in pH, reaction time, and metal sulfide catalysts modulate product yields but do not prevent the parallel emergence of both molecular classes. These findings support a scenario in which proto-metabolic chemistry and molecular precursors of genetic information could have arisen simultaneously within a shared geochemical setting. The results provide experimental support for a coupled origin of metabolism and transcriptional building blocks, offering a potential resolution to the dichotomy between metabolism-first and information-first models of early life. Full article

The dry period is a critical window of susceptibility to intramammary infection in dairy cows, yet the metabolic environment of the mammary gland during this phase remains largely uncharacterized. The objective of this study was to profile the metabolome of bovine dry secretion fluid from quarters with subclinical mastitis (SCM; SCC ≥ 200,000 cells/mL) and healthy quarters (H; SCC < 200,000 cells/mL) on day 2 (D2) and day 21 (D21) of the dry period (n = 10 per group quarters per group, drawn from a cohort of 41 enrolled Holstein dairy cows) using high-performance chemical isotope labeling liquid chromatography–mass spectrometry (CIL-LC–MS). A total of 474 metabolites were positively identified. At D2, 186 metabolites differed significantly between SCM and H quarters, with dipeptides dominating the upregulated metabolites, indicating active proteolysis in infected quarters. Norepinephrine was the most significantly depleted metabolite (FC = 0.27, p = 3.37 × 10⁻⁷), pointing to local catecholamine exhaustion. By D21, only 36 metabolites remained altered, representing an 80.6% attenuation of the SCM signature. Interestingly, temporal changes from D2 to D21 far exceeded disease-related differences, with 316 metabolites shifting in both SCM and healthy quarters, establishing mammary involution as the dominant metabolic event during the dry period. Principal component and PLS-DA analyses confirmed that time, not disease status, was the primary driver of metabolic variation. Pathway analysis revealed significant perturbations in amino acid metabolism, glyoxylate and dicarboxylate metabolism, and tryptophan metabolism. These findings provide the first comprehensive metabolomic map of bovine dry secretion, reveal that subclinical mastitis superimposes a proteolytic and neuroimmune disruption onto the involution process, and identify candidate biomarkers for early detection of intramammary infection during the dry period. Full article