Analysis Of Metabolites Research Articles

Untargeted Metabolomics Based on Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Orbitrap High-Resolution Mass Spectrometry for Differential Metabolite Analysis of Pinelliae Rhizoma and Its Adulterants

Untargeted Metabolomics Based on Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Orbitrap High-Resolution Mass Spectrometry for Differential Metabolite Analysis of Pinelliae Rhizoma and Its Adulterants

Strigolactones promote flowering by inducing the miR319-LA-SFT module in tomato

Strigolactones are a class of phytohormones with various functions in plant development, stress responses, and in the interaction with (micro)organisms in the rhizosphere. While their effects on vegetative development are well studied, little is known about their role in reproduction. We investigated the effects of genetic and chemical modification of strigolactone levels on the timing and intensity of flowering in tomato (Solanum lycopersicum L.) and the molecular mechanisms underlying such effects. Results showed that strigolactone levels in the shoot, whether endogenous or exogenous, correlate inversely with the time of anthesis and directly with the number of flowers and the transcript levels of the florigen-encoding gene SINGLE FLOWER TRUSS (SFT) in the leaves. Transcript quantifications coupled with metabolite analyses demonstrated that strigolactones promote flowering in tomato by inducing the activation of the microRNA319-LANCEOLATE module in leaves. This, in turn, decreases gibberellin content and increases the transcription of SFT. Several other floral markers and morpho-anatomical features of developmental progression are induced in the apical meristems upon treatment with strigolactones, affecting floral transition and, more markedly, flower development. Thus, strigolactones promote meristem maturation and flower development via the induction of SFT both before and after floral transition, and their effects are blocked in plants expressing a miR319-resistant version of LANCEOLATE. Our study positions strigolactones in the context of the flowering regulation network in a model crop species.

Abstract PO5-27-06: Development of TTX-MC138, a First-In-Class miRNA-10b-Targeted Therapeutic Against Metastatic Cancers of Diverse Primary Disease Origins

Abstract Conventional therapies targeted towards the primary tumor cell oftentimes do not affect the metastatic cell and, in fact, may promote metastasis. For these reasons, our research has focused on developing therapies specific to unique properties of metastatic tumor cells. In our earlier work, we identified miRNA-10b as a master regulator of the viability of metastatic tumor cells. We determined that miRNA-10b not only promotes the capacity of tumor cells to migrate and invade surrounding tissue, but, importantly, serves as a powerful master regulator of the viability of cancer cells. This knowledge allowed us to develop a therapeutic miR-10b inhibitor, named TTX-MC138, which is effectively delivered to metastatic tumor cells and can elicit complete responses and life-long disease remissions in preclinical models of breast and pancreatic adenocarcinoma. As a critical step towards de-risking further clinical development of TTX-MC138, we have embarked on a Phase 0 clinical trial with Cu64-labeled TTX-MC138. The trial involves microdose injection of Cu64-labeled TTX-MC138 into stage IV breast cancer patients, followed by positron emission tomography-magnetic resonance imaging (PET-MRI). The study seeks to determine the pharmacokinetics of TTX-MC138 and its uptake in metastatic lesions. The impact of this work is three-fold. First it can establish that TTX-MC138, which is so effective in mice, will also accumulate in human metastases. This greatly de-risks the clinical development of the therapeutic because it shows drug delivery is indeed feasible. Second, the Phase 0 studies will reveal the pharmacokinetic behavior of TTX-MC138 which will allow one to establish dosing during therapy. Third, once TTX-MC138 reaches late-stage clinical trials, one can use the radiolabeled therapeutic to select patients for treatment, based on which patients’ metastases accumulate the drug. Studies that we present in support of the Phase 0 trial include dosimetry/PK and tissue distribution studies, as well as metabolite analysis in non-human primates. In non-human primates injected with Cu64-labeled TTX-MC138 at a dose of 100 microg, the organs with the highest uptake were the liver, heart, lung and spleen. The mean whole blood half-life was 12.2 ± 2.3 h (mean ± standard deviation) and the mean plasma blood half-life was 11.5 ± 1.8 h. RadioHPLC analysis of plasma samples showed that the drug is very stable with respect to metabolism. Combined, these studies support first-in-human testing of TTX-MC138 for the treatment of metastatic cancer and, by addressing the issue of drug delivery, enable the clinical development of a wide array of TTX-based therapeutics. Citation Format: Zdravka Medarova, Neil Robertson, Subrata Ghosh, Andreas Varkaris, Peter Caravan, Susan Duggan. Development of TTX-MC138, a First-In-Class miRNA-10b-Targeted Therapeutic Against Metastatic Cancers of Diverse Primary Disease Origins [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-06.

Abstract PO3-06-08: Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways

Abstract Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways Haleigh Parker1, Kayla Haberman1, Alexander Kornienko2, Antonio Evidente3, Daniel Romo4, Benny Abraham Kaipparettu5,6, Joseph Taube1,6 1 Department of Biology, Baylor University, Waco, TX, USA 2 Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, USA 3 Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Naples, Italy 4 Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA 5 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 6 Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA Background Metastatic progression in patients with triple negative breast cancer (TNBC) occurs in approximately half of all patients, reducing median overall survival. Metastasis may be facilitated through the epithelial to mesenchymal transition (EMT), which generates cancer cells with enhanced metastatic capacity and resistance to chemotherapeutics, features which are partially mediated by alterations in metabolic pathways and mitochondrial function. Here, we show a drug-like small molecule possesses EMT-specific cytotoxic activity via EMT-imparted variations in metabolic and mitochondrial functions. The fungus-derived sesterterpenoid, Ophiobolin A (OpA), possesses nanomolar cytotoxic activity and a high therapeutic index, though its target and mechanism of action remain unknown. Objectives The objectives of this study are to characterize alterations in metabolic pathways and mitochondrial function in the context of EMT, determine OpA induced alterations in specific metabolic pathways involved in metastasis and identify metabolic targets for the treatment of chemotherapeutic-resistant, metastatic triple negative breast cancer. Methods This study identifies distinct, novel metabolic alterations associated with EMT status and specific changes in mitochondrial and metabolic function associated with OpA treatment via analysis of intermediate metabolites, rates of oxidative phosphorylation and glycolysis, metabolic pathway capacity and dependency, protein expression, iron localization, reactive oxygen species accumulation and transmission electron microscopy. Results Our analysis indicates that OpA acts in a mitochondria-specific manner to cause a loss of membrane potential in EMT-positive, but not EMT-negative, cells with specific effects on complex III of the electron transport chain and oxidative phosphorylation as a whole. Furthermore, distinct shifts in glycolysis not only support previous data, but also provides insight as to mitochondrial functionality. Cells were also found to have distinct metabolic pathway capacities/dependencies and ROS and iron accumulation, depending on EMT status and OpA treatment. Finally, specific morphological alterations were uncovered via TEM image analysis. Therefore, we conclude that EMT imparts alterations in mitochondrial function and metabolic pathways, conferring sensitivity to the cytotoxic effects of OpA. Citation Format: Haleigh Parker, Kayla Haberman, Joseph Taube, Alexander Kornienko, Antonio Evidente, Daniel Romo, Benny Kaipparettu. Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-08.

Abstract PO1-26-04: Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients

Abstract Patient-derived organotypic tissue cultures (PD-OTC) are unique models for probing how individual patient’s tumor microenvironment (TME) influences cancer development and treatment responses. They retain the patient tumor architecture and TME, that are difficult to recapitulate in other models, while affording flexible treatment options and comparison of cancer (CA) versus matched non-cancer (NC) tissue responses to treatments. We have developed a culturing method for establishing PD-OTC of breast cancer patients that exhibit CA tissue growth and extensive metabolic reprogramming between CA versus matched NC tissues while enabling patient-derived organoids (PDO) and fibroblasts to be isolated. Pairs of CA and NC tissues freshly resected from six patients bearing early-stage estrogen receptor/progesterone receptor positive ductal carcinoma were sliced at 750 µm, embedded in Matrigel, and cultured in a 6-well plate with a Biopore membrane insert and custom medium at 37°C/5% CO2 with gentle rocking (16-40 mg wet weight; n=3). Excess tissue slices were cryopreserved and kept at -196 °C. Culture media were sampled periodically for metabolite analysis by NMR. Microscopic examination revealed significant tissue, organoid-like, and fibroblast outgrowth after 1-1.5 month of culturing. Two to three days prior to the harvest of two patients’ (CZ16 and 17) OTC, the culture medium was replaced with [U-2H]-glucose + [U-13C,15N]-glutamine-containing medium to allow the two stable isotope tracers to be metabolized, followed by Stable Isotope-Resolved Metabolomic (SIRM) analysis by NMR and ion chromatography coupled with ultra high-resolution Fourier transform mass spectrometry (IC-UHRFTMS). Two separate pieces of the OTC were cut and each was assayed for glucose uptake and mitochondrial membrane potential by live fluorescence spectroscopy via a portable custom-built fluorescence microscope, and by histochemical analysis. Histological analysis showed that both CA and NC OTC of all patients maintained their structural integrity. SIRM analysis of two patients’ OTC revealed active catabolic and anabolic metabolism including glucose uptake/glycolysis, the Krebs cycle/mitochondrial membrane potential, the pentose phosphate pathway (PPP), gluconeogenesis, glycogen synthesis, and purine/pyrimidine/sugar nucleotides synthesis. The CA OTC of both patients showed enhanced glucose uptake, glycolysis, mitochondrial membrane potential/Krebs cycle, PPP, glycogen synthesis, and purine nucleotide synthesis, when compared with the matched NC OTC. Treatment-naïve CA OTC of CZ16 showed much more significant metabolic reprogramming (NAD+ metabolism, in particular) than CA OTC of CZ17 who has undergone neoadjuvant treatment, chemotherapy, and hormone therapy prior to surgery. Distinct metabolic features of breast CA tissues can be exploited as therapeutic targets. Organoids and fibroblasts were isolated from CA and/or NC OTC cultures and their characterization is in progress. When revived, cryopreserved OTC exhibited active metabolism and tissue/organoid/fibroblast outgrowth similarly to fresh OTC. In conclusion, our culturing method enables PD-OTC models to be established efficiently for early-stage breast cancer patients for downstream functional studies including live cell imaging and SIRM analysis. Organoids and fibroblasts can also be derived from < 20 mg of tissues for further studies. Future direction includes therapeutic studies on these patient-derived models to better understand and predict individual patient’s responses to therapy. Citation Format: Teresa Fan, Carlos Goncalves, Jing Yan, Jahid Islam, Penghui Lin, Mohamed Kaddah, Richard Higashi, Andrew Lane, Caigang Zhu. Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-04.

Abstract PO1-03-10: Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer

Abstract Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer Background: It has previously been shown that microbial metabolic pathways are differentially activated in immunotherapy resistance, hence can serve as potential targets for therapeutic intervention. With the advent of Immune Checkpoint Blockers in the neoadjuvant setting of locally advanced (LA) TNBC, we hypothesized that similar microbial metabolite mediated resistance may be at play. We are presenting the real world data for neoadjuvant treatment in early TNBC along with a subset of tumor metabolite analysis. Method: 35 patients who received neoadjuvant pembrolizumab and chemotherapy for LA TNBC were identified at Moffitt Cancer Center between July 2019 to January 2023. These included 22 Caucasian, 5 African American, 2 Hispanic and 6 patients with undisclosed race. The age range was 33-78 years. The weight and Body Mass Index range was 55.3 kg-127.3 kg and 20.96-45.43 kg/m2 respectively. Nine patients had stage III disease while 24 had stage II breast cancer. The response was assessed in 33 patients; 20 patients (60.6%) achieved pathological complete response (pCR) and 13 (39.4%) had residual disease (RD). 1 patient developed severe immune mediated toxicity with colitis, hepatitis, myasthenia gravis leading to death while 1 patient had disease progression leading to drug discontinuation. Six patients developed immune mediated adverse events. We conducted untargeted metabolomic profiling on 24 patients to identify differentially abundant metabolites associated with pCR (ypT0/is ypN0) vs RD; 16 vs 8 patients respectively. Formalin fixed paraffin embedded (FFPE) slides from tumor tissue vs tumor bed were retrieved and histopathological evaluation of tumor cells was performed. 20 μm sections of FFPE block were used for untargeted metabolomics via liquid chromatography/mass spectrometry. Among the 200 metabolites detected, 63 were differentially abundant between pCR and tumor samples with RD. After correcting for false discovery rate, we observed striking differential activation of two microbial metabolic pathways. Samples with RD had significantly elevated kynurenine (OR-3.64 X1034, P-1.94 X 10-74 and kynurenine/Tryptophan ratio). The tryptophan, kynurenine pathway has been known to be overexpressed in patients resistant to immunotherapy and modulated by the microbiome. Similarly, the purine metabolic pathway, specifically microbiota-derived immunostimulatory metabolites- inosine, hypoxanthine, uric acid were significantly depleted in patients with RD (Inosine OR-0.54, p-0.007, Hypoxanthine OR-0.5, p-0.00039, uric acid OR: 0.41, P-0.003). Previously Inosine has been shown to increase the anti-tumor effects of immune checkpoint blockade by enhancing CD8+ effector T-cell function. Hence depletion of inosine, hypoxanthine and uric acid signals lack of an effector CD8 T cell response. Conclusion: Despite being a small study, it corroborates pCR rates noted in KEYNOTE 522 in the real world setting but shows significant concern for serious immune mediated adverse effects. This highlights the importance of stratifying patients who might be resistant to ICB or develop significant toxicities, thus deriving little to no benefit from standard neoadjuvant treatment. This underscores the need to develop precision-based treatment algorithms. The study represents one of the first investigations of differential abundance of microbial metabolites with immunotherapy resistance in LA TNBC. Our results highlight two microbially mediated mechanisms that could be targeted to improve pCR rates in LA TNBC. We are working to identify microbial metabolic pathways associated with immune mediated toxicity. Citation Format: Humaira Sarfraz, Brian Czerniecki, Zena Jameel, Michael Jaglal, Mostafa Eysha, Turkan Ozkul, Jameel Muzaffar, Aditi Saha, Sara Sarfraz, Fatima Zahra, Min Liu, Khushali Jhaveri, Melissa Sur, Adrianna Bagos-Estevez, Shahla Bari. Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-03-10.

UDP-glycosyltransferase UGT96C10 functions as a novel detoxification factor for conjugating the activated dinitrotoluene sulfonate in switchgrass.

Dinitrotoluene sulfonates (DNTSes) are highly toxic hazards regulated by the Resource Conservation and Recovery Act (RCRA) in the United States. The trinitrotoluene (TNT) red water formed during the TNT purification process consists mainly of DNTSes. Certain plants, including switchgrass, reed and alfalfa, can detoxify low concentrations of DNTS in TNT red water-contaminated soils. However, the precise mechanism by which these plants detoxify DNTS remains unknown. In order to aid in the development of phytoremediation resources with high DNTS removal rates, we identified and characterized 1-hydroxymethyl-2,4-dinitrobenzene sulfonic acid (HMDNBS) and its glycosylated product HMDNBS O-glucoside as the degradation products of 2,4-DNT-3-SO3Na, the major isoform of DNTS in TNT red water-contaminated soils, in switchgrass via LC-MS/MS- and NMR-based metabolite analyses. Transcriptomic analysis revealed that 15 UDP-glycosyltransferase genes were dramatically upregulated in switchgrass plants following 2,4-DNT-3-SO3Na treatment. We expressed, purified and assayed the activity of recombinant UGT proteins in vitro and identified PvUGT96C10 as the enzyme responsible for the glycosylation of HMDNBS in switchgrass. Overexpression of PvUGT96C10 in switchgrass significantly alleviated 2,4-DNT-3-SO3Na-induced plant growth inhibition. Notably, PvUGT96C10-overexpressing transgenic switchgrass plants removed 83.1% of 2,4-DNT-3-SO3Na in liquid medium after 28 days, representing a 3.2-fold higher removal rate than that of control plants. This work clarifies the DNTS detoxification mechanism in plants for the first time, suggesting that PvUGT96C10 is crucial for DNTS degradation. Our results indicate that PvUGT96C10-overexpressing plants may hold great potential for the phytoremediation of TNT red water-contaminated soils.

Impact of Drying Method and Solvent Extraction on Ethiopian Verbascum sinaiticum (Qetetina) Leaves: Metabolite Profiling and Evaluation of Antioxidant Capacity

The aim of this study was to evaluate the effects of different drying methods on bioactive compounds and to analyze their composition in Verbascum sinaiticum (V. sinaiticum) leaf extracts using UHPLC-ESI-QTOF-MS/MS. V. sinaiticum is traditionally used as an herbal medicine, yet it has undergone limited scientific investigations regarding its secondary metabolites. V. sinaiticum leaves were dried using oven dryers at 50 °C, 60 °C, and 70 °C, as well as a freeze dryer. The leaves were then extracted using 50% and 70% aqueous ethanol and 100% aqueous solutions. The results showed that the highest contents of TPC and TFC were observed when 70% aqueous ethanol was used during freeze drying, reaching 181.73 mg GAE/g dw and 78.57 mg CE/g dw, respectively. The strongest correlations were observed between the TFC and DPPH radical scavenging activity (0.9082), followed by TPC and ABTS assays (0.8933) and TPC and DPPH (0.8272). In the FTIR analysis, freeze drying exhibited a lower intensity of the phenolic -OH functional groups, contrasting with significant denaturation observed during oven drying at 70 °C. Metabolite analysis identified 29 compounds in V. sinaiticum leaves, further confirming the presence of 14 phenolic and flavonoid compounds, including kaempferol, catechin, gallic acid, and myricetin derivatives, consistent with the experimentally observed antioxidant capacity. This study highlights the impact of drying methods on the bioactive composition of V. sinaiticum and underscores its potential as a source of antioxidants for food, nutraceutical, and pharmaceutical applications.

Connection of pre-competition anxiety with gut microbiota and metabolites in wrestlers with varying sports performances based on brain-gut axis theory

ObjectiveThe purpose of this study is to investigate the connection of pre-competition anxiety with gut microbiota and metabolites in wrestlers with different sports performances.MethodsOne week prior to a national competition, 12 wrestlers completed anxiety questionnaires. Faecal and urine samples were collected for the analysis of gut microbiota and metabolites through the high-throughput sequencing of the 16 S rRNA gene in conjunction with untargeted metabolomics technology. The subjects were divided into two groups, namely, achievement (CP) and no-achievement (CnP) wrestlers, on the basis of whether or not their performances placed them in the top 16 at the competition. The relationship amongst the variations in gut microbiota, metabolites, and anxiety indicators was analyzed.Results(1) The CP group exhibited significantly higher levels of “state self-confidence,” “self-confidence,” and “somatic state anxiety” than the CnP group. Conversely, the CP group displayed lower levels of “individual failure anxiety” and “sports competition anxiety” than the CnP group. (2) The gut microbiota in the CP group was more diverse and abundant than that in the CnP group. Pre-competition anxiety was linked to Oscillospiraceae UCG_005, Paraprevotella, Ruminococcaceae and TM7x. (3) The functions of differential metabolites in faeces and urine of the CP/CnP group were mainly enriched in caffeine metabolism, lipopolysaccharide biosynthesis and VEGF and mTOR signaling pathways. Common differential metabolites in feces and urine were significantly associated with multiple anxiety indicators.ConclusionsWrestlers with different sports performance have different pre-competition anxiety states, gut microbiota distribution and abundance and differential metabolites in faeces and urine. A certain correlation exists between these psychological and physiological indicators.

Exploring low-dose gamma radiation effects on monoterpene biosynthesis in Thymus vulgaris: insights into plant defense mechanisms.

Thymus vulgaris, commonly known as thyme, is a plant renowned for producing monoterpenes. This study aimed to understand the effects of low-dose gamma radiation, specifically in the range of 1-5Gy, on various traits of Thymus vulgaris, providing context on its importance in agricultural and medicinal applications. The research explored morpho-physiological, biochemical, and gene-expression responses in thyme plants under no gamma- and gamma-ray exposure conditions. The study revealed complex relationships between gamma-ray doses and plant characteristics. In particular, shoot and root lengths initially increased with low doses (1-3Gy) but decreased at higher doses (5Gy), suggesting a dose-dependent threshold effect. Similarly, shoot and root fresh weights displayed an initial increase followed by a decline with increasing doses. Biochemical parameters showed dose-dependent responses, with low to moderate doses (1-3Gy) stimulating enzyme activities and high doses (5Gy) inhibiting them. Gene expression analysis was focused on the following specific genes: thymol synthase, γ-terpinene synthase, and carvacrol synthase. Low to moderate doses increased the expression of these genes, resulting in increased production of bioactive compounds. However, higher doses had diminished effects or suppressed gene expression. Metabolite analysis demonstrated dose-dependent responses, with moderate doses enhancing secondary metabolite production, while higher doses provided limited benefits. These findings underscore the implications of using gamma radiation to enhance secondary metabolite production in plants and its potential applications in agriculture, medicine, and environmental science. The study emphasizes the potential of gamma radiation as an external stressor to influence plant responses and highlights the importance of understanding such effects in various fields.

Analysis of differential metabolites in serum metabolomics of patients with aortic dissection

BackgroundPathogenesis and diagnostic biomarkers of aortic dissection (AD) can be categorized through the analysis of differential metabolites in serum. Analysis of differential metabolites in serum provides new methods for exploring the early diagnosis and treatment of aortic dissection.ObjectivesThis study examined affected metabolic pathways to assess the diagnostic value of metabolomics biomarkers in clients with AD.MethodThe serum from 30 patients with AD and 30 healthy people was collected. The most diagnostic metabolite markers were determined using metabolomic analysis and related metabolic pathways were explored.ResultsIn total, 71 differential metabolites were identified. The altered metabolic pathways included reduced phospholipid catabolism and four different metabolites considered of most diagnostic value including N2-gamma-glutamylglutamine, PC(phocholines) (20:4(5Z,8Z,11Z,14Z)/15:0), propionyl carnitine, and taurine. These four predictive metabolic biomarkers accurately classified AD patient and healthy control (HC) samples with an area under the curve (AUC) of 0.9875. Based on the value of the four different metabolites, a formula was created to calculate the risk of aortic dissection. Risk score = (N2-gamma-glutamylglutamine × -0.684) + (PC (20:4(5Z,8Z,11Z,14Z)/15:0) × 0.427) + (propionyl carnitine × 0.523) + (taurine × -1.242). An additional metabolic pathways model related to aortic dissection was explored.ConclusionMetabolomics can assist in investigating the metabolic disorders associated with AD and facilitate a more in-depth search for potential metabolic biomarkers.

Pure-Shift-Based Proton Magnetic Resonance Spectroscopy for High-Resolution Studies of Biological Samples

Proton magnetic resonance spectroscopy (1H MRS) presents a powerful tool for revealing molecular-level metabolite information, complementary to the anatomical insight delivered by magnetic resonance imaging (MRI), thus playing a significant role in in vivo/in vitro biological studies. However, its further applications are generally confined by spectral congestion caused by numerous biological metabolites contained within the limited proton frequency range. Herein, we propose a pure-shift-based 1H localized MRS method as a proof of concept for high-resolution studies of biological samples. Benefitting from the spectral simplification from multiplets to singlet peaks, this method addresses the challenge of spectral congestion encountered in conventional MRS experiments and facilitates metabolite analysis from crowded NMR resonances. The performance of the proposed pure-shift 1H MRS method is demonstrated on different kinds of samples, including brain metabolite phantom and in vitro biological samples of intact pig brain tissue and grape tissue, using a 7.0 T animal MRI scanner. This proposed MRS method is readily implemented in common commercial NMR/MRI instruments because of its generally adopted pulse-sequence modules. Therefore, this study takes a meaningful step for MRS studies toward potential applications in metabolite analysis and disease diagnosis.

GC–IMS facilitates identification of carbapenem-resistant Klebsiella pneumoniae in simulated blood cultures

This study aimed to identify carbapenem-resistant Klebsiella pneumoniae (CRKP) based on changes in levels of its volatile organic compounds (VOCs) in simulated blood cultures (BCs) using the gas chromatography–ion mobility spectrometry (GC–IMS) technique. A comprehensive analysis of volatile metabolites produced by Klebsiella pneumoniae (K. pneumoniae) in BC bottles was conducted using GC–IMS. Subsequently, the released VOCs were analyzed to examine differences in VOC release between CRKP and carbapenem-susceptible Klebsiella pneumoniae (CSKP). A total of 54 VOCs were detected, of which 18 (6 VOCs found in both monomer and dimer forms) were successfully identified. The VOCs produced by K. pneumoniae in BC bottles (BacT/ALERT® SA) were primarily composed of organic acids, alcohols, esters, and ketones. The content of certain VOCs was significantly different between CRKP and CSKP after the addition of imipenem (IPM). Moreover, the inclusion of carbapenemase inhibitors facilitated the identification of carbapenemase-producing K. pneumoniae based on the variations in VOCs. This study demonstrates the utility of GC–IMS technology in identifying CRKP, and reveals that changes in VOCs are closely related to the growth and metabolism of K. pneumoniae, indicating that they can be leveraged to promote early identification of CRKP bacteremia. However, further in-depth studies and experiments are needed to validate our findings.

Metabolomic profiles predict clinical severity in patients with obstructive sleep apnea hypopnea syndrome.

Obstructive sleep apnea hypopnea syndrome (OSAHS) poses a significant health hazard, as intermittent hypoxia inflicts damage throughout the body and is considered a critical risk factor for metabolic disorders. The aim of this study was to establish a metabolic profile for patients with OSAHS using nontargeted metabolomics detection techniques, providing a basis for OSAHS diagnosis and novel biological marker identification. Forty-five patients with OSAHS composed the OSAHS group, and 44 healthy volunteers composed the control group. Nontargeted metabolomics technology was used to analyze participants' urinary metabolites. Differentially abundant metabolites were screened and correlated through hierarchical clustering analysis. We constructed a composite metabolite diagnostic model using a random forest model. Simultaneously, we analyzed the relationships between 20 metabolites involved in model construction and OSAHS severity. The urinary metabolomics pattern of the OSAHS group exhibited significant changes, demonstrating noticeable differences in metabolic products. Urinary metabolite analysis revealed differences between the mild-moderate OSAHS and severe OSAHS groups. The composite metabolite model constructed in this study demonstrated excellent diagnostic performance not only in distinguishing healthy control participants from patients with mild-moderate OSAHS (AUC = 0.78) and patients with severe OSAHS (AUC = 0.78), but also in discriminating between patients with mild-moderate and severe OSAHS (AUC = 0.71). This study comprehensively analyzed the urinary metabolomic characteristics of patients with OSAHS. The established composite metabolite model provides robust support for OSAHS diagnosis and severity assessment. Twenty metabolites associated with OSAHS disease severity offer a new perspective for diagnosis.

Exhaled Volatile Organic Compounds for Asthma Control Classification in Children with Moderate to Severe Asthma: Results from the SysPharmPediA Study.

Early identification of children with poorly controlled asthma is imperative for optimizing treatment strategies. The analysis of exhaled volatile organic compounds (VOCs) is an emerging approach to identify prognostic and diagnostic biomarkers in pediatric asthma. To assess the accuracy of gas chromatography-mass spectrometry based exhaled metabolite analysis to differentiate between controlled and uncontrolled pediatric asthma. This study encompassed a discovery (SysPharmPediA) and validation phase (U-BIOPRED, PANDA). Firstly, exhaled VOCs that discriminated asthma control levels were identified. Subsequently, outcomes were validated in two independent cohorts. Patients were classified as controlled or uncontrolled, based on asthma control test scores and number of severe attacks in the past year. Additionally, potential of VOCs in predicting two or more future severe asthma attacks in SysPharmPediA was evaluated. Complete data were available for 196 children (SysPharmPediA=100, U-BIOPRED=49, PANDA=47). In SysPharmPediA, after randomly splitting the population into training (n=51) and test sets (n=49), three compounds (acetophenone, ethylbenzene, and styrene) distinguished between uncontrolled and controlled asthmatics. The area under the receiver operating characteristic curve (AUROCC) for training and test sets were respectively: 0.83 (95% CI: 0.65-1.00) and 0.77 (95% CI: 0.58-0.96). Combinations of these VOCs resulted in AUROCCs of 0.74 ±0.06 (UBIOPRED) and 0.68 ±0.05 (PANDA). Attacks prediction tests, resulted in AUROCCs of 0.71 (95% CI 0.51-0.91) and 0.71 (95% CI 0.52-0.90) for training and test sets. Exhaled metabolites analysis might enable asthma control classification in children. This should stimulate further development of exhaled metabolites-based point-of-care tests in asthma.

Metabolomics-driven discovery of therapeutic targets for cancer cachexia.

Cancer cachexia (CC) is a devastating metabolic syndrome characterized by skeletal muscle wasting and body weight loss, posing a significant burden on the health and survival of cancer patients. Despite ongoing efforts, effective treatments for CC are still lacking. Metabolomics, an advanced omics technique, offers a comprehensive analysis of small-molecule metabolites involved in cellular metabolism. In CC research, metabolomics has emerged as a valuable tool for identifying diagnostic biomarkers, unravelling molecular mechanisms and discovering potential therapeutic targets. A comprehensive search strategy was implemented to retrieve relevant articles from primary databases, including Web of Science, Google Scholar, Scopus and PubMed, for CC and metabolomics. Recent advancements in metabolomics have deepened our understanding of CC by uncovering key metabolic signatures and elucidating underlying mechanisms. By targeting crucial metabolic pathways including glucose metabolism, amino acid metabolism, fatty acid metabolism, bile acid metabolism, ketone body metabolism, steroid metabolism and mitochondrial energy metabolism, it becomes possible to restore metabolic balance and alleviate CC symptoms. This review provides a comprehensive summary of metabolomics studies in CC, focusing on the discovery of potential therapeutic targets and the evaluation of modulating specific metabolic pathways for CC treatment. By harnessing the insights derived from metabolomics, novel interventions for CC can be developed, leading to improved patient outcomes and enhanced quality of life.

Widely Targeted Metabolomics and Network Pharmacology Reveal the Nutritional Potential of Yellowhorn (Xanthoceras sorbifolium Bunge) Leaves and Flowers.

Yellowhorn (Xanthoceras sorbifolium Bunge) is a unique oilseed tree in China with high edible and medicinal value. However, the application potential of yellowhorn has not been adequately explored. In this study, widely targeted metabolomics (HPLC-MS/MS and GC-MS) and network pharmacology were applied to investigate the nutritional potential of yellowhorn leaves and flowers. The widely targeted metabolomics results suggested that the yellowhorn leaf contains 948 non-volatile metabolites and 638 volatile metabolites, while the yellowhorn flower contains 976 and 636, respectively. A non-volatile metabolite analysis revealed that yellowhorn leaves and flowers contain a variety of functional components beneficial to the human body, such as terpenoids, flavonoids, alkaloids, lignans and coumarins, phenolic acids, amino acids, and nucleotides. An analysis of volatile metabolites indicated that the combined action of various volatile compounds, such as 2-furanmethanol, β-icon, and 2-methyl-3-furanthiol, provides the special flavor of yellowhorn leaves and flowers. A network pharmacology analysis showed that various components in the flowers and leaves of yellowhorn have a wide range of biological activities. This study deepens our understanding of the non-volatile and volatile metabolites in yellowhorn and provides a theoretical basis and data support for the whole resource application of yellowhorn.

Optimization of Ultrasound-Assisted Extraction of Verbascum sinaiticum Leaves: Maximal Phenolic Yield and Antioxidant Capacity.

Verbascum sinaiticum (Qetetina or yeahya Joro) is a medicinal plant with secondary metabolites such as phenolics, flavonoids, glycosides, saponins, and alkaloids. This study was designed to optimize the ultrasonic-assisted extraction (UAE) parameters to enhance the phenolic content and characterize the phenolic compounds using ultra-high-performance liquid chromatography, coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-QTOF-MS/MS), and antioxidant activities in Verbascum sinaiticum extract. Extraction time, sample-to-solvent ratio, and extraction temperature were considered for UAE optimization. It was found that UAE generated the highest extraction yield (21.6%), total phenolic content (179.8 GAE mg/g), total flavonoid content (64.49 CE mg/g), DPPH (61.85 µg/mL), and ABTS (38.89 µg/mL) when compared to maceration extraction. Metabolite analysis in this study showed the detection of 17 phenolic compounds, confirming antioxidant capacities. The optimization parameters have significant effects on phenolic compounds. Scanning electron microscopy showed the presence of structural changes when UAE was used over the maceration method. The optimized UAE parameters for extraction temperature (41.43 °C), sample-to-solvent ratio (36.32 g/mL), and extraction time (33.22 min) for TPC were obtained. This study shows the potential application for UAE of Verbascum sinaiticum leaves in the development of pharmaceutical and nutraceutical products.

Effects of geographical origin and post-harvesting processing on the bioactive compounds and sensory quality of Brazilian specialty coffee beans

Specialty coffee beans are those produced, processed, and characterized following the highest quality standards, toward delivering a superior final product. Environmental, climatic, genetic, and processing factors greatly influence the green beans’ chemical profile, which reflects on the quality and pricing. The present study focuses on the assessment of eight major health-beneficial bioactive compounds in green coffee beans aiming to underscore the influence of the geographical origin and post-harvesting processing on the quality of the final beverage. For that, we examined the non-volatile chemical profile of specialty Coffea arabica beans from Minas Gerais state, Brazil. It included samples from Cerrado (Savannah), and Matas de Minas and Sul de Minas (Atlantic Forest) regions, produced by two post-harvesting processing practices. Trigonelline, theobromine, theophylline, chlorogenic acid derivatives, caffeine, caffeic acid, ferulic acid, and p-coumaric acid were quantified in the green beans by high-performance liquid chromatography with diode array detection. Additionally, all samples were roasted and subjected to sensory analysis for coffee grading. Principal component analysis suggested that Cerrado samples tended to set apart from the other geographical locations. Those samples also exhibited higher levels of trigonelline as confirmed by two-way ANOVA analysis. Samples subjected to de-pulping processing showed improved chemical composition and sensory score. Those pulped coffees displayed 5.8% more chlorogenic acid derivatives, with an enhancement of 1.5% in the sensory score compared to unprocessed counterparts. Multivariate logistic regression analysis pointed out altitude, ferulic acid, p-coumaric acid, sweetness, and acidity as predictors distinguishing specialty coffee beans obtained by the two post-harvest processing. These findings demonstrate the influence of regional growth conditions and post-harvest treatments on the chemical and sensory quality of coffee. In summary, the present study underscores the value of integrating target metabolite analysis with statistical tools to augment the characterization of specialty coffee beans, offering novel insights for quality assessment with a focus on their bioactive compounds.

Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer.

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [11C]CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [11C]CS1P1 detected a radiometabolite peak that is more lipophilic than [11C]CS1P1. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [11C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [18F]TZ82121 by the oxidation of [18F]FS1P1. Radio-HPLC analysis confirmed that [18F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [18F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [18F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [11C]TZ82121, detected in human plasma post injection of [11C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [11C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.