Solanaceous plants, such as Solanum dulcamara, produce steroidal glycosides (SGs). Leaf SG profiles vary among S. dulcamara individuals, leading to distinct phytochemical phenotypes (\'chemotypes\') and intraspecific phytochemical diversity (\'chemodiversity\'). However, if and how SG chemodiversity varies among organs and across ontogeny, and how this relates to SG metabolism gene expression is unknown. Among organs and across ontogeny, S. dulcamara plants with saturated (S) and unsaturated (U) SG leaf chemotypes were selected and clonally propagated. Roots, stems and leaves were harvested from vegetative and flowering plants. Extracts were analysed using untargeted LC-MS. Expression of candidate genes in SG metabolism (SdGAME9, SdGAME4, SdGAME25, SdS5αR2 and SdDPS) was analysed using RT-qPCRs. Our analyses showed that SG chemodiversity varies among organs and across ontogeny in S. dulcamara; SG richness (Dmg) was higher in flowering than vegetative plants. In vegetative plants, Dmg was higher for leaves than for roots. Lack of SdGAME25 expression in U-chemotype leaves, while readily expressed in roots and stems, suggests a pivotal role for SdGAME25 in differentiation of leaf chemotypes in vegetative and flowering plants. By acting as an ontogeny-dependent chemotypic switch, differential regulation of SdGAME25 enables adaptive allocation of SGs, thereby increasing SG chemodiversity in leaves. This indicates that differential expression and/or regulation of glycoalkaloid metabolism genes, rather than their presence or absence, explains observed chemotypic variation in SG chemodiversity among organs and across ontogeny.

Tryptophan (an essential amino acid) and its clinically important metabolite-kynurenine contribute to several fundamental biological processes and methods that allow their determination in biological samples are in demand. The novelty of the work was a demonstration of the utility of two polymers: 4-vinylpyridine crosslinked with trimethylolpropane trimethacrylate (poly(4VP-co-TRIM)) or 1,4-dimethacryloyloxybenzene (poly(4VP-co-14DMB))-in terms of human serum clean-up for simultaneous LC-MS determination of tryptophan and kynurenine. The goal was to achieve a reduction of the matrix effect, which is responsible for signal suppression, with minimal capture of analytes. The adsorption properties of the polymeric beads were studied by evaluating the adsorption kinetics and isotherms in model matrices. Therefore, the adsorption capacities of both molecules were not efficient, the tested 4-vinylpyridine-based copolymers have shown great promise (especially poly(4VP-co-TRIM)) as sorbents for serum clean-up. In the model human serum matrix, poly(4VP-co-TRIM) provided good recoveries of tryptophan and kynurenine (76% and 87%, respectively) and allowed for the reduction of the matrix effect. Performances of both copolymers were compared to those of commercially available sorbents (octadecylsilane, activated charcoal, and primary secondary amine).

BACKGROUND: The human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection presents significant challenges due to the complex interplay between these diseases, leading to exacerbated metabolic disturbances. Understanding these metabolic profiles is crucial for improving diagnostic and therapeutic approaches.
OBJECTIVE: This study aimed to characterise the urinary acylcarnitine and amino acid profiles, including 5-hydroxyindoleacetic acid (5-HIAA), in patients co-infected with HIV and TB using targeted liquid chromatography mass spectrometry (LC-MS) metabolomics.
METHODS: Urine samples, categorised into HIV, TB, HIV/TB co-infected, and healthy controls, were analysed using HPLC-MS/MS. Statistical analyses included one-way ANOVA and a Kruskal-Wallis test to determine significant differences in the acylcarnitine and amino acid profiles between groups.
RESULTS: The study revealed significant metabolic alterations, especially in TB and co-infected groups. Elevated levels of medium-chain acylcarnitines indicated increased fatty acid oxidation, commonly associated with cachexia in TB. Altered amino acid profiles suggested disruptions in protein and glucose metabolism, indicating a shift towards diabetes-like metabolic states. Notably, TB was identified as a primary driver of these changes, affecting protein turnover, and impacting energy metabolism in co-infected patients.
CONCLUSIONS: The metabolic profiling of HIV/TB co-infection highlights the profound impact of TB on metabolic pathways, which may exacerbate the clinical complexities of co-infection. Understanding these metabolic disruptions can guide the development of targeted treatments and improve management strategies, ultimately enhancing the clinical outcomes for these patients. Further research is required to validate these findings and explore their implications in larger, diverse populations.

Extracellular proteolysis critically regulates cellular and tissue responses and is often dysregulated in human diseases. The crosstalk between proteolytic processing and other major post-translational modifications (PTMs) is emerging as an important regulatory mechanism to modulate protease activity and maintain cellular and tissue homeostasis. Here, we focus on matrix metalloproteinase (MMP)-mediated cleavages and N-acetylgalactosamine (GalNAc)-type of O-glycosylation, two major PTMs of proteins in the extracellular space. We investigated the influence of truncated O-glycan trees, also referred to as Tn antigen, following the inactivation of C1GALT1-specific chaperone 1 (COSMC) on the general and MMP9-specific proteolytic processing in MDA-MB-231 breast cancer cells. Quantitative assessment of the proteome and N-terminome using terminal amine isotopic labelling of substrates (TAILS) technology revealed enhanced proteolysis by MMP9 within the extracellular proteomes of MDA-MB-231 cells expressing Tn antigen. In addition, we detected substantial modifications in the proteome and discovered novel ectodomain shedding events regulated by the truncation of O-glycans. These results highlight the critical role of mature O-glycosylation in fine-tuning proteolytic processing and proteome homeostasis by modulating protein susceptibility to proteolytic degradation. These data suggest a complex interplay between proteolysis and O-GalNAc glycosylation, possibly affecting cancer phenotypes.

BACKGROUND: Infertility is a major health issue, affecting 15% of reproductive-age couples with male factors contributing to 50% of cases. Asthenozoospermia (AS), or low sperm motility, is a common cause of male infertility with complex aetiology, involving genetic and metabolic alterations, inflammation and oxidative stress. However, the molecular mechanisms behind low motility are unclear. In this study, we used a metabolomics approach to identify metabolic biomarkers and pathways involved in sperm motility.
METHODS: We compared the metabolome and lipidome of spermatozoa of men with normozoospermia (n = 44) and AS (n = 22) using untargeted LC-MS and the metabolome of seminal fluid using 1H-NMR. Additionally, we evaluated the seminal fluid redox status to assess the oxidative stress in the ejaculate.
RESULTS: We identified 112 metabolites and 209 lipids in spermatozoa and 27 metabolites in the seminal fluid of normozoospermic and asthenozoospermic men. PCA analysis of the spermatozoa\'s metabolomics and lipidomics data showed a clear separation between groups. Spermatozoa of asthenozoospermic men presented lower levels of several amino acids, and increased levels of energetic substrates and lysophospholipids. However, the metabolome and redox status of the seminal fluid was not altered inAS.
CONCLUSIONS: Our results indicate impaired metabolic pathways associated with redox homeostasis and amino acid, energy and lipid metabolism in AS. Taken together, these findings suggest that the metabolome and lipidome of human spermatozoa are key factors influencing their motility and that oxidative stress exposure during spermatogenesis or sperm maturation may be in the aetiology of decreased motility in AS.

The United States (US) Food and Drug Administration\'s (FDA) regulatory oversight over electronic cigarettes (e-cigs) includes access restriction for persons <21 years of age and flavor restrictions for \"cartridge-based\" products. Despite the restrictions, consumption by US youth perseveres. Studies on youth e-cig use are limited by the reliability and accuracy of self-reports. As an alternative to self-reports, the current study examined nicotine, cannabinoid, and unlabeled e-cigs and other vaping products confiscated from Virginia public schools to characterize trends among students. Findings highlight a shift from JUUL and pod-based products to single use disposable e-cigs following the FDA flavor restrictions on cartridge-based e-cigs. Chemical analysis of e-liquids by gas chromatography-mass spectrometry identified a wide variety of flavorants and an increase in the prevalence of synthetic coolants. Most confiscated products were nicotine salt formulations, but the prevalence of cannabinoid-based vaping products increased. The popularity of flavored disposable e-cigs highlights the need for further restrictions to reduce youth consumption. The increasing use of synthetic coolants instead of menthol may suggest that manufacturers are employing tactics to bypass regulations. Continued youth access to e-cigs and the abundance of cannabinoid-based products is problematic from health and safety perspectives. Continued research incorporating confiscated product analysis can be used to understand youth access to vaping products and evolutions in manufacturing practices.

Capsule phase microextraction (CPME) is an efficient bioanalytical technique that streamlines the sample preparation by integrating the filtration and stirring mechanism directly into the device. A novel composite sorbent designed to be selective towards the target analytes consisting of mixed-mode sorbent chemistry synthesized by sol-gel technology is found promising and superior to the conventional C18 sorbents. Herein we describe the encapsulation of an ionic liquid (IL)/Carbowax 20M-functionalized sol-gel sorbent (sol-gel IL/Carbowax 20 M) in the lumen of porous polypropylene tubes for the capsule phase microextraction of three phosphodiesterase-5 inhibitors namely avanafil, sildenafil, and tadalafil in human serum and urine samples. The CPME device was characterized by Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FT-IR). The experimental parameters of CPME procedure (e.g. sample pH and ionic strength, extraction time, stirring rate, elution solvent and volume) were carefully optimized to achieve the highest possible extraction efficiency for the analytes. Method validation was conducted in terms of precision, linearity, accuracy, matrix effect, lower limits of quantification, and limits of detection (LOD). The method linearity was investigated in the range of 50-1000 ng mL-1 for all analytes while the precision was less than 11.8 % in all cases. For all analytes, the LOD values were 17 ng mL-1. The IL/CW 20M-functionalized microextraction capsules could be reused at least 25 times both for urine and serum samples. The green character and the applicability of the proposed method were evaluated using the ComplexGAPI and BAGI indexes. The optimized CPME protocol exhibited reduced consumption of organic solvent and generation of waste, cost-effectiveness, and simplicity. Finally, the proposed method was successfully applied to the analysis of sildenafil in human urine after administration of drug-containing formulation.

BACKGROUND: Accurately identifying and quantifying polar metabolites using untargeted metabolomics has proven challenging in comparison to mid to non-polar metabolites. Hydrophilic interaction chromatography and gas chromatography-mass spectrometry are predominantly used to target polar metabolites.
OBJECTIVE: This study aims to demonstrate a simple one-step extraction combined with liquid chromatography-mass spectrometry (LC-MS) that reliably retains polar metabolites.
METHODS: The method involves a MilliQ + 10% trichloroacetic acid extraction from 6 healthy individuals serum, combined with porous graphitic carbon liquid chromatography-mass spectrometry (LC-MS). The coefficient of variation (CV) assessed retention reliability of polar metabolites with logP as low as - 9. QreSS (Quantification, Retention, and System Suitability) internal standards determined the method\'s consistency and recovery efficiency.
RESULTS: The method demonstrated reliable retention (CV < 0.30) of polar metabolites within a logP range of - 9.1 to 5.6. QreSS internal standards confirmed consistent performance (CV < 0.16) and effective recovery (70-130%) of polar to mid-polar metabolites. Quality control dilution series demonstrated that ~ 80% of annotated metabolites could be accurately quantified (Pearson\'s correlation coefficient > 0.80) within their concentration range. Repeatability was demonstrated through clustering of repeated extractions from a single sample.
CONCLUSIONS: This LC-MS method is better suited to covering the polar segment of the metabolome than current methods, offering a reliable and efficient approach for accurate quantification of polar metabolites in untargeted metabolomics.

Cinnamomi ramulus (CR) is a common Chinese herbal medicine with a long history. It is often used to treat exogenous wind-cold diseases in clinic, but its chemical compositions remain to be studied. In this study, CR was extracted with 75% ethanol, and UPLC-Q-Orbitrap-MS combined with data post-processing method was used to identify the chemical components in the extract. Through this technology, the components in CR can be separated and accurately identified. A total of 61 compounds were identified, including 14 simple phenylpropanoids, 3 coumarins, 5 lignans, 14 flavonoids, 10 benzoic acids, 8 organic acids, and 7 others. This study confirmed the existence of these compounds in CR and speculated the cleavage pathways of each compound, which enriched the mass spectrometry data and cleavage rules. This study can provide a reference for CR and other research.

Kisspeptin-10 is a peptide hormone capable of increasing circulating follicle-stimulating hormone, luteinizing hormone and testosterone levels in humans. Clinically, these effects suggest its use as a treatment for infertility. However, its testosterone-increasing effect indicates potential misuse in sports. As such, it is included in the 2024 World Anti-Doping Agency Prohibited List. This work describes the successful validation of an initial testing procedure (screening) and a confirmation procedure for kisspeptin-10 in urine using liquid chromatography-mass spectrometry. Additionally, kisspeptin-10 was incubated in human serum to mimic endogenous metabolism to improve method sensitivity, as previous research had demonstrated a rapid elimination time of only 30 min after injection (in rats). Four metabolites, corresponding to peptide fragments y9, y8, y7 and y5, were found and added to the ITP in full scan mode. A degradation product discovered during early experimentation was found to probably be caused by oxidation of the tryptophan residue into a kynurenine residue. Further research should elucidate the kinetic parameters of the reaction to improve product stability. Using the validated confirmation procedure, a black-market vial of kisspeptin-10 was analysed. The product contained no unexpected impurities, although it appeared to have undergone more degradation than the purchased reference standard.