Liquid Chromatography-tandem Mass Spectrometry Research Articles

RNAseq and targeted metabolomics implicate RIC8 in regulation of energy homeostasis, amino acid compartmentation, and asexual development in Neurospora crassa

ABSTRACT Heterotrimeric G protein signaling pathways control growth and development in eukaryotes. In the multicellular fungus Neurospora crassa , the guanine nucleotide exchange factor RIC8 regulates heterotrimeric Gα subunits. In this study, we used RNAseq and liquid chromatography-mass spectrometry (LC-MS) to profile the transcriptomes and metabolomes of N. crassa wild type, the Gα subunit mutants Δ gna-1 and Δ gna-3 , and Δ ric8 strains. These strains exhibit defects in growth and asexual development (conidiation), with wild-type and Δ gna-1 mutants producing hyphae in submerged cultures, while Δ gna-3 and Δ ric8 mutants develop conidiophores, particularly in the Δ ric8 mutant. RNAseq analysis showed that the Δ gna-1 mutant possesses 159 mis-regulated genes, while Δ gna-3 and Δ ric8 strains have more than 1,000 each. Many of the mis-regulated genes are involved in energy homeostasis, conidiation, or metabolism. LC-MS revealed changes in levels of primary metabolites in the mutants, with several arginine metabolic intermediates impacted in Δ ric8 strains. The differences in metabolite levels could not be fully explained by the expression or activity of pathway enzymes. However, transcript levels for two predicted vacuolar arginine transporters were affected in Δ ric8 mutants. Analysis of arginine and ornithine levels in transporter mutants yielded support for altered compartmentation of arginine and ornithine between the cytosol and vacuole in Δ ric8 strains. Furthermore, we validated previous reports that arginine and ornithine levels are low in wild-type conidia. Our results suggest that RIC8 regulates asexual sporulation in N. crassa at least in part through altered expression of vacuolar transporter genes and the resultant mis-compartmentation of arginine and ornithine. IMPORTANCE Resistance to inhibitors of cholinesterase-8 (RIC8) is an important regulator of heterotrimeric Gα proteins in eukaryotes. In the filamentous fungus Neurospora crassa , mutants lacking ric8 undergo inappropriate asexual development (macroconidiation) during submerged growth. Our work identifies a role for RIC8 in regulating expression of transporter genes that retain arginine and ornithine in the vacuole (equivalent of the animal lysosome) and relates this function to the developmental defect. Arginine is a critical cellular metabolite, both as an amino acid for protein synthesis and as a precursor for an array of compounds, including proline, ornithine, citrulline, polyamines, creatine phosphate, and nitric oxide. These results have broad relevance to human physiology and disease, as arginine modulates immune, vascular, hormonal, and other functions in humans.

Apixaban removal during emergency surgery for type a acute aortic dissection. a prospective cohort study

Background: Acute type A aortic dissection (ATAAD) has a high mortality, and acute aortic repair is the only curative treatment. In patients treated with factor Xa (FXa) inhibitors, the risk of severe disease-related complications such as cardiac tamponade and hemodynamic shock must be balanced against the potential for severe perioperative bleeding. The aim was to study intraoperative changes in plasma levels of the FXa inhibitor apixaban when using hemoadsorption during acute thoracic aortic repair. Materials and Methods: This is a single-center prospective cohort study. Eight apixaban-treated patients presenting with ATAAD underwent acute thoracic aortic repair with intraoperative hemoadsorption with CytoSorb®. Apixaban concentrations were measured at the start of cardiopulmonary bypass (CPB), and after 5, 15, 30, 60 and 90 minutes of CPB, at CPB weaning, 30 minutes after CPB weaning and 24 hours postoperatively, using ultraperformance liquid chromatography mass spectrometry (UPLC-MS). Results: After 30 minutes of CPB with hemoadsorption, mean apixaban concentration (± SD) was reduced by 59% from 108 (± 69) µg/L to 44 (± 20) µg/L (P = 0.009). There was a further reduction to 37 (± 17) µg/L at CPB weaning (P = 0.008). Apixaban concentration displayed an increase to 56 (± 29) µg/L 24 hours postoperatively (P = 0.01). In-hospital mortality was 25%. Mean 24H chest tube drainage volume was 621 (± 136) mL. Conclusion: Intraoperative hemoadsorption lowers apixaban levels in patients undergoing emergency surgery for ATAAD. Further research is needed to determine its impact on perioperative bleeding complications and mortality.

Blood metabolomic profile in patients with type 2 diabetes mellitus with diabetic peripheral neuropathic pain.

This study aimed to identify metabolic markers for diabetic peripheral neuropathic pain (DPNP) in patients with type 2 diabetes mellitus (T2DM). Blood metabolite levels in the amino acid, biogenic amine, sphingomyelin, phosphatidylcholine (PC), carnitines, and hexose classes were analyzed in nondiabetic control (n = 27), T2DM without DPNP (n = 58), and T2DM with DPNP (n = 29) using liquid chromatography tandem mass spectrometry. Variable importance projection (VIP) evaluation by partial least squares discriminant analysis was performed on clinical parameters and metabolites. Sixteen variables with VIP > 1.0 (P < 0.05) were identified across all patient groups, and 5 variables were identified to discriminate between the two T2DM groups. DPNP patients showed elevated fasting blood glucose, glutamate, PC aa C36:1, lysoPC a C18:1, and lysoPC a C18:2, while low-density lipoprotein cholesterol, phenylalanine, and tryptophan were reduced. Glutamate, lysoPC a C18:1, and lysoPC a C18:2 discriminated T2DM with DPNP from those without DPNP with an AUC of 0.671. The AUC was improved to 0.765 when ratios of metabolite pairs were considered. Blood metabolites include glutamate, and phospholipid-related metabolites implicated in neuropathic pain may have the potential as biomarkers for DPNP. Further investigation is required to understand the mechanism of action of these altered metabolites in DPNP.

Endocannabinoid dysregulation and PTSD in urban adolescents: Associations with anandamide concentrations and FAAH genotype.

The endocannabinoid system, which regulates fear- and anxiety-related behaviors, is dysregulated in adults with posttraumatic stress disorder (PTSD), as indicated by higher circulating anandamide (AEA) concentrations. The C385A (rs324420) polymorphism in the fatty acid amide hydrolase (FAAH) gene, which catabolizes AEA, is linked to higher AEA concentrations and greater PTSD symptoms in adults. Given that adolescence is a critical period during which trauma and psychiatric disorders emerge, understanding this relationship in youth is essential. This study examines PTSD symptoms, AEA concentrations, and FAAH genotype in a diverse adolescent sample. This study included 102 Detroit-area adolescents (M ± SD = 13.33 ± 2.21 years, 54.9% female) and their parents/guardians. The sample consisted of 40.2% White Non-Hispanic, 34.3% Black Non-Hispanic, 6.9% White Hispanic, 4.9% Asian/Pacific Islander, and 12.7% Biracial adolescents. Trauma exposure and PTSD symptoms were assessed using the UCLA PTSD Reaction Index for DSM-5. Plasma concentrations of AEA were measured by liquid chromatography-tandem mass spectrometry, and FAAH genotype was determined from saliva samples and high-throughput screening. The majority (90%) of adolescents reported trauma exposure, and 20% met PTSD criteria. Higher AEA concentrations were associated with more severe PTSD symptoms (p = 0.009), especially hyperarousal. The FAAH A-allele (present in 52.5% of participants) was associated with higher AEA concentrations (2.11 ± 0.69 pmol/ml, p = 0.013) and greater PTSD severity (22.65 ± 15.931, p = 0.027), particularly those with the reexperiencing cluster, compared to the CC genotype (1.79 ± 0.66 pmol/ml and 15.87 ±+ 13.043, respectively). Elevated AEA concentrations and the FAAH A-allele were associated with greater PTSD symptom severity in urban adolescents. These findings suggest endocannabinoid dysregulation may play a role in adolescent PTSD, highlighting the need for further research and targeted interventions.

Phosphorylation of Zearalenone Retains Its Toxicity.

Microbial biotransformation of Zearalenone (ZEN) is a promising deactivation approach. The residual toxicity and stability of Zearalenone-14-phosphate (ZEN-14-P) and Zearalenone-16-phosphate (ZEN-16-P), two novel microbial phosphorylation products of ZEN, remain unknown. We investigated the cytotoxicity, oxidative stress, proinflammatory, and estrogenic activity of phosphorylated ZENs using porcine intestinal cells, uterine explants, and human endometrial cells and traced their metabolic fate by liquid chromatography-tandem mass spectrometry (LC-MS)/MS analysis. The phosphorylated ZENs significantly decreased the viability of the IPEC-J2 and Ishikawa cells. Similar to ZEN, phosphorylation products induced significant oxidative stress, activated the expression of proinflammatory cytokines, and demonstrated estrogenic activity through upregulation of estrogen-responsive genes, activation of alkaline phosphatase, and proliferation of endometrial glands. LC-MS/MS analysis pointed out that although phosphorylated ZENs are partially hydrolyzed to ZEN, their respective metabolic pathways differ. We conclude that phosphorylation might not be sufficient to detoxify ZEN, leaving its cytotoxic, proinflammatory, and estrogenic properties intact.

Altered colonic microflora and its metabolic profile in mice with acute viral myocarditis induced by coxsackievirus B3.

Mounting evidence suggests that the gut-heart axis is critical in the pathogenesis of cardiovascular diseases. The gut serves as the primary pathway through which Coxsackievirus B3 (CVB3) infects its host, leading to acute viral myocarditis (AVMC). However, little is known about the role of gut microflora and its metabolites in the development of AVMC. The AVMC model was established by intraperitoneal injection of CVB3 in mice. Then, 16S ribosomal RNA (16S rRNA) gene sequencing and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) untargeted metabolomics profiling were performed to analyze the microflora composition and metabolic profile of colonic contents. Compared to the Control mice, the AVMC mice displayed a significant reduction in gut microflora richness and diversity, as revealed by an increased abundance of Proteobacteria and a decreased abundance of Cyanobacteria and Desulfobacterota. LEfSe analysis indicated that the main genera differing between the two groups were Escherichia-Shigella, Lactobacillus, Clostridium_sensu_stricto_1, Prevotellaceae_UCG-001, and Odoribacter. Based on the criterion of OPLS-DA VIP ≥ 1.0 and p-value < 0.05, a total of 198 differential metabolites (DMs) were identified in the gut, including 79 upregulated and 119 downregulated metabolites, of which lipids and lipid-like molecules accounted for the largest proportion. Notably, both altered gut bacterial taxa and metabolites were significantly enriched in the Lipid metabolism pathway, with Traumatic acid (TA), Alpha-Linolenic acid (ALA), Eicosapentaenoic acid (EPA), and Docosahexaenoic acid (DHA) being the key DMs in the pathway. Additionally, significant positive correlations (|r| > 0.80 and p < 0.05) were found between TA levels and Anaerotruncus and Bilophila abundance, between EPA levels and Clostridium_sensu_stricto_1 abundance, and between DHA levels and Escherichia-Shigella abundance, respectively. CVB3 infection leads to notable alterations in gut microflora composition and its metabolic profile, which may participate in AVMC development. Our findings provide important clues for future in-depth studies on AVMC etiology.

Detection of "smoke powder" etomidate and its metabolite etomidate acid in blood and urine by UHPLC-MS-MS: application in authentic cases.

Recently, etomidate has been widely used as an alternative in illicit drug market. It is usually added to regular cigarette tobacco (commonly known as "cigarette powder") or mixed in e-cigarette oil sold through the Internet, retail stores, or entertainment outlets and other channels. An ultra-high performance liquid chromatography tandem mass spectrometry method was developed to quantify etomidate and etomidate acid in human blood and urine. The limit of detection (LOD) of etomidate and etomidate acid in blood is 0.5 and 2 ng/mL, respectively, and the lower limit of quantification (LLOQ) is 1 and 5 ng/mL, respectively. The LOD of etomidate and etomidate acid in urine is 1 and 2 ng/mL, respectively, and the LLOQ is 2 and 5 ng/mL, respectively. The precision, accuracy, recoveries, and matrix effects of etomidate and etomidate acid determinations in blood and urine met the requirements for methodological validation. The method was successfully applied to the identification and quantification of etomidate and etomidate acid in blood and urine of 62 forensic cases. The concentration of etomidate ranged from 1.52 to 8.41 ng/mL (positive cases, n = 5) and the concentration of etomidate acid ranged from 2.76 to 112 ng/mL (positive cases, n = 5) in blood. The concentrations of etomidate and etomidate acid in urine samples were 2.64-79,300 ng/mL (positive cases, n = 59) and 6.11-518,000 ng/mL (positive cases, n = 60), respectively. Therefore, the concentration of etomidate in blood and urine is mostly higher than that of etomidate.

Advancing Targeted Metabolomics Using Cyanopropyl-Based Liquid Chromatography Tandem Mass Spectrometry.

The change of metabolic pathways is recognized as the key to disease discovery prompting the development of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based quantitative platforms to explore the dynamic metabolite profiles of organisms. In this study, a liquid chromatography method based on cyanopropyl (CN) was developed. By adjusting the pH environment of the column, we achieved the elution of 51 metabolites spanning the most comprehensive set of biological pathways currently known. Offering rapid chromatography, efficient separation, and green chemistry benefits, the method encompasses nucleosides and nucleotides, the oxidative-redox metabolome, the glycolysis pathway, the pentose phosphate pathway, the purine de novo pathway, amino acids, and neurological disorder-related metabolites. The mass spectrometry was equipped with electrospray ionization in both positive and negative modes with scheduled multiple reactions monitoring. The validation of the method involved a comprehensive assessment of linearity, accuracy, precision, and matrix effect. The linear range was from 1.0 to 2000 ng mL-1 with a high correlation coefficient (r > 0.99). The LOD ranged from 0.1 to 10 ng mL-1, and the LOQ ranged from 0.1 to 25 ng mL-1. The overall recovery ranged from 81.3% to 117.8%, with RSD < 15.1%. Subsequently, an analysis of metabolites was conducted in dSH-SY5Y neuroblastoma cells with 6-hydroxydopamine, a commonly used neurotoxin in neurodegenerative diseases. The results demonstrate that neurotoxin-induced mitochondrial damage significantly altered related analytes, corroborating previous estimates and validating the feasibility and reliability of the bioanalytical platform.

Comparative Analysis of Liquid Chromatography-Tandem Mass Spectrometry and Radioimmunoassay in Determining Plasma Aldosterone Concentration and Plasma Renin Activity for Primary Aldosteronism Screening.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) accurately measures plasma aldosterone concentration (PAC), but its correlation with radioimmunoassay (RIA), equivalent RIA levels, and optimal cutoff for PAC and aldosterone-to-renin ratio (ARR) in primary aldosteronism (PA) screening have not been determined in a Korean population. Our study of 127 patients who underwent diagnostic testing for PA showed that the LC-MS/MS and RIA methods have good correlation, with a mean bias of 29.3% for PAC. An LC-MS/MS PAC level of 11.7 ng/dL was equivalent to an RIA PAC level of 15 ng/dL. Receiver operating characteristic curve analysis showed that an LC-MS/MS PAC level of 10.3 ng/dL and LC-MS/MS ARR level of 20.0 provided sensitivity of 73.1% with a specificity of 57.3% and sensitivity of 92.3% with a specificity of 14.7%, respectively. When the LC-MS/MS method is used for PA screening, an adjustment of cutoff values is necessary.

A novel miniaturized potentiometric electrode based on carbon nanotubes and molecularly imprinted polymer for the determination of lidocaine.

A novel miniaturized, solid-contact potentiometric screen-printed electrode was developed for highly sensitive and selective determination of lidocaine anesthetic. The electrode integrated single-walled carbon nanotubes as a solid-contact material and a molecularly imprinted polymer as a recognition sensory material. The performance characteristics of the electrode were evaluated and optimized to display a Nernstian slope of 58.92 ± 0.98mV/decade over a linear concentration range of 4.53 × 10-7 to 6.18 × 10-3mol/l within < 6s. Thedetection limit was 7.75 × 10-8mol/l (18.16ng/ml) of lidocaine. The use of the molecularly imprinted polymer significantly enhanced the selectivity of the electrode, and carbon nanotubes increased the sensitivity, accuracy, and potential stability. The electrode was successfully used for determining lidocaine in pharmaceutical preparations and human urine. The results favorably compared with data obtained by liquid chromatography-tandem mass spectrometry.

The human plasma lipidome response to exertional heat tolerance testing.

The year of 2023 displayed the highest average global temperatures since it has been recorded-the duration and severity of extreme heat are projected to increase. Rising global temperatures represent a major public health threat, especially to occupations exposed to hot environments, such as construction and agricultural workers, and first responders. Despite efforts of the scientific community, there is still a need to characterize the pathophysiological processes leading to heat related illness and develop biomarkers that can predict its onset. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics analysis was performed on plasma from male and female subjects who underwent exertional heat tolerance testing (HTT), consisting of a 2-h treadmill walk at 5km/h with 2.0% incline at a controlled temperature of 40ºC. From HTT, heat tolerance was calculated using the physiological strain index (PSI). Nearly half of all 995 detected lipids from 27 classes were responsive to HTT. Lipid classes related to substrate utilization were predominantly affected by HTT, with a downregulation of triacylglycerols and upregulation of free fatty acids and acyl-carnitines (CARs). Even chain CAR 4:0, 14:0 and 16:1, suggested by-products of incomplete beta oxidation, and diacylglycerols displayed the highest correlation to PSI. PSI did not correlate with plasma lactate levels, suggesting that correlations between even chain CARs and PSI are related to metabolic efficiency versus physical exertion. Overall, HTT displays a strong impact on the human plasma lipidome and lipid metabolic inefficiencies may underlie reduced heat tolerance.

Global metabolomic profiling of tumor tissue and paired serum samples to identify biomarkers for response to neoadjuvant FOLFIRINOX treatment of human pancreatic cancer.

Neoadjuvant chemotherapy (NAT) is increasingly used for the treatment of non-metastatic pancreatic ductal adenocarcinoma (PDAC) and is established as a standard of care for borderline resectable and locally advanced PDAC. However, full exploitation of its clinical benefits is limited by the lack of biomarkers that assess treatment response. To address this unmet need, global metabolomic profiling was performed on tumor tissue and paired serum samples from patients with treatment-naïve (TN; n = 18) and neoadjuvant leucovorin calcium (folinic acid), fluorouracil, irinotecan hydrochloride and oxaliplatin (FOLFIRINOX)-treated (NAT; n = 17) PDAC using liquid chromatography mass spectrometry. Differentially abundant metabolites (DAMs) in TN versus NAT groups were identified and their correlation with various clinical parameters was assessed. Metabolomics profiling identified 40 tissue and five serum DAMs in TN versus NAT PDAC. In general, DAMs associated with amino acid andnucleotide metabolism were lower in NAT compared to TN. Four DAMs-3-hydroxybutyric acid (BHB), 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), glycochenodeoxycholate and citrulline-were common to both tissue and serum and showed a similar pattern of differential abundance in both groups. A strong positive correlation was observed between serum carbohydrate 19-9 antigen (CA 19-9) and tissue carnitines (C12, C18, C18:2) and N8-acetylspermidine. The reduction in CA 19-9 following NAT correlated negatively with serum deoxycholate levels, and the latter correlated positively with survival. This study revealed neoadjuvant-chemotherapy-induced changes in metabolic pathways in PDAC, mainly amino acid and nucleotide metabolism, and these correlated with reduced CA 19-9 following neoadjuvant FOLFIRINOX treatment.

Systematic characterization of extracellular vesicles from potato (Solanum tuberosum cv. Laura) roots and peels: biophysical properties and proteomic profiling

IntroductionExtracellular vesicles (EVs) facilitate inter and intra-species/kingdom communication through biomolecule transfer, including proteins and small RNAs. Plant-derived EVs, a hot topic in the field, hold immense capability both as a potential biomarker to study plant physiology and as a biomaterial that can be mass-produced to be used in various industries ranging from cosmetics and food additives to biological pesticides. However, a systematic characterization of plant EVs is required to establish a foundation for further applications and studies.MethodsIn this study, EVs were enriched from hydroponically cultivated potato plants (Solanum tuberosum, cv. Laura). We isolated EVs from root exudates and the apoplastic wash of potato peels using vacuum infiltration. These EVs were then systematically characterized for their biophysical and chemical properties to compare with standard EV characteristics and to explore their roles in plant physiology.ResultsBiophysical and chemical analyses revealed morphological similarities between potato root and peel-derived EVs. The average diameter of root-derived EVs (164.6 ± 7.3 nm) was significantly larger than that of peel-derived EVs (132.2 ± 2.0 nm, p &amp;lt; 0.004). Liquid chromatography–mass spectrometry (LC-MS) demonstrated substantial protein enrichment in purified EVs compared to crude samples, with a 42% enrichment for root EVs and 25% for peel EVs. Only 11.8% of the identified proteins were common between root and peel EVs, with just 2% of significantly enriched proteins shared. Enriched pathways in both EV proteomes were associated with responses to biotic and abiotic stress, suggesting a defensive role of EVs in plants.DiscussionWith further experimentation to elucidate the specific methods of communication, these findings increase the details known about plant EVs in terms of their physical and chemical characteristics and their potential functions, aiding in sustainable agricultural waste utilization for large-scale EV production, aligning with the concept of “valorization”.

Association between overweight or obesity and vitamin D status in preschool children: an epidemiological survey in Beijing, China, 2021-2023.

Previous studies have shown that vitamin D deficiency is a risk factor for obesity in children and adolescents, but most focus on school-age children and adolescents, with little attention paid preschoolers. To explore the relationship between overweight or obesity in the context of vitamin D intake among preschool children in Beijing, and analyze the correlation. A total of 51,640 preschoolers (26,775 boys; 24,865 girls), aged 1-6 years, were recruited for physical examination. Overweight or obesity was defined according to the World Health Organization Child Growth and Development Standards (2006 edition). Serum 25-hydroxy (OH) vitamin D levels were determined using standardized liquid chromatography-tandem mass spectrometry (LC-MS/MS), and vitamin D status was evaluated based on the Practice Guide on Clinical Issues Related to Vitamin D Nutrition in Chinese Children (2022). No significant change was observed in the incidence of overweight (7.72, 7.51, and 7.24 %) or obesity (2.75, 2.63, and 2.40 %) from 2021 to 2023. Among boys, 7.3 % were overweight and 2.8 % were obese. Among girls, 7.6 % were overweight and 2.3 % were obese. Vitamin D deficiency (2.10, 1.70, and 1.01 %) and insufficiency rate (24.09, 18.42, and 15.44 %) showed a decreasing trend. Deficiency or insufficiency was most common in children aged 36-59months, and serum 25- (OH) vitamin D levels were significantly lower in winter compared to other seasons, with the highest levels in summer. Time spent outdoors was significantly less among children with vitamin D deficiency or insufficiency, and preschoolers who spent more than 2.94 h/day had higher serum vitamin D level. Serum 25- (OH) vitamin D levels were negatively correlated with body mass index (BMI), with overweight or obesity preschoolers showing significantly lower than their normal weight group. After adjusting for age, gender and season, family economic status, guardian educational level and time spent outdoors, the odds of vitamin D deficiency and insufficiency in overweight or obesity in preschoolers were 1.025 (95 % Cl: 1.002-1.174), 1.218 (95 % Cl: 1.099-1.708), respectively. From 2021 to 2023, there was no significant change in the incidence of overweight or obesity among preschool children in Beijing. Furthermore, the rate of vitamin D deficiency or insufficiency decreased year by year. Vitamin D deficiency or insufficiency associates with factors such as age, season of the year, and time spent outdoors, and there is an association between overweight or obesity and vitamin D status among preschoolers.

Development and Clinical Validation of Liquid Chromatography-Tandem Mass Spectrometry for Measuring Ruxolitinib in Steroid-Refractory Graft-Versus-Host Disease: A First Step Towards Optimized Treatment.

This non-interventional, prospective, single-center study aimed to develop a technique to measure ruxolitinib (RUX) concentrations and provide preliminary data on the distribution of plasma drug levels in patients with steroid refractory (SR) GvHD. Between April 2023 and May 2024, we analyzed 48 blood samples from 29 patients with SR-GvHD. Median individual plasma concentrations varied across different RUX doses and largely overlapped: 39.2 ng/mL at 10 mg b.i.d (range: 0-73), 13.1 ng/mL at 10-5 mg (range, 6.1-35.6), and 31.6 ng/mL at 5 mg b.i.d (range: 0.7-99.9). Samples taken under non-optimal temperature conditions showed a lower median concentration of 0.77 ng/mL (range: 0-7.4 ng/mL). The four patients who did not respond at days +28 and +180 after RUX initiation (3 with lower gastrointestinal aGvHD, and 1 with ocular, hepatic, and pulmonary cGvHD) showed a median concentration of only (7.4 ng/mL (range, 0-29) ng/mL) with full dosing. The introduction and validation of a liquid chromatography-tandem mass spectrometry method for quantifying plasma RUX concentrations was feasible in our center. Administering predetermined and fixed doses of RUX in patients with SR-GvHD showed highly variable and overlapping plasma drug concentrations. This underscores the potential importance of RUX- pharmacokinetics (PK) monitoring.

Jinwu Jiangu capsule attenuates rheumatoid arthritis via the SLC7A11/GSH/GPX4 pathway in M1 macrophages.

JinWu JianGu capsule (JWJGC) is a Chinese herbal medicine that alleviates the clinical manifestations of rheumatoid arthritis (RA). However, the mechanism of action requires further investigation.. This study aimed to investigate the anti-inflammatory effects of JWJGC on RA via the regulation of reactive oxygen species (ROS) and ferroptosis. JWJGC was administered to rats with collagen-induced arthritis (CIA) via oral gavage for 28 days. In vitro, M1 macrophages were pre-treated with JWJGC-containing serum. After assessing joint swelling and physiologic, the M1/M2 macrophage ratio was detected in CIA rats. The levels of ROS markers were assessed in the serum and cell supernatants. Liquid chromatography-tandem mass spectrometry analyses were employed to check lipid metabolism, and changes in mitochondrial morphology during ferroptosis were detected by transmission electron microscopy. Western blotting, immunofluorescence, immunohistochemistry, and qRT-PCR were performed to validate these results. JWJGC ameliorated CIA by reducing ROS levels in rats. It also restored the balance of the M1/M2 macrophage ratio and reduced the levels of macrophage-related inflammatory markers. Additionally, JWJGC affected lipid metabolism to alleviate inflammation by downregulating lipids associated with ferroptosis. It attenuated ferroptosis by modulating glutathione (GSH)/ Glutathione peroxidase 4(GPX4) expression in CIA rats. In vitro, JWJGC targeted M1 macrophages via the solute carrier family 7a member 11 (SLC7A11)/GSH/GPX4 signaling pathway. This study showed that JWJGC improved RA, primarily through the integrated regulation of the SLC7A11/GSH/GPX4 pathway in M1 macrophages. These findings provide an innovative therapeutic basis for RA treatment and expanding the clinical applications of JWJGC.

P63 affects distinct metabolic pathways during keratinocyte senescence, evaluated by metabolomic profile and gene expression analysis.

Unraveling the molecular nature of skin aging and keratinocyte senescence represents a challenging research project in epithelial biology. In this regard, depletion of p63, a p53 family transcription factor prominently expressed in human and mouse epidermis, accelerates both aging and the onset of senescence markers in vivo animal models as well as in ex vivo keratinocytes. Nonetheless, the biochemical link between p63 action and senescence phenotype remains largely unexplored. In the present study, through ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) and gas chromatography/mass spectrometry (GC/MS) metabolomic analysis, we uncover interesting pathways linking replicative senescence to metabolic alterations during p63 silencing in human keratinocytes. Integration of our metabolomic profiling data with targeted transcriptomic investigation empowered us to demonstrate that absence of p63 and senescence share similar modulation profiles of oxidative stress markers, pentose phosphate pathway metabolites and lyso-glycerophospholipids, the latter due to enhanced phospholipases gene expression profile often under p63 direct/indirect gene control. Additional biochemical features identified in deranged keratinocytes include a relevant increase in lipids production, glucose and pyruvate levels as confirmed by upregulation of gene expression of key lipid synthesis and glycolytic enzymes, which, together with improved vitamins uptake, characterize senescence phenotype. Silencing of p63 in keratinocytes instead, translates into a blunted flux of metabolites through both glycolysis and the Krebs cycle, likely due to a p63-dependent reduction of hexokinase 2 and citrate synthase gene expression. Our findings highlight the potential role of p63 in counteracting keratinocyte senescence also through fine regulation of metabolite levels and relevant biochemical pathways. We believe that our research might contribute significantly to the discovery of new implications of p63 in keratinocyte senescence and related diseases.

Multi-omics with dynamic network biomarker algorithm prefigures organ-specific metastasis of lung adenocarcinoma.

Efficacious strategies for early detection of lung cancer metastasis are of significance for improving the survival of lung cancer patients. Here we show the marker genes and serum secretome foreshadowing the lung cancer site-specific metastasis through dynamic network biomarker (DNB) algorithm, utilizing two clinical cohorts of four major types of lung cancer distant metastases, with single-cell RNA sequencing (scRNA-seq) of primary lesions and liquid chromatography-mass spectrometry data of sera. Also, we locate the intermediate status of cancer cells, along with its gene signatures, in each metastatic state trajectory that cancer cells at this stage still have no specific organotropism. Furthermore, an integrated neural network model based on the filtered scRNA-seq data is successfully constructed and validated to predict the metastatic state trajectory of cancer cells. Overall, our study provides an insight to locate the pre-metastasis status of lung cancer and primarily examines its clinical application value, contributing to the early detection of lung cancer metastasis in a more feasible and efficacious way.

Multi-class cyanobacterial toxin analysis using hydrophilic interaction liquid chromatography-mass spectrometry.

Cyanobacteria produce diverse classes of toxins including microcystins, nodularins, anatoxins, cylindrospermopsins and saxitoxins, encompassing a range of chemical properties and mechanisms of toxicity. Comprehensive analysis of these toxins in cyanobacterial, environmental and biological samples generally requires multiple methods of extraction and analysis. In this work, a method was developed for the major classes of cyanotoxins, which comprised of a three-step liquid-solid extraction method using 75 % CH3CN with 0.1 % HCOOH and a hydrophilic interaction liquid chromatography (HILIC) elution gradient that provided retention of the less polar microcystins through to the highly polar saxitoxins. Detection was performed by tandem mass spectrometry in selected reaction monitoring mode with positive and negative polarity switching. Identification criteria included matching retention times and product ion ratios with available standards. In-house validation demonstrated good performance of the method including precision ranging from 1.5 (microcystin-LA) to 5.8 (gonyautoxin-2) % RSDs, and detection limits ranging from 0.01 (cylindrospermopsin) to 0.99 (gonyautoxin-3) µg/g in freeze dried material cyanobacteria. Recovery was assessed using spiked non-toxic cyanobacterial samples (Aphanizomenon sp.) and ranged from 83 (neosaxitoxin) to 107 % ([Dha7]microcystin-LR). As a demonstration of application, toxin profiles in cyanobacterial cultures, benthic and planktonic cyanobacteria field samples, and shellfish reference materials were successfully evaluated. The procedure is also amenable for extension to other polar toxin classes including domoic acid and guanitoxin. With increasing reports of cyanobacterial blooms globally, the method represents a powerful quantitative screening tool for measuring cyanotoxins across a broad range of samples.

Depletion by Hemodialysis of the Antioxidant Ergothioneine

Background: Hemodialysis may excessively remove valuable solutes. Untargeted metabolomics data from a prior study suggested that ergothioneine was depleted in the plasma of hemodialysis subjects. Ergothioneine is a dietary-derived solute with antioxidant properties. The presence of a highly specific ergothioneine uptake transporter suggests that it is valuable. Ergothioneine levels are high in tissues susceptible to oxidative stress, particularly erythrocytes. We compared erythrocyte and plasma ergothioneine levels in subjects receiving hemodialysis to those in subjects with advanced chronic kidney disease (CKD) and subjects without known kidney disease (controls). We further examined the extent to which indiscriminate removal by hemodialysis could contribute to ergothioneine depletion. Methods: Liquid chromatography tandem mass spectrometry with stable isotope dilution was used to measure the erythrocyte and plasma levels of ergothioneine in 12 control, 12 CKD, and 11 hemodialysis subjects. We also measured the urinary excretion of ergothioneine in control and CKD subjects, and the dialytic removal of ergothioneine in hemodialysis subjects. Results: Erythrocyte ergothioneine levels were markedly reduced in CKD and hemodialysis subjects. Erythrocyte levels in CKD subjects were on average 24% of the levels in control subjects and were even lower in hemodialysis subjects, averaging 8% of control subjects. Plasma ergothioneine levels were also reduced in CKD and hemodialysis subjects but to a lesser extent than the erythrocyte levels. Kidney tubular reabsorption of ergothioneine was avid. In contrast, hemodialysis cleared ergothioneine at a rate of 146±36 ml/min so that removal of ergothioneine by hemodialysis greatly exceeded the amount excreted in the urine in both CKD and control subjects. Conclusions: Ergothioneine, a potentially valuable antioxidant, is severely depleted in people maintained on hemodialysis. Future studies are required to assess the consequences of ergothioneine depletion.