Liquid Chromatography-tandem Mass Spectrometry Research Articles

Fecal metabolomic analysis of the role of gut microbiota and short-chain fatty acids in the therapeutic mechanism of Timosaponin AIII in Sjögren's syndrome.

Sjogren's syndrome (SS) is a chronic inflammatory and difficult-to-treat autoimmune disease. Timosaponin AIII (TAIII), a plant-derived steroidal saponin, effectively inhibits cell proliferation, induces apoptosis, and exhibits anti-inflammatory properties. This study explored the mechanisms of action of TAIII in SS treatment by studying gut microbiota and short-chain fatty acids (SCFAs) using fecal metabolomics. The model group used non-obese diabetic (NOD) mice. The treatment group was classified into TAIII and hydroxychloroquine groups. The gut microbiota, SCFAs, and metabolites were analyzed using 16S rRNA sequencing, gas chromatography-mass spectrometry analysis, and liquid chromatography-mass spectrometry, respectively. TAIII effectively alleviated dry mouth in NOD mice, slowed the progression of salivary gland tissue injury, reduced inflammatory factor expression, and increased the levels of aquaporins 1 and 5. TAIII regulated SCFA content and tryptophan metabolism by altering the abundance of the Rikenellaceae_RC9_gut_group, thereby reducing the inflammatory response. TAIII can improve imbalances in the gut microbiota and the metabolic levels of related SCFAs and tryptophan, thereby reducing the level of inflammation. The significant differences observed in the abundance of the Rikenellaceae_RC9_gut_group between the treatment and control groups indicated the potential relationship between bacteria and metabolites in SS. Key Points • The safe and effective treatment of SS with traditional Chinese medicine • Multi-means study on intestinal flora, short-chain fatty acids, and metabonomics.

Proteomic Profiling Reveals Alterations in Metabolic and Cellular Pathways in Severe Obesity and Following Metabolic Bariatric Surgery.

In this study, we investigated the impact of bariatric surgery on the adipose proteome to better understand the metabolic and cellular mechanisms underlying weight loss following the procedure. A total of 46 patients with severe obesity were included, with samples collected both before and after bariatric surgery. Additionally, 15 healthy, non-obese individuals who did not undergo surgery served as controls and were studied once. We utilized quantitative liquid chromatography tandem mass spectrometry analysis to conduct a large-scale proteomic study on abdominal subcutaneous biopsies obtained from the study participants. Our proteomic profiling revealed that among the 2254 compared proteins, 46 were upregulated, and 34 were downregulated 6 months post-surgery compared to baseline (FDR < 0.01). We observed a downregulation of proteins associated with mitochondrial integrity, amino acid catabolism, and lipid metabolism in the patients with severe obesity compared to the controls. Bariatric surgery was associated with an upregulation in pathways related to mitochondrial function, protein synthesis, folding and trafficking, actin cytoskeleton regulation, and DNA binding and repair. These findings emphasize the significant changes in metabolic and cellular pathways following bariatric surgery, highlighting the potential mechanisms underlying the observed health improvements post-bariatric surgery. The data provided alongside this paper will serve as a valuable resource for the development of targeted therapeutic strategies for obesity and related metabolic complications.

Cross-sectional and longitudinal associations between serum vitamin D and continuous metabolic syndrome score among children and adolescents: roles of levels of inflammation in peripheral blood

BackgroundSerum vitamin D deficiency is intricately linked to metabolic disorders, however, evidence on its association with continuous metabolic risk in children and adolescents remains insufficient. This study aims to elucidate the relationship between serum vitamin D levels and continuous metabolic risk.MethodsThe cross-sectional analysis involved 4490 participants aged 6 ~ 18, and the longitudinal investigation included 1398 individuals aged 6 ~ 12 years. Serum 25(OH)D concentrations were quantified using liquid chromatography-mass spectrometry. Continuous Metabolic syndrome risk score (CMSRS), incorporating waist, blood pressure, blood lipid levels, and glucose metabolism as four components, utilizes age- and gender-specific Z scores to evaluate metabolic risk. Restricted cubic splines (RCS) were used to visualize dose–response relationships and generalized linear models (GLM) were used to estimate potential associations. Mediation analysis was used to evaluate the mediating role of levels of Neutrophil-to-lymphocyte ratio (NLR).ResultsThe RCS indicated a negative linear association between serum 25(OH)D levels and CMSRS (P-overall = 0.0066, P-nonlinear = 0.1393). GLM revealed that compared to Q1, with the quartiles of serum 25(OH)D concentrations increase, the β value ranged from 0.028 (95% CI: − 0.093, 0.037) to 0.001(95%CI: − 0.067, 0.069), and then to -0.074 (95%CI: -0.146, -0.003, P for trend = 0.0659). For every 10 ng/mL increase in serum 25(OH)D concentration corresponded to the β value change -0.058 (95%CI: -0.098, -0.017). This association was more pronounced in younger or overweight/obese individuals. Furthermore, in the longitudinal study, as the baseline quartile of serum 25(OH)D concentration increased, the estimated change of subsequent CMSRS indicated a decreasing trend, ranging from -0.085 (95%CI: -0.203, 0.032) to -0.166 (95%CI: − 0.285, − 0.046), and then to − 0.174 (95%CI: − 0.296, -0.053, P for trend = 0.0031). The mediating proportion of levels of NLR was 7.2%.ConclusionsHigher serum 25(OH)D concentration is significantly associated with reduced CMSRS in children and adolescents, and adequate serum vitamin D levels play a prominent role in preventing long-term metabolic disorders, partly meditating by inflammation in peripheral blood.

Investigation into Drug-Induced Liver Damage Using Multimodal Mass Spectrometry Imaging.

Drug toxicity during the development of candidate pharmaceuticals is the leading cause of discontinuation in preclinical drug discovery and development. Traditionally, the cause of the toxicity is often determined by histological examination, clinical pathology, and the detection of drugs and/or metabolites by liquid chromatography-mass spectrometry (LC-MS). While these techniques individually provide information on the pathological effects of the drug and the detection of metabolites, they cannot provide specific molecular spatial information without additional experiments. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful, label-free technique for the simultaneous detection of pharmaceuticals, metabolites, and endogenous chemical species in tissue sections, which makes it suitable for mechanistic toxicological studies to directly correlate the distribution of the drug and its metabolites with histological findings. This capability was demonstrated by the analysis of the liver from dogs dosed with discontinued drug compound B and its N-desmethyl metabolite, compound A. Histological examination showed multifocal hepatocellular necrosis, bile duct hyperplasia, periportal fibrosis, and chronic inflammation. MALDI-MSI analysis of liver tissue dosed with only compound A indicated that liver lesions were associated exclusively with the parent compound, whereas liver lesions with compound B showed the presence of the parent compound and its two metabolites (compound A and an N-oxide metabolite). Using both positive and negative ion modes, simultaneous detection and identification of endogenous molecular markers of the connective tissue, blood vessels, liver parenchyma, and bile duct epithelium was achieved, allowing optimal visualization of histological lesions by mass spectrometry imaging.

Comprehensive analysis of metabolomics and transcriptomics reveals varied tepal pigmentation across Gloriosa varieties

Gloriosa L. possesses exceptional ornamental value, with its floral hues exhibiting a wide range of variations. In this study, we employed sophisticated colorimetry, Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS), and transcriptome sequencing to investigate the phenotypic expression of tepal colors, the composition of carotenoids and anthocyanins, and the differential gene expression in four Gloriosa varieties during their full bloom phase. Our findings revealed that the redness of the tepals, indicated by higher a* values, increased with the intensity of the red hue, while lighter colors corresponded to higher L* values. Metabolomic analysis identified 50 carotenoids and 60 anthocyanins. It was observed that carotenoids primarily influence the yellow and orange color of Gloriosa tepals, with β-carotene, lutein, and zeaxanthin being the predominant carotenoids. Anthocyanins serve as the principal coloring agents in the orange, red and purple tepals of Gloriosa. High levels and proportions of cyanidins and pelargonidins are key contributors to the formation of red and purple tepals, while high levels and proportions of peonidins also play a significant role in purple coloration. In contrast, the presence of high levels and proportions of pelargonidins alone is a crucial factor in the formation of orange tepals. Transcriptomic data unearthed 57 and 92 candidate differentially expressed genes (DEGs) belong to carotenoid and anthocyanin biosynthesis pathway, respectively, with PSY, PDS, DFR, and ANS genes considered as critical genes for the differential accumulation of pigments of Gloriosa tepals. Weighted gene co-expression network analysis (WGCNA) revealed significant co-expression patterns between 217 transcription regulatory factors (TFs) and 8 carotenoid biosynthesis genes, and between 194 TFs and 41 anthocyanin biosynthesis genes. qRT-PCR verified the expression patterns of four carotenoid biosynthesis-related genes, eight anthocyanin biosynthesis-related genes, and three transcription regulatory factors. It was found that Cluster-121969.6 (MYB) gene is specifically expressed in the tepals of the four varieties (compared to stems and leaves) and shows a high consistency with the trend of anthocyanin content changes. This research provides new insights into the mechanisms underlying the formation of diverse floral colors in Gloriosa tepals.

An accessible workflow for high-sensitivity proteomics using parallel accumulation-serial fragmentation (PASEF).

Deep and accurate proteome analysis is crucial for understanding cellular processes and disease mechanisms; however, it is challenging to implement in routine settings. In this protocol, we combine a robust chromatographic platform with a high-performance mass spectrometric setup to enable routine yet in-depth proteome coverage for a broad community. This entails tip-based sample preparation and pre-formed gradients (Evosep One) combined with a trapped ion mobility time-of-flight mass spectrometer (timsTOF, Bruker). The timsTOF enables parallel accumulation-serial fragmentation (PASEF), in which ions are accumulated and separated by their ion mobility, maximizing ion usage and simplifying spectra. Combined with data-independent acquisition (DIA), it offers high peak sampling rates and near-complete ion coverage. Here, we explain how to balance quantitative accuracy, specificity, proteome coverage and sensitivity by choosing the best PASEF and DIA method parameters. The protocol describes how to set up the liquid chromatography-mass spectrometry system and enables PASEF method generation and evaluation for varied samples by using the py_diAID tool to optimally position isolation windows in the mass-to-charge and ion mobility space. Biological projects (e.g., triplicate proteome analysis in two conditions) can be performed in 3 d with ~3 h of hands-on time and minimal marginal cost. This results in reproducible quantification of 7,000 proteins in a human cancer cell line in quadruplicate 21-min injections and 29,000 phosphosites for phospho-enriched quadruplicates. Synchro-PASEF, a highly efficient, specific and novel scan mode, can be analyzed by Spectronaut or AlphaDIA, resulting in superior quantitative reproducibility because of its high sampling efficiency.

Identification of Oligopeptides in the Distillates from Various Rounds of Soy Sauce-Flavored Baijiu and Their Effect on the Ester–Acid–Alcohol Profile in Baijiu

Endogenous peptides in Baijiu have primarily focused on finished liquor research, with limited attention given to the peptides in base liquor prior to blending. Liquid chromatography–tandem mass spectrometry (LC-MS) was employed to identify endogenous peptides in the distillates from the first to seventh rounds of soy sauce-flavored Baijiu. Two hundred and five oligopeptides were identified from these distillates, all of which had molecular weights below 1000 Da and were composed of amino acid residues associated with flavor (sweet, sour, and bitter) and biological activity. Furthermore, full-wavelength scanning, content determination of the main compounds, and molecular docking were performed to analyze these oligopeptides’ effect on the ester–acid–alcohol profile in Baijiu. This determination revealed a negative correlation between the peptide content and total ester content (r = −0.691), as well as the total acid content (r = −0.323), and a highly significant negative correlation with ethanol content (r = −0.916). Notably, the screened peptides (TRH, YHY, RQTQ, PLDLTSFVLHEAI, KHVS, LPQRHRMVYSLL, and NEWH) had specific interactions with the major flavor substances via hydrogen bonds, including esters (ethyl acetate, ethyl butanoate, ethyl hexanoate, and ethyl lactate), acids (acetate acid, butanoate acid, hexanoate acid, lactate acid), and alcohols (ethanol, 1-propanol, 1-butanol, and 1-hexanol). These findings elucidate the distribution and dynamic changes of endogenous peptides in the distillates from various rounds of soy sauce-flavored Baijiu, providing a theoretical foundation for further investigation into their interaction mechanisms associated with flavor compounds.

Renin–angiotensin–aldosterone system activation in plasma as marker for prognosis in critically ill patients with COVID-19: a prospective exploratory study

BackgroundAcute respiratory distress syndrome (ARDS) associated with coronavirus infectious disease (COVID)-19 has been a challenge in intensive care medicine for the past three years. Dysregulation of the renin–angiotensin system (RAS) is linked to COVID-19, but also to non-COVID-19 ARDS. It is still unclear whether changes in the RAS are associated with prognosis of severe COVID-19.MethodsIn this prospective exploratory study, blood samples of 94 patients with COVID-19 were taken within 48 h of admission to a medical ward or an ICU. In ICU patients, another blood sample was taken seven days later. Angiotensin (Ang) I-IV, Ang 1–7, Ang 1–5 and aldosterone concentrations were measured with liquid chromatography tandem mass spectrometry (LC–MS/MS) followed by calculation of markers for activities of renin (PRA-S) and ACE (ACE-S), alternative RAS activation (ALT-S) as well as the ratio of aldosterone to Ang II (AA2R). Angiotensin-converting enzyme (ACE) and ACE2 concentrations were measured by LC–MS/MS-based assays. All RAS parameters were evaluated as predictors of 28-day and 60-day survival using receiver operating characteristic and multivariate logistic regression analysis.ResultsAA2R at inclusion was a predictor of 60-day survival for ICU patients with an AUROC of 0.73. Ang II and active ACE2 were inversely associated with survival (OR 0.07; 95%CI 0.01, 0.39 and OR 0.10; 95%CI 0.01, 0.63) while higher Ang 1–7 predicted favorable outcome (OR 6.8; 95%CI 1.5, 39.9). ICU patients showed higher concentrations of all measured angiotensin metabolites, PRA-S, ALT-S and active ACE2, and lower ACE-S and AA2R than patients in the medical ward at inclusion. After seven days in the ICU, Ang I, Ang II, Ang III and Ang IV concentrations decreased, while ACE and ACE2 levels increased. Ang I, PRA-S, Ang 1–7 and Ang 1–5 concentrations correlated with the SOFA score both at the time of inclusion and after seven days, and driving pressure after seven days.ConclusionsAA2R at inclusion predicted 60-day survival with moderate sensitivity, revealing a dissociation between unchanged aldosterone and increased Ang II levels in the most severely ill COVID-19 patients. After adjustment for confounders, Ang 1–7 as the final metabolite of alternative RAS was predictive for survival.

Conjugation of Short-Chain Fatty Acids to Bicyclic-Amines for Analysis by Liquid Chromatography Tandem Mass Spectroscopy

Conjugation of Short-Chain Fatty Acids to Bicyclic-Amines for Analysis by Liquid Chromatography Tandem Mass Spectroscopy

Genome-Guided Identification and Characterisation of Broad-Spectrum Antimicrobial Compounds of Bacillus velezensis Strain PD9 Isolated from Stingless Bee Propolis.

The emergence of multidrug-resistant pathogens presents a significant global health challenge, which is primarily fuelled by overuse and misuse of antibiotics. Bacteria-derived antimicrobial metabolites offer a promising alternative strategy for combating antimicrobial resistance issues. Bacillus velezensis PD9 (BvPD9), isolated from stingless bee propolis, has been reported to have antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). This study aimed to characterise and identify the antimicrobial compounds (AMCs) synthesised by BvPD9 through integration of genome mining and liquid chromatography-mass spectrometry (LC-MS) analysis. The whole-genome sequence of BvPD9 contained 4,263,351 base pairs and 4101 protein-coding sequences, with 12 potential AMC biosynthetic gene clusters. Comparative genomic analysis highlighted the unique profile of BvPD9 that possesses the largest number of unknown proteins, indicating significant potential for further exploration. The combined genomics-metabolic profiling uncovered five AMCs in BvPD9 extract, including bacillibactin, bacilysin, surfactin A, fengycin A, and bacillomycin D. The extract exhibited a broad antibacterial spectrum against 25 pathogenic bacteria, including both Gram-positive and Gram-negative bacteria, with the lowest minimum inhibitory concentration (MIC, 0.032mg/ml) against S. epidermidis ATCC 12228, and the lowest minimum bactericidal concentration (MBC; 0.128mg/ml) against MRSA ATCC 700699 and Aeromonas hydrophilia. The robust stability of BvPD9 extract was demonstrated at high temperatures, over a wide range of pH conditions (6 to 12) and in the presence of various hydrolytic enzymes. Additionally, the extract showed 50% haemolytic and cytotoxicity activity at 0.158 and 0.250mg/ml, respectively. These characteristics suggest potential applications of BvPD9 metabolites for tackling antimicrobial resistance and its applicability across diverse industries.

Population pharmacokinetic model of tranexamic acid in patients who undergo cardiac surgery with cardiopulmonary bypass.

Tranexamic acid (TXA) is widely used as an antifibrinolytic drug. However, studies to determine the optimal blood concentration of TXA have produced inconsistent results. During cardiac surgery, cardiopulmonary bypass (CPB) has serious effects on drug distribution, elimination, and plasma concentration. Therefore, we aimed to establish a population pharmacokinetics model of TXA in patients undergoing cardiac surgery with CPB that considers renal function as a covariate, thereby facilitating personalized treatment. In total, 453 TXA plasma samples were prospectively collected from 77 patients who underwent cardiac surgery with CPB. Plasma concentrations were determined by ultra-performance liquid chromatography-tandem mass spectrometry. The population pharmacokinetic model of TXA was analyzed using nonlinear mixed-effects modeling. The two-compartment-based model with combined errors was determined as the best. The final model included the effect of bodyweight and CLcr may be summarized as V1 (L) = 12.77 × (bodyweight / 61.4)0.911, V2 (L) = 6.857, CL1 (L/h) = 3.263 × [CLcr (L/h) / 61.0]0.752, CL2 (L/h) = 2.859. Patients who undergo cardiac surgery with CPB may require an adjusted dose of TXA tailored to CPB due to lower CL1 and increased V1. Our TXA population pharmacokinetic model may be useful for developing individualized dosing designs for TXA in patients who undergo cardiac surgery with CPB.

Relationship Between the Salivary Microbiome and Oral Malodor Metabolites in Older Thai Individuals with Periodontitis and the Cytotoxic Effects of Malodor Compounds on Human Oral Squamous Carcinoma (HSC-4) Cells

Background/Objectives: Halitosis is primarily caused by the activity of oral microorganisms. In this study, we employed metagenomic sequencing and metabolomic approaches to investigate the differences in salivary microbiota and metabolite profiles between individuals with halitosis and periodontitis and healthy controls. Additionally, we expanded the study to examine how oral malodorous compounds interact with human oral squamous carcinoma (HSC-4) cells. Methods: Saliva samples were collected and analyzed using Ultra-High Performance Liquid Chromatography–Mass Spectrometry (UHPLC-MS) to identify metabolites. We then assessed the correlations between the microbiota and metabolites. Furthermore, the impact of oral malodorous substances on HSC-4 cells was investigated by evaluating apoptosis, antioxidant activity, and inflammatory properties. Results: The microbiota and metabolite profiles showed significant differences between the halitosis with periodontitis group and the periodontally healthy group. The halitosis with periodontitis group exhibited significantly higher relative abundances of eight genera: Tannerella, Selenomonas, Bacteroides, Filifactor, Phocaeicola, Fretibacterium, Eubacterium saphenum, and Desulfobulbus. In contrast, the periodontally healthy group showed significantly higher relative abundances of Family XIII UCG-001, Haemophilus, and Streptobacillus. Two metabolites, 2,3-dihydro-1H-indole and 10,11-dihydro-12R-hydroxy-leukotriene E4, were significantly higher in individuals with halitosis and periodontitis. In the treatment of HSC-4 cells with metabolites, dimethyl sulfide (DMS) did not show significant effects while indole appeared to induce cell death in HSC-4 cells by triggering apoptotic pathways. Additionally, both indole and DMS affected the inflammatory and antioxidant properties of HSC-4 cells. Conclusions: This study provides insights into the mechanisms of halitosis by exploring the correlations between microbiota and metabolite profiles. Furthermore, oral metabolites were shown to impact the cellular response of HSC-4 cells.

Direct Evidence of Microbial Sunscreen Production by Scum-Forming Cyanobacteria in the Baltic Sea.

Mycosporine-like amino acids are water-soluble secondary metabolites that protect photosynthetic microorganisms from ultraviolet radiation. Here, we present direct evidence for the production of these compounds in surface scums of cyanobacteria along the Baltic Sea coast. We collected 59 environmental samples from the southern coast of Finland during the summers of 2021 and 2022 and analysed them using high-resolution liquid chromatography-mass spectrometry. Our results revealed the presence of microbial sunscreens in nearly all surface scum samples. Mycosporine-like amino acids are synthesised through the coordinated action of four biosynthetic enzymes encoded in a compact biosynthetic pathway. Bioinformatics analysis of the mysB biosynthetic gene from a surface scum indicated that the cyanobacteria responsible for production belonged to the Anabaena/Dolichospermum/Aphanizomenon species complex. We mapped the distribution of biosynthetic enzymes onto a phylogenomic tree, utilising 120 bacterial single-copy conserved genes from 101 draft or complete genomes within the species complex. This analysis showed that 48% of identified species possess the ability to produce these compounds, with biosynthetic pathways being most common in Dolichospermum and Aphanizomenon strains. We detected the production of porphyra-334 and shinorine, two widely reported family members, in Dolichospermum strains isolated from the Gulf of Finland's surface layer. The estimated content of porphyra-334 in Dolichospermum sp. UHCC 0684 was 7.4 mg per gram dry weight. Our results suggest that bloom-forming cyanobacteria could be a potential source of these compounds for cosmetic and biotechnological applications and may play a significant role in cyanobacterial bloom formation.

Intracellular tenofovir-diphosphate concentrations in HIV pre-exposure prophylaxis users who underwent bariatric surgery.

To measure concentrations of tenofovir diphosphate (TFV-DP) in dried blood spots (DBS) among individuals taking tenofovir disoproxil fumarate plus emtricitabine (TDF/FTC) or tenofovir alafenamide plus emtricitabine (TAF/FTC) who were scheduled to undergo or had already undergone bariatric surgery. We enrolled pre-exposure prophylaxis (PrEP) users attending clinics in Toronto or Ottawa who were undergoing or had undergone bariatric surgery. After participants completed a minimum of 7 days of consecutive PrEP dosing, we collected DBS samples immediately before they administered their next daily dose of PrEP. Participants who had already undergone bariatric surgery before enrolment provided a single sample at baseline only. One participant undergoing planned bariatric surgery provided samples preoperatively and on postoperative days 7, 28 and 84. TFV-DP was measured by liquid chromatography tandem mass spectrometry. We compared results against the population range TFV-DP at varying degrees of adherence and stratified by chronology of bariatric surgery, type of bariatric surgery and PrEP regimen. Of seven eligible participants, all were gay, cis-gender men. Median age was 48 years (Q1-Q3: 44-51). Six participants underwent bariatric surgery before enrolment: four Roux-en-Y gastric bypass (RYGB) and two sleeve gastrectomy (SG). Four were taking TDF/FTC and two were taking TAF/FTC. All had therapeutic TFV-DP concentrations, except for one TDF/FTC participant who underwent SG. One participant taking TAF/FTC enrolled before receiving RYGB and displayed a slight decrease in TFV-DP over time, although all concentrations remained in the therapeutic range. Tenofovir diphosphate concentrations were at or near therapeutic values in this small sample of men using oral PrEP who underwent RYGB or SG.

Berberine ameliorates coronary artery endothelial cell injury in Kawasaki disease through complement and coagulation cascades

To explore the role of berberine (BBR) in ameliorating coronary endothelial cell injury in Kawasaki disease (KD) by regulating the complement and coagulation cascade. Human coronary artery endothelial cells (HCAEC) were divided into a healthy control group, a KD group, and a BBR treatment group (n=3 for each group). The healthy control group and KD group were supplemented with 15% serum from healthy children and KD patients, respectively, while the BBR treatment group received 15% serum from KD patients followed by the addition of 20 mmol/L BBR. Differential protein expression was analyzed and identified using isobaric tags for relative and absolute quantitation technology and liquid chromatography-tandem mass spectrometry, followed by GO functional enrichment analysis and KEGG signaling pathway enrichment analysis of the differential proteins. Western blot was used to detect differential protein expression. A total of 518 differential proteins were identified between the KD group and the healthy control group (300 upregulated proteins and 218 downregulated proteins). A total of 422 differential proteins were identified between the BBR treatment group and the KD group (221 upregulated proteins and 201 downregulated proteins). Bioinformatics analysis showed that compared to the healthy control group, the differential proteins in the KD group were enriched in the complement and coagulation cascade and ribosome biogenesis in eukaryotes. Compared to the KD group, the differential proteins in the BBR treatment group were also enriched in the complement and coagulation cascade and ribosome biogenesis in eukaryotes. Western blot results indicated that compared to the healthy control group, the expression of complement C1q subcomponent subunit C (C1QC), kininogen-1 (KNG1), complement C1s subcomponent (C1S), and C4b-binding protein alpha chain (C4BPA) was increased in the KD group (P<0.05). Compared to the KD group, the expression of KNG1, C1S, C1QC, and C4BPA was decreased in the BBR treatment group (P<0.05). The complement and coagulation cascade may be involved in the regulation of BBR treatment for coronary injury in KD, and C1QC, KNG1, C1S, and C4BPA may serve as biomarkers for this treatment.

Phytochemical Profile, Acute and Subacute Toxicity of Spray-Dried Hydroethanolic Extract from Punica granatum Leaves.

This study aimed to provide a comprehensive understanding of the acute and subacute safety and phytochemical profile of pomegranate leaves, aligning with the growing interest in sustainable, plant-based therapeutics. The phytochemical composition, the acute and subacute toxicity of a spray-dried hydroethanolic extract from pomegranate leaves (SDE) were investigated using experimental animal models. Utilizing UV-visible spectrophotometry and liquid chromatography-mass spectrometry (LC-MS), a diverse array of tannins and flavonoids, totaling 38 compounds, was identified. The findings revealed that SDE exhibited no adverse effects across various tests, including macroscopic, biochemical, hematological, and histological evaluations, even at high doses (2000mg/kg for single doses and 1000mg/kg for repeated doses). Furthermore, SDE significantly (p<0.05) reduced serum total cholesterol levels in both acute and subacute toxicity evaluations, suggesting a positive impact on lipid metabolism. This research not only confirms the safety of pomegranate leaf spray-dried extract at significant concentrations but also highlights its potential as a source of bioactive compounds with therapeutic benefits. The absence of toxicity, coupled with its cholesterol-lowering properties, supports the use of pomegranate leaves in medicinal, nutritional, and food additive applications. Additionally, this study provides essential phytochemical and safety data, paving the way for future research exploring its potential as an active ingredient.

Comparative Efficacy of Melatonin and Brassinolide in Mitigating the Adverse Effects of Cadmium on Wolffia arrhiza

Melatonin (MT) and brassinolide (BL) are phytohormones that regulate various physiological processes in plants. This study investigates their effects on Wolffia arrhiza when exposed to cadmium (Cd). Plant hormones were quantified using liquid chromatography-mass spectrometry, while photosynthetic pigments and phytochelatins (PCs) were analyzed through high-performance liquid chromatography. Protein, monosaccharide levels, and antioxidant activities were also spectrophotometrically measured. The findings reveal that MT and BL treatment decreased Cd accumulation in W. arrhiza compared to plants only exposed to Cd. MT was particularly effective in reversing Cd-induced growth inhibition and reducing stress markers more significantly than BL. It also enhanced antioxidant activity and maintained higher levels of photosynthetic pigments, proteins, and sugars. Although BL was less effective in these aspects, it promoted greater synthesis of glutathione and PCs in Cd-exposed duckweed. Overall, both MT and BL alleviate the negative impact of Cd on W. arrhiza, confirming their crucial role in supporting plant health under metal stress conditions.

First-trimester metabolic profiling of gestational diabetes mellitus: insights into early-onset and late-onset cases compared with healthy controls

IntroductionGestational diabetes mellitus (GDM) is a global health concern with significant short and long-term complications for both mother and baby. Early prediction of GDM, particularly late-onset, is crucial for implementing timely interventions to mitigate adverse outcomes. In this study, we conducted a comprehensive metabolomic analysis to explore potential biomarkers for early GDM prediction.MethodsPlasma samples were collected during the first trimester from 60 women: 20 with early-onset GDM, 20 with late-onset GDM, and 20 with normal glucose tolerance. Using advanced analytical techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS), we profiled over 150 lipid species and central carbon metabolism intermediates.ResultsSignificant metabolic alterations were observed in both early- and late-onset GDM groups compared to healthy controls, with a specific focus on glycerolipids, fatty acids, and glucose metabolism. Key findings revealed a 4.0-fold increase in TG(44:0), TG(46:0), TG(46:1) with p-values &amp;lt;0.001 and TG(46:2) with 4.7-fold increase and p-value &amp;lt;0.0001 as well as changes in several phospholipids as PC(38:3), PC(40:4) with 1.4-fold increase, p &amp;lt; 0.001 and PE(34:1), PE(34:2) and PE(36:2) with 1.5-fold change, p &amp;lt; 0.001 in late-onset GDM.DiscussionObserved lipid changes highlight disruptions in energy metabolism and inflammatory pathways. It is suggested that lipid profiles with distinct fatty acid chain lengths and degrees of unsaturation can serve as early biomarkers of GDM risk. These findings underline the importance of integrating metabolomic insights with clinical data to develop predictive models for GDM. Such models could enable early risk stratification, allowing for timely dietary, lifestyle, or medical interventions aimed at optimizing glucose regulation and preventing complications such as preeclampsia, macrosomia, and neonatal metabolic disorders. By focusing on metabolic disruptions evident in the first trimester, this approach addresses a critical window for improving maternal and fetal outcomes. Our study demonstrates the value of metabolomics in understanding the metabolic perturbations associated with GDM. Future research is needed to validate these biomarkers in larger cohorts and assess their integration into clinical workflows for personalized pregnancy care.

Identification of compounds against atherosclerosis induced by ox-LDL based on cell extraction/UPLC–MS/MS from mulberry twigs and their mechanistic analysis

BackgroundMulberry twigs, a traditional Chinese medicinal and agricultural byproduct, contain bioactive compounds with anti-atherosclerotic potential. This study aims to identify and evaluate the effects of key compounds in mulberry twig extracts (MTEs) on oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs), with a focus on understanding how these compounds modulate oxidative stress and related signaling pathways.MethodsBiospecific cell extraction and ultra-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) were employed to screen and identify these compounds. Protective effects were assessed by measuring cell viability, malondialdehyde (MDA), and superoxide dismutase (SOD) levels, along with detecting intracellular reactive oxygen species (ROS) using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidine (DHE) probes. Real-time qPCR and Western blotting were used for mRNA and protein level analysis.ResultsTwo novel active compounds, Kuwanon H and Morusin, and the known Kuwanon G, were identified. They significantly reduced MDA and ROS levels while increasing SOD activity in ox-LDL-treated HUVECs. Kuwanon H was particularly effective, enhancing nuclear factor erythroid 2-related factor 2 (Nrf-2) activity and upregulating its target genes Heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO-1).ConclusionIn conclusion, Kuwanon H, Morusin, and Kuwanon G effectively protected HUVECs from ox-LDL-induced oxidative injury, with Kuwanon H showing the strongest protective effects via the Nrf-2/HO-1 signaling pathway. These compounds hold potential in treating atherosclerosis and related oxidative stress diseases.Graphical

MpactR: an R adaptation of the metabolomics peak analysis computational tool (MPACT) for use in reproducible data analysis pipelines.

mpactR automates pre-processing of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data from microbiological samples to correct mispicked peaks, resolve inter-sample variation in abundance across technical replicates, account for in-source ion fragmentation, and remove background noise to yield high-quality mass spectrometry features. The package is available through CRAN and GitHub.