Mass Spectrometry-based Metabolomics Research Articles

Exploration of urine metabolic biomarkers for new-onset, untreated pediatric epilepsy: A gas and liquid chromatography mass spectrometry-based metabolomics study

ObjectiveThe discovery of objective indicators for recent epileptic seizures will help confirm the diagnosis of epilepsy and evaluate therapeutic effects. Past studies had shortcomings such as the inclusion of patients under treatment and those with various etiologies that could confound the analysis results significantly. We aimed to minimize such confounding effects and to explore the small molecule biomarkers associated with the recent occurrence of epileptic seizures using urine metabolomics. MethodsThis is a multicenter prospective study. Subjects included pediatric patients aged 2 to 12 years old with new-onset, untreated epilepsy, who had had the last seizure within 1 month before urine collection. Controls included healthy children aged 2 to 12 years old. Those with underlying or chronic diseases, acute illnesses, or recent administration of medications or supplements were excluded. Targeted metabolome analysis of spot urine samples was conducted using gas chromatography (GC)- and liquid chromatography (LC)-tandem mass spectrometry (MS/MS). ResultsWe enrolled 17 patients and 21 controls. Among 172 metabolites measured by GC/MS/MS and 41 metabolites measured by LC/MS/MS, only taurine was consistently reduced in the epilepsy group. This finding was subsequently confirmed by the absolute quantification of amino acids. No other metabolites were consistently altered between the two groups. ConclusionsUrine metabolome analysis, which covers a larger number of metabolites than conventional biochemistry analyses, found no consistently altered small molecule metabolites except for reduced taurine in epilepsy patients compared to healthy controls. Further studies with larger samples, subjects with different ages, expanded target metabolites, and the investigation of plasma samples are required.

Citraconate promotes the malignant progression of colorectal cancer by inhibiting ferroptosis.

Metastasis is a principal factor in the poor prognosis of colorectal cancer. Recent studies have found microbial metabolites regulate colorectal cancer metastasis. By analyzing metabolomics data, we identified an essential fecal metabolite citraconate that potentially promotes colorectal cancer metastasis. Next, we tried to reveal its effect on colorectal cancer and the underlying mechanism. Firstly, the response of colorectal cancer cells (HCT116 and MC38 cells) to citraconate was assessed by Cell Counting Kit-8 assay, clonogenic assay, transwell migration and invasion assay. Moreover, we utilized an intra-splenic injection model to evaluate the effect of citraconate on colorectal cancer liver metastasis in vivo. Then molecular approaches were employed, including RNA sequencing, mass spectrometry-based metabolomics, western blot, quantitative real-time PCR, cell ferrous iron colorimetric assay and intracellular malondialdehyde measurement. In vitro, citraconate promotes the growth of colorectal cancer cells. In vivo, citraconate aggravated liver metastasis of colorectal cancer. Mechanistically, downstream genes of NRF2, NQO1, GCLC, and GCLM high expression induced by citraconate resulted in resistance to ferroptosis of colorectal cancer cells. In summary, citraconate promotes the malignant progression of colorectal cancer through NRF2-mediated ferroptosis resistance in colorectal cancer cells. Furthermore, our study indicates that fecal metabolite may be crucial in colorectal cancer development.

The Beneficial Effects of Dietary Astragali Radix Are Related to the Regulation of Gut Microbiota and Its Metabolites.

Astragali Radix (AR) or its extract has been used as an herbal medicine and dietary supplement in China, Europe, and the United States. The gut microbiota could provide new insights for exploring dietary supplements' underlying mechanism on organisms. However, no reports have focused on the regulatory effect of AR on the gut microbiota as a dietary supplement. In this study, healthy ICR mice of either sex were divided into AR and control (CON) groups and given AR water extract (4.55 mg/kg·day-1) or saline by gavage for 14 days, respectively. Then 16S rRNA gene sequencing and ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry-based fecal metabolomics were integrated to investigate the benefits of dietary AR. Weighted gene coexpression network analysis was also introduced to investigate the metabolites with highly synergistic changes. AR supplementation influenced the structure of intestinal microflora, especially enriching short-chain fatty acid-producing bacteria g_Coprobacillus, g_Prevotella, and g_Parabacteroides. AR also significantly altered the fecal metabolome, mainly related to amino acid metabolism, nucleotide metabolism, and bile acid (BA) metabolism. Moreover, the increased secondary BAs and BA-sulfates might closely relate to intestinal microflora. These findings provide valuable insights for future research of dietary AR as a functional food.

Branch Chain Amino Acid Metabolism Promotes Brain Metastasis of NSCLC through EMT Occurrence by Regulating ALKBH5 activity.

Metabolic reprogramming is one of the essential features of tumors that may dramatically contribute to cancer metastasis. Employing liquid chromatography-tandem mass spectrometry-based metabolomics, we analyzed the metabolic profile from 12 pairwise serum samples of NSCLC brain metastasis patients before and after CyberKnife Stereotactic Radiotherapy. We evaluated the histopathological architecture of 144 surgically resected NSCLC brain metastases. Differential metabolites were screened and conducted for functional clustering and annotation. Metabolomic profiling identified a pathway that was enriched in the metabolism of branched-chain amino acids (BCAAs). Pathologically, adenocarcinoma with a solid growth pattern has a higher propensity for brain metastasis. Patients with high BCAT1 protein levels in lung adenocarcinoma tissues were associated with a poor prognosis. We found that brain NSCLC cells had elevated catabolism of BCAAs, which led to a depletion of α-KG. This depletion, in turn, reduced the expression and activity of the m6A demethylase ALKBH5. Thus, ALKBH5 inhibition participated in maintaining the m6A methylation of mesenchymal genes and promoted the occurrence of epithelial-mesenchymal transition (EMT) in NSCLC cells and the proliferation of NSCLC cells in the brain. BCAA catabolism plays an essential role in the metastasis of NSCLC cells.

High-Resolution Mass Spectrometry-Based Metabolomics for Increased Grape Juice Metabolite Coverage.

The composition of the juice from grape berries is at the basis of the definition of technological ripeness before harvest, historically evaluated from global sugar and acid contents. If many studies have contributed to the identification of other primary and secondary metabolites in whole berries, deepening knowledge about the chemical composition of the sole flesh of grape berries (i.e., without considering skins and seeds) at harvest is of primary interest when studying the enological potential of widespread grape varieties producing high-added-value wines. Here, we used non-targeted DI-FT-ICR-MS and RP-UHPLC-Q-ToF-MS analyses to explore the extent of metabolite coverage of up to 290 grape juices from four Vitis vinifera grape varieties, namely Chardonnay, Pinot noir, Meunier, and Aligoté, sampled at harvest from 91 vineyards in Europe and Argentina, over three successive vintages. SPE pretreatment of samples led to the identification of more than 4500 detected C,H,O,N,S-containing elemental compositions, likely associated with tens of thousands of distinct metabolites. We further revealed that a major part of this chemical diversity appears to be common to the different juices, as exemplified by Pinot noir and Chardonnay samples. However, it was possible to build significant models for the discrimination of Chardonnay from Pinot noir grape juices, and of Chardonnay from Aligoté grape juices, regardless of the geographical origin or the vintage. Therefore, this metabolomic approach opens access to a remarkable holistic molecular description of the instantaneous composition of such a biological matrix, which is the result of complex interplays among environmental, biochemical, and vine growing practices.

Chemical and microbial diversity of a tropical intertidal ascidian holobiont

The tropical ascidian Eudistoma vannamei, endemic to the northeastern coast of Brazil, is considered a prolific source of secondary metabolites and hosts Actinomycetota that produce bioactive compounds. Herein, we used an omics approach to study the ascidian as a holobiont, including the microbial diversity through 16S rRNA gene sequencing and metabolite production using mass spectrometry-based metabolomics. Gene sequencing analysis revealed all samples of E. vannamei shared about 50% of the observed ASVs, and Pseudomonadota (50.7%), Planctomycetota (9.58%), Actinomycetota (10.34%), Bacteroidota (12.05%) were the most abundant bacterial phyla. Analysis of tandem mass spectrometry (MS/MS) data allowed annotation of compounds, including phospholipids, amino acids, and pyrimidine alkaloids, such as staurosporine, a member of a well-known chemical class recognized as a microbial metabolite. Isolated bacteria, mainly belonging to Streptomyces and Micromonospora genera, were cultivated and extracted with ethyl acetate. MS/MS analysis of bacterial extracts allowed annotation of compounds not detected in the ascidian tissue, including marineosin and dihydroergotamine, yielding about 30% overlapped ions between host and isolated bacteria. This study reveals E. vannamei as a rich source of microbial and chemical diversity and, furthermore, highlights the importance of omic tools for a comprehensive investigation of holobiont systems.

Metabolomic Insights on Potassium Excretion, Blood Pressure, and Glucose Homeostasis: The African-PREDICT Study

BackgroundLow-potassium intake is associated with a higher risk of type 2 diabetes and hypertension. Both conditions occur more frequently in Black populations, who also consume less potassium-rich foods. ObjectivesUsing metabolomics to identify dysregulated metabolic pathways associated with low-potassium excretion may procure more accurate entry points for nutritional prevention and intervention for type 2 diabetes and hypertension. MethodsA total of 440 White and 350 Black adults from the African-PREDICT study (aged 20–30 y) were included. Twenty-four-hour blood pressure (BP) was measured. Potassium, sodium, and fasting glucose concentrations were analyzed in 24-h urine and plasma samples. Liquid chromatography-tandem mass spectrometry-based metabolomics included the analyses of amino acids and acylcarnitines in spot urine samples. ResultsBlack participants had lower urinary potassium concentrations than Whites (36.6 compared with 51.1 mmol/d; P < 0.001). In White but not Black adults, urinary potassium correlated positively with 2-aminoadipic acid (2-AAA) (r = 0.176), C3-[propionyl]carnitine (r = 0.137), C4-[butyryl]carnitine (r = 0.169) and C5-[isovaleryl]carnitine (r = 0.167) in unadjusted and 2-AAA (r = 0.158) and C4-carnitine (r = 0.160) in adjusted analyses (all P < 0.05 and q < 0.05). Elevated C0-, C3-, and C5-carnitine in turn were positively associated with systolic BP (Black and White groups), diastolic BP (Black group), and glucose (White group) (all P < 0.05). ConclusionsRacial differences are an important consideration when investigating nutrient–metabolite relationships and the role thereof in cardiovascular disease. Only in White adults did urinary potassium associate with 2-AAA and short-chain acylcarnitines. These metabolites were positively related to BP and fasting plasma glucose concentrations. In White adults, the metabolomic profiles related to potassium excretion may contribute to BP regulation and glucose homeostasis.This trial was registered at clinicaltrials.gov as NCT03292094.

Metabolite profiling of Trichinella spiralis adult worms and muscle larvae identifies their excretory and secretory products.

Human trichinellosis is a parasitic infection caused by roundworms belonging to the genus Trichinella, especially Trichinella spiralis. Early and accurate clinical diagnoses of trichinellosis are required for efficacious prognosis and treatment. Current drug therapies are limited by antiparasitic resistance, poor absorption, and an inability to kill the encapsulating muscle-stage larvae. Therefore, reliable biomarkers and drug targets for novel diagnostic approaches and anthelmintic drugs are required. In this study, metabolite profiles of T. spiralis adult worms and muscle larvae were obtained using mass spectrometry-based metabolomics. In addition, metabolite-based biomarkers of T. spiralis excretory-secretory products and their related metabolic pathways were characterized. The metabolic profiling identified major, related metabolic pathways involving adenosine monophosphate (AMP)-dependent synthetase/ligase and glycolysis/gluconeogenesis in T. spiralis adult worms and muscle larvae, respectively. These pathways are potential drug targets for the treatment of the intestinal and muscular phases of infection. The metabolome of larva excretory-secretory products was characterized, with amino acid permease and carbohydrate kinase being identified as key metabolic pathways. Among six metabolites, decanoyl-l-carnitine and 2,3-dinor-6-keto prostaglandin F1α-d9 were identified as potential metabolite-based biomarkers that might be related to the host inflammatory processes. In summary, this study compared the relationships between the metabolic profiles of two T. spiralis growth stages. Importantly, the main metabolites and metabolic pathways identified may aid the development of novel clinical diagnostics and therapeutics for human trichinellosis and other related helminthic infections.

Abstract A021: Nutrient competition in the tumor microenvironment alters NK cell metabolism in Pancreatic Cancer

Abstract Tumor cells present constant nutritional demand of macronutrients and micronutrients for their rapid growth needs. Often this nutrient limitation hampers the function of anti-tumor immune cells, which need basal energy levels to execute cytotoxic effects against malignant cells. Hence, we investigated nutrient limitation-induced metabolic alterations in NK cells that regulate the anti-tumor activity of NK cells. Using 2D cultures and PDAC organoid models, we demonstrate that pancreatic tumor cells exhaust vitamin B6 (VB6) in the co-culture milieu, causing reduced killing activity of NK cells. Additionally, we demonstrate that PDAC patients have significantly reduced plasma VB6 as compared to the healthy counterparts. This data aligns with the epidemiological studies showing that VB6 intake reduces the risk of pancreatic cancer incidence. Moreover, we noted a significant reduction in circulating VB6 in the pancreatic tumor-bearing mice as compared to healthy counterparts. Utilizing mass spectrometry-based metabolomics we noted that limitation of the VB6 prevents glycogen breakdown in NK cells. This was validated by follow up electron microscopy-based studies that demonstrated glycogen depots in NK cells that were very prominent upon inhibiting glycogenolysis. Metabolic tracing studies demonstrated that NK cells require VB6 for intracellular glycogen breakdown. Correspondingly, knocking down glycogen phosphorylase, a key enzyme involved in glycogen breakdown abrogated anti-tumoral NK cell function. Notably, supplementation of co-cultures in 2D and organoid models and in vivo orthotopic mouse models, with VB6 restored NK cell function against pancreatic tumor cells. In parallel, we observed dependence of tumor cell metabolism on VB6 for sustaining growth. Accordingly, we observed that tumor cells actively deplete VB6 in tumor microenvironments, as observed by comparing VB6 levels in circulation and in tumor interstitial fluids. Finally, we observed that VB6 supplementation in combination with inhibitors targeting VB6-driven metabolic pathway dependencies in tumor cells effectively enhances NK cell frequency and diminishes tumor burden in vivo. These studies demonstrate a novel role of glycogen breakdown as a critical energy source for activated NK cells and demonstrate, for the first time a key role of glycogenolysis in NK cell cytotoxic activities. Our results expand the understanding of the critical role of micronutrients (vitamin B6) in regulating cancer progression and anti-tumor immunity and open new avenues for developing NK cell-based immunotherapies for PDAC patients. Citation Format: Kamiya Mehla. Nutrient competition in the tumor microenvironment alters NK cell metabolism in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A021.

Monitoring of cellulose-rich biowaste co-digestion with 3D fluorescence spectroscopy and mass spectrometry-based metabolomics

Anaerobic digestion (AD) is a promising waste management strategy that reduces landfilling while generating biogas. Anaerobic co-digestion involves mixing two or more substrates to enhance the nutrient balance required for microorganism growth and thus improve the degradation. Monitoring AD is crucial for comprehending the biological process, optimizing process stability, and achieving efficient biogas production. In this work, we have used three dimensional excitation emission fluorescence spectroscopy and mass spectrometry metabolomics, two complementary techniques, to monitor the anaerobic co-digestion (AcoD) of cellulose, ash wood or oak wood with food waste. The two approaches were compared together and to the biogas production records. Results of this experiment demonstrated the complementarity of both analytical techniques with the measurement of the biogas production since 3D fluorescence spectroscopy and MS metabolomics revealed the earlier molecular changes occurring in the bioreactors, mainly associated with the hydrolysis step, whereas the biogas production data reflected the biological activity in the last step of the digestion. Moreover, in all cases, the three data sets effectively delineated the differences among the substrates. While the two wood substrates were poorly degradable as they were richer in aromatic compounds, cellulose was highly degradable and was characterized by the production of several glycolipids. Then, the three tested AcoDs resulted in a similar 3D EEM fluorescence and metabolomics profiles, close to the one observed for the AD of food waste alone, indicating that the incorporation of the food waste drove the molecular degradation events in the AcoDs. Substrate-specific differences were appreciated from the biogas production data. The overall results of this research are expected to provide insight into the design of guidelines for monitoring AcoD.

Novel Humanized G6PD Deficient Mice Reveal Mechanisms of Increased Tolerance to Exercise As Gleaned By Multi-Omics in Models of Critical Speed

INTRODUCTION: Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is the most common enzymatic deficiency in humans, affecting ~500 million people or ~6% of mankind. G6PD is the rate limiting enzyme of the pentose phosphate pathway (PPP), the main antioxidant pathway in mature red blood cells (RBCs). Activation of the PPP generates NADPH, an essential reducing equivalent for antioxidant metabolism that protects against intra- and extra-vascular hemolysis in response to oxidant stress. Impaired G6PD activity makes RBCs susceptible to hemolysis induced by oxidant challenges. Exercise-induced oxidant stress is a well-established concept in the field of physiology. Individuals with G6PD deficiency are recommended to refrain from strenuous exercise, as they are deemed to be at increased risk for hemolytic events in response to exercise-induced oxidant stress. While biochemically sound, this rationale has never been tested mechanistically, owing to ethical and technical limitations. As such, knowledge is limited regarding molecular impacts of exercise activity in G6PD deficient individuals. METHODS: Here we generated novel humanized mouse models carrying either (i) human canonical G6PD (normal activity), or two of the most common G6PD variants (ii) African (V68M - &amp;lt; 10% residual enzymatic activity) or (iii) Mediterranean (S188F - &amp;lt;1% residual enzymatic activity). We leveraged these mouse models to assess critical speed (CS), a functional measurement of the speed-to-duration relationship. To expand the molecular resolution of genetically associated adaptations to exercise, CS tests were combined with measurements of hemodynamics - including cardiac output, imaging (e.g., scanning electron microscopy of RBCs before and after exercise) and multi-omics (mass spectrometry-based metabolomics, lipidomics and proteomics) characterization of plasma, RBCs, and multiple tissue matrices including liver, heart, kidney, lung, spleen, and muscles (gastrocnemius and soleus). RESULTS: First of all, we confirmed a significant decrease in G6PD activity in RBCs from mice carrying the African and Mediterranean variants ( Figure 1). Unexpectedly, G6PD deficient mice showed ~10% faster CS compared to mice carrying the canonical human G6PD (p&amp;lt;0.0001 and p&amp;lt;0.02, Figure 1). While scanning electron microscopy showed differences before and after exercise, with accumulation of reversibly modified morphologies across all groups after the run, no significant differences were observed across groups. No significant changes in hemolysis markers were observed in plasma samples after exercise, neither at the metabolic (e.g., heme catabolites) or proteomics level (e.g., hemoglobin chains, RBC-derived proteins). Within hemodynamics, the hG6PD Med- mice are showing a higher efficiency in their cardiac function through cardiac function (p&amp;lt;0.005 - Figure 1). Pathway analysis of metabolomics data generated from RBC and plasma fractions showed significant decreases in arginine metabolism, increased glycolysis, and elevated Krebs cycle intermediates, in addition to expected differences in PPP. Specifically, in hG6PD A- RBC acylcarnitines accumulate after running. In hG6PD Med-, there are clear left ventricular differences highlighted in energy metabolism ( Figure 1). Multiomics analyses identified five major tissue clusters between heart, muscle, liver, kidney, and spleen, with G6PD function having the most significant metabolic impact on liver and soleus. RBC proteomics results highlighted deceased ferroportin (key iron transporter) in both the deficient groups, with markedly significant changes in the hG6PD Med- (p&amp;lt;0.0001). CONCLUSION: Novel humanized G6PD deficient mice maintain faster CS compared to mice. Omics approaches afford the opportunity to identify metabolic correlates to measurements of performance in relevant animal models, such as the CS model, which offer expanded mechanistic understanding of molecular bases of CS and the associated impact of G6PD deficiency. Our results not only challenge the assumption that exercise-induced oxidant stress predisposes the ~500M individuals with G6PD deficiency worldwide to increased hemolytic propensity, but rather indicate a metabolic advantage of functional polymorphisms to G6PD with respect to exercise performance, an observation that could contribute explaining the prevalence of such mutation in human populations.

Multi-Omics Analysis of the Impact of Long Distance Trail Running on Red Blood Cells Highlights Acylcarnitine Mediated Lipid Damage Repair Mechanisms

Red blood cells (RBC) play critical roles in the maintenance of vascular tone and metabolite transfer in addition to their well characterized role in gas exchange. In this capacity, they play a central role in maintaining systemic activity during stressful physiological situations such as exercise. At the far end of the spectrum for endurance sports, the popularity of long-distance running has been increasing in recent years. However, literature on the impact of marathon and ultramarathon running on red blood cell physiology is limited. To characterize molecular responses in RBC to long-distance running, blood was sampled from 11 runners before and after the Martigny-Combes à Chamonix (MCC, distance: 40 km, elevation gain: 2300 m) or 12 runners before and after the Ultra - Trail du Mont Blanc (UTMB, distance: 171 km, elevation gain: 10,000 m) and separated into plasma and RBC components. Hematological parameters and RBC deformability were assessed before and after each race. 1 Here, the same samples were analyzed using mass spectrometry-based metabolomics, lipidomics, proteomics, and elemental analysis ( Figure 1A). While RBC after both races had decreased mean cell volume (MCV), RBC exhibited higher amounts of exposed phosphatidylserine (PS) on the outer membrane only after the MCC, which serves as a signal for eryptosis. However, hematocrit (Hct) remained unchanged after this race, but was significantly lower after the UTMB. 1 Although decreased Hct can result from hemodilution, the plasma levels of albumin, sodium, and potassium were not significantly altered in either race. In addition, RBC microparticles increased after the UTMB with concomitant decreases in the deformability of intact RBC at high shear stresses, effects of which were not observed after the MCC. Intracellular metabolic correlates with deformability were enriched with acylcarnitines (AC - Figure 1A), which inversely correlated with RBC deformability at 30 Pa. This compound class was also positively associated with the level of IL-6 in addition to kynurenine, an immunomodulatory metabolite which has also been linked to IL-6 level and RBC damage in COVID19. 2 Within RBC, that are largely devoid of mitochondria, AC play an intermediary role in membrane lipid repair ( Figure 1A). While AC and fatty acids were significantly higher after both races, the magnitude of increase was higher after the MCC ( Figure 1B). However, the ratios of lysophosphatidylcholines or lysophosphatidylethanolamines to their respective intact lipid counterparts were predominantly lower after the UTMB, indicating more active membrane lipid repair in these cells ( Figure 1B). In support, the coenzyme A precursor pantothenate was consumed after both races, though carnitine accumulated only after the UTMB possibly due to increased acyl-CoA utilization for lipid repair rather than interconversion into AC. Increased oxidative stress was also apparent in the proteome, which demonstrated that the proportion of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with irreversibly oxidized active site cysteine was significantly higher after the UTMB. As this protein plays a key regulatory role in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion, 3 increased damage to its active site may partially explain a shift in glucose catabolism away from glycolysis and towards the PPP to generate NADPH reducing equivalents to cope with accumulating oxidative stress. In consideration of the varying temporal exposures to physiological stress between the MCC and UTMB, these results provide an in vivo snapshot into the accumulation of damage, repair, and removal of RBC as they circulate throughout the body during exercise. 1. Robert M, Stauffer E, Nader E, et al. Impact of Trail Running Races on Blood Viscosity and Its Determinants: Effects of Distance. Int. J. Mol. Sci. 2020;21(22):8531. 2. Thomas T, Stefanoni D, Dzieciatkowska M, et al. Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients. J. Proteome Res. 2020;19(11):4455-4469. 3. Reisz JA, Wither MJ, Dzieciatkowska M, et al. Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells. Blood. 2016;128(12):e32-e42.

Results from a Phase (Ph) 1 Clinical Study of the All-Oral Regimen of CC-486 and Venetoclax for Relapsed and Refractory Acute Myeloid Leukemia

Background: Venetoclax (Ven) with azacitidine (Aza) is the standard of care for newly diagnosed AML patients who are unfit for intensive induction chemotherapy. It is well known that this treatment also has activity in the relapsed and refractory (R/R) setting. However, this regimen requires 7 consecutive daily subcutaneous or intravenous doses of Aza each month, which continues indefinitely. The relative inconvenience of this, and its negative impact on quality of life, may lead to its early discontinuation, which may decrease the efficacy or durability of this regimen. A more convenient all-oral regimen may decrease visits to an infusion center, improve quality of life, and potentially increase the efficacy of the regimen due to improved compliance. CC-486 is the oral formulation of Aza that is currently approved for post-induction chemotherapy maintenance in AML. Methods: This is a single center open label, Ph I study investigating CC-486 and Ven in R/R AML patients. In the dose escalation phase, subjects received CC-486 at one of two cohorts (200 mg PO days 1-14 and 300 mg PO days 1-14). Ven was given at the 400 mg/day PO regimen, for 28 days, after an initial intra-patient dose escalation per the standard of care. Using a 3+3 study design for these two cohorts, we aimed to determine the maximum tolerated dose (MTD) of CC-486 in combination with Ven. An expansion cohort is planned for 10 additional patients treated at the MTD. To assess whether CC-486 exerts similar effects in the leukemia stem cell population (LSCs) compared to conventional IV Aza, we retrospectively analyzed patient samples from cohort 1 with a patient sample treated with Ven/IV Aza outside of the study. We performed mass spectrometry-based metabolomics analyses in LSCs isolated from patient samples as well as single-cell RNA sequencing. Results: A total of 9 patients were accrued for this Ph I study (Figure 1A). The average age at the time of enrolment was 66, and median prior treatment regimens was 1 (range 1-3). Five patients had received Ven/Aza in prior treatments. There were no dose-limiting toxicity (DLT) events and the MTD was determined to be 300mg. The most common hematologic toxicities were Gr 3-4 neutropenia (n = 8, 89%), Gr 3-4 anemia (n = 7, 78%), and Gr 3-4 thrombocytopenia (n = 5, 56%). The most common non-hematologic toxicities were Gr 1-2 nausea (n = 5, 56%), fatigue (n = 2, 22%), and diarrhea (n = 2, 22%). Febrile neutropenia was also seen in two subjects (Gr 1-2 n = 1, 11%, Gr 3-4 n= 1, 11%). Best responses were complete remission (CR) (n = 2, 22%) partial remission (PR) (n = 1, 11%), and stable disease (SD) (n= 3, 33%). Three patients proceeded to an allogeneic stem cell transplant. Overall survival was 288 days, 95% CI: (39 days, 424 days). To determine whether CC-486/Ven exerts similar effects on the LSC population compared to conventional IV Aza/Ven, LSCs were isolated from the bone marrow of a responder and non-responder, and these were compared to LSCs from a patient who underwent treatment and responded to conventional IV Aza/Ven. Given subjects who achieved a CR in the study cleared their blasts by day 4 rendering no leukemia cells for analyses, samples used were from Subject 1 (stable disease with unchanged blasts), and Subject 2 (stable disease with significant blast reduction). Metabolomics assays performed in LSCs at day 4 and day 28 suggested mitochondrial dysfunction, decreased electron transport chain complex II activity and decreased glutathione levels in Subject 2 and conventional IV Aza/Ven but not on Subject 1. These findings are consistent with Pollyea et al. 2018 suggesting CC-486/Ven targets LSCs by perturbing complex II activity and OXPHOS in LSCs. Further, scRNA seq performed in Subject 1 and 2 showed gene expression patterns similar to those seen in patients who respond to Aza/Ven vs not, including a significant shift of BCL-2 to MCL-1 expression in the cell population harboring LSCs (Figure 1B). Conclusion: CC-486 and Ven is an all-oral regimen currently being investigated for the treatment of R/R AML. Our Ph I dose escalation demonstrated a favorable safety profile for this regimen with a MTD of 300mg CC-486. Responses were seen in both dose cohorts including 2 CRs and 1 PR, and patients have been successfully bridged to hematopoietic stem cell transplant. Analyses performed in patient samples from this study suggested a similar effect in LSCs in CC-486/Ven compared to conventional IV Aza/Ven.

Polyamines Are Required for AML LSC Function through Their Role in Regulating eIF5A Dependent Protein Synthesis

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapies to fully eradicate disease initiating leukemia stem cells (LSCs) (Shlush et al., 2017). Many studies have demonstrated that LSCs have unique metabolic properties compared to AML blasts and normal hematopoietic stem and progenitor cells (HSPCs) (Jones et al., 2021). In this study we sought to quantify metabolite levels in LSCs compared to HSPCs using an unbiased metabolites screen. To examine metabolite levels in enriched LSCs and HSPCs we used mass spectrometry-based metabolomics analysis on enriched LSCs from 18 AML patients and HSPCs from 5 normal bone marrow (BM) specimens. Pathway analysis showed that arginine metabolism and biosynthesis were the most significantly enriched pathways in LSCs compared to HSPCs. Clinical studies have shown limited efficacy of targeting arginine levels using arginine degrading enzymes; therefore, we sought to target pathways downstream of arginine. To identify the pathways that arginine metabolism supports in LSCs, we used stable isotope labeled tracing analysis which showed arginine was metabolized through the urea cycle into polyamines in LSCs. Interestingly, the polyamine spermidine was the most significantly enriched metabolite in LSCs compared to HSPCs in our metabolomics analysis. Moreover, expression of SAT1, the enzyme catalyzing the export of polyamines from the cells, was decreased in AML compared to normal BM. Based on these observations, we sought to determine if the polyamine pathway represents a metabolic vulnerability that can be exploited to target LSCs. Polyamines are cationic compounds which have been previously shown to be important for the growth of various cancers but their role in AML LSCs has yet to be determined. To investigate if polyamines are essential for LSC survival and function, we treated LSCs and HSPCs with N 1, N 11-diethylnorspermine (DENSpm), a polyamine analog causing polyamine depletion through increased SAT1 expression and subsequent cellular export. DENSpm treatment decreased the viability of LSCs in 10 out of 12 of patients tested but had no impact on HSPCs viability. Further, treatment with DENSpm resulted in decreased engraftment potential of 5 AML specimens but did not alter the engraftment of HSPCs from 3 normal BM specimens, suggesting that targeting polyamines represents a promising approach to kill LSCs while sparing HSPCs. We then investigated the mechanisms by which polyamines are essential in LSCs. Using RNA-sequencing analysis on enriched LSCs from 5 AML patients and HSPCs from 3 normal BM specimens we observed an enrichment in myeloid differentiation signatures in DENSpm-treated LSCs but not in DENSpm-treated HSPCs. In line with this, expression of myeloid markers CD11b and CD15 were increased in patient-derived xenograft models engrafted with DENSpm-treated LSCs. As we did not observe such dysregulation in mice engrafted with DENSpm-treated HSPCs, these data suggest polyamine depletion promotes differentiation of LSCs but not HSPCs. Our transcriptomic analysis also unveiled decreased expression of genes involved in protein synthesis in DENSpm-treated LSCs but not in normal HSPCs. Protein synthesis and its regulation are critical for AML survival (Chen et al., 2019; Messling et al., 2022) but the role of polyamines in protein synthesis in LSCs has not been explored. Polyamines stimulate translation notably by serving as precursors for the hypusination of eukaryotic translation initiation factor 5A (eIF5A) (Park et al., 2010). Accordingly, DENSpm led to decreased hypusination of eIF5A in AML cells. This was associated with reduced protein synthesis level in AML cell lines and LSCs but not in normal HSPCs. Strikingly, eIF5A hypusination, protein synthesis and viability were rescued upon co-treatment with spermidine. Moreover, knockdown of eIF5A using siRNA resulted in impaired colony forming potential of AML and altered engraftment in patient-derived xenograft models. Altogether, these results suggest that DENSpm alters AML cell and LSC viability and function at least in part through decreased eIF5A-mediated protein translation. Altogether, our data demonstrates that depletion of polyamines decreased LSC function while sparing normal HSPCs. Mechanistically, our data suggest that LSC targeting is accomplished through decreased eIF5A-mediated synthesis of proteins crucial for LSCs.

Liquid Chromatography-Tandem Mass Spectrometry-Based Metabolomics Analysis of Indigo Naturalis Treatment of Ulcerative Colitis in Mice.

Ulcerative colitis (UC), often known as UC, is an inflammatory disease of the intestines that has frequent and long-lasting flare-ups. It is unknown precisely how the traditional Chinese drug Indigo Naturalis (IN) heals inflammatory bowel disease, despite its long-standing use in China and Japan. Finding new metabolite biomarkers linked to UC could improve our understanding of the disease, speed up the diagnostic process, and provide insight into how certain drugs work to treat the condition. Our work is designed to use a metabolomic method to analyze potential alterations in endogenous substances and their impact on metabolic pathways in a mouse model of UC. To determine which biomarkers and metabolisms are more frequently connected with IN's effects on UC, liquid chromatography-tandem mass spectrometry analysis of the serum metabolomics of UC mice and normal mice was performed. The outcomes demonstrated that IN boosted the health of UC mice and reduced the severity of their metabolic dysfunction. In the UC model, it was also found that IN changed the way 17 biomarkers and 3 metabolisms functioned.

Polygoni Cuspidati Rhizoma et Radix extract attenuates high-fat diet-induced NAFLD in rats: Impact on untargeted serum metabolomics and liver lipidomics

BackgroundPolygoni Cuspidati Rhizoma et Radix (PCRR; the root and rhizome of Polygonum cuspidatum Sieb. et Zucc) has long been used to treat atherosclerosis, gastrointestinal disorders, and liver disease. However, the underlying mechanism of PCRR against liver steatosis remains unclear. PurposeThe aim of this study was to determine the potential therapeutic targets and molecular mechanisms of PCRR in improving liver steatosis. MethodsA high-fat diet (HFD)-induced simple steatosis model in rats was used to evaluate the therapeutic effect of polyphenol-rich water extract from PCRR. Its underlying mechanisms were revealed using untargeted mass spectrometry-based serum metabolomics, hepatic lipidomic profiling approaches, qPCR, and Western blotting assays. ResultsOur study showed that PCRR-treated rats displayed significant reduction of serum TC and LDL-C, hepatic TG, TC levels, and total liver lipid content under a HFD feeding condition. Untargeted metabolomics showed that PCRR significantly restored the overall levels of nine lipid classes in liver tissues and elevated the bile acid levels in the serum samples. We demonstrated that PCRR prevented HFD-induced hepatic steatosis by regulating the expressions of TG metabolism proteins, including p-AMPK, p-ACC, PPARα, and FAS, as well as the expressions of TC metabolism proteins, including p-AMPK, LDLR, and CYP7A1. Moreover, resveratrol and/or polydatin­-derived metabolites were solely detected in the serum samples of the PCRR-treated group, indicating these microbial transformed metabolites might partly contribute to the beneficial effects of PCRR. ConclusionPCRR can be employed as a therapeutic agent to improve non-alcoholic fatty liver disease via regulating lipid metabolism.

Altered Gut Microbiota and Short-chain Fatty Acids in Chinese Children with Constipated Autism Spectrum Disorder

Gastrointestinal symptoms are more prevalent in children with autism spectrum disorder (ASD) than in typically developing (TD) children. Constipation is a significant gastrointestinal comorbidity of ASD, but the associations among constipated autism spectrum disorder (C-ASD), microbiota and short-chain fatty acids (SCFAs) are still debated. We enrolled 80 children, divided into the C-ASD group (n = 40) and the TD group (n = 40). In this study, an integrated 16S rRNA gene sequencing and gas chromatography–mass spectrometry-based metabolomics approach was applied to explore the association of the gut microbiota and SCFAs in C-ASD children in China. The community diversity estimated by the Observe, Chao1, and ACE indices was significantly lower in the C-ASD group than in the TD group. We observed that Ruminococcaceae_UCG_002, Erysipelotrichaceae_UCG_003, Phascolarctobacterium, Megamonas, Ruminiclostridium_5, Parabacteroides, Prevotella_2, Fusobacterium, and Prevotella_9 were enriched in the C-ASD group, and Anaerostipes, Lactobacillus, Ruminococcus_gnavus_group, Lachnospiraceae_NK4A136_group, Ralstonia, Eubacterium_eligens_group, and Ruminococcus_1 were enriched in the TD group. The propionate levels, which were higher in the C-ASD group, were negatively correlated with the abundance of Lactobacillus taxa, but were positively correlated with the severity of ASD symptoms. The random forest model, based on the 16 representative discriminant genera, achieved a high accuracy (AUC = 0.924). In conclusion, we found that C-ASD is related to altered gut microbiota and SCFAs, especially decreased abundance of Lactobacillus and excessive propionate in faeces, which provide new clues to understand C-ASD and biomarkers for the diagnosis and potential strategies for treatment of the disorder. This study was registered in the Chinese Clinical Trial Registry (www.chictr.org.cn; trial registration number ChiCTR2100052106; date of registration: October 17, 2021).

Genetic Polymorphisms in the Ferrireductase STEAP3 Regulate a Ferroptosis-like Process of Lipid Peroxidation-Induced Hemolysis in Murine and Human Red Blood Cells

Red blood cell (RBC) storage in the blood bank induces a series of biochemical and morphological alterations, collectively denoted as the storage lesion(s). Membrane damage by lipid peroxidation is a hallmark of the storage lesion, a process thought to be triggered by oxidant stress. To identify the genetics underlying this phenomenon, we leveraged a novel murine system, the Jackson lab Diversity Outbred (JDO) mouse colony, which was obtained by extensively cross-breeding 8 founder mouse strains with extreme genetically heterogeneity ( Figure 1.A). A total of 350 fresh and stored (up to 7 days) murine RBC samples were tested by mass spectrometry-based metabolomics and lipidomics. At the end of storage, post-transfusion recovery (PTR), which measures intra- and extravascular hemolysis and is a gold standard for determining RBC storage quality, was determined by quantifying the percentage of stored RBCs that were still circulating 24h after their transfusion into Ubi-GFP+ recipient mice ( Figure 1.A). Genome wide-association studies identified a strong association between PTR and a polymorphic region on chromosome 1 ( Figure 1.A), which encodes STEAP3, a ferrireductase. This region was associated with altered metabolism of stored RBCs ( Figure 1.A), especially an elevation of lipid peroxidation products (hive plot in Figure 1.A). Mechanistically, these data support a role for STEAP3 in promoting Fenton and Haber-Weiss chemistry in iron-loaded RBCs. Specifically, ferrous iron participates in this chemistry, becoming oxidized to its ferric state with concomitant generation of hydroxyl or hydroperoxyl radicals; these in turn attack fatty acids (preferentially, poly- and highly-unsaturated fatty acids, such as octadecadienoic and eicosatetraenoic acid), thereby promoting lipid peroxidation (to hydroxyoctadecadienoic (HODE) and hydroxyeicosatetraenoic (HETE) acids, respectively). By reducing Fe 3+ to Fe 2+, STEAP3 can shift the balance of this reaction by increasing the availability of the reactant, thereby favoring the generation of product radicals; this process is analogous to ferroptosis, as described in other biological systems. To extend the relevance of these findings to humans, we performed metabolomics on end-of-storage (i.e., Day 42) packed RBCs from 13,091 human donors in the Recipient Epidemiology and Donor Evaluation Study (REDS RBC Omics; Figure 1.B), enrolled in four blood centers across the United States. We stratified donors based on oxylipin levels (e.g., acids - HETEs - Figure 1.B) and common STEAP3 polymorphisms (e.g., a representative non-synonymous coding single nucleotide polymorphism (SNP), rs17013371, was observed with ~10% prevalence in these donors), along with 37 other common SNPs observed in this study ( Figure 1.B). When correlating metabolic and hematological parameters to common STEAP3 non-synonymous coding SNPs, we found strong associations of such polymorphisms with RBC susceptibility to hemolysis (either osmotic or oxidative), lipid peroxidation, RBC numbers, and hemoglobin levels ( Figure 1.B). These associations held true for a subset of the original donors with extreme hemolytic propensity (n=643) who donated a second RBC unit that was again stored for 42 (i.e., the “recalled donor population”). In the initial (n=13,091) and recalled donor (n=643) populations, lipid peroxidation products, HETEs, and HODEs ranked as the top predictors of hemolysis and vesiculation. In summary, we identified a role for the STEAP3 ferrireductase in contributing to a ferroptosis-like phenotype of lipid peroxidation that is associated with increased susceptibility to hemolysis in vitro and in vivo in murine and human RBCs following storage under blood bank conditions. Figure 1 - A. Jackson Lab diversity outbred (JDO) mice were bred from 8 founder mouse strains with extreme genetic heterogeneity. A total of 350 fresh and stored murine red blood cells (RBCs) were tested for metabolomics, lipidomics, and post-transfusion recovery (PTR). A polymorphic region on chromosome 1, coding for the ferrireductase STEAP3 was associated with heterogeneous PTR and elevation in lipid peroxidation products. B. Metabolomics analyses of 13,091 donors from the REDS RBC Omics identified an association between the end of storage levels of oxylipins and common polymorphisms for STEAP3, both linked to an increased RBC susceptibility to hemolysis (either osmotic or oxidative.

Discovering Nature's shield: Metabolomic insights into green zinc oxide nanoparticles Safeguarding Brassica parachinensis L. from cadmium stress

Heavy metal cadmium (Cd) hinders plants' growth and productivity by causing different morphological and physiological changes. Nanoparticles (NPs) are promising for raising plant yield and reducing Cd toxicity. Nonetheless, the fundamental mechanism of nanoparticle-interfered Cd toxicity in Brassica parachineses L. remains unknown. A novel ZnO nanoparticle (ZnO-NPs) was synthesized using a microalgae strain (Chlorella pyrenoidosa) through a green process and characterized by different standard parameters through TEM, EDX, and XRD. This study examines the effect of different concentrations of ZnO-NPs (50 and 100 mgL−1) in B. parachineses L. under Cd stress through ultra-high-performance liquid chromatography/high-resolution mass spectrometry-based untargeted metabolomics profiling. In the presence of Cd toxicity, foliar spraying with ZnO-NPs raised Cu, Fe, Zn, and Mg levels in the roots and/or leaves, improved seedling development, as demonstrated by increased plant height, root length, and shoot and root fresh weight. Furthermore, the ZnO-NPs significantly enhanced the photosynthetic pigments and changed the antioxidant activities of the Cd-treated plants. Based on a metabolomics analysis, 481 untargeted metabolites were accumulated in leaves under normal and Cd-stressed conditions. These metabolites were highly enriched in producing organic acids, amino acids, glycosides, flavonoids, nucleic acids, and vitamin biosynthesis. Surprisingly, ZnO-NPs restored approximately 60% of Cd stress metabolites to normal leaf levels. Our findings suggest that green synthesized ZnO-NPs can balance ions’ absorption, modulate the antioxidant activities, and restore more metabolites associated with plant growth to their normal levels under Cd stress. It can be applied as a plant growth regulator to alleviate heavy metal toxicity and improve crop yield in heavy metal-contaminated regions.

Metabolic Reprogramming By PRDM16 Drives Cytarabine Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients with high PRDM16 expression frequently experience induction failure and have a poor prognosis. However, the underlying molecular mechanisms are poorly understood. Here, we show that AML cells with high PRDM16 expression acquire resistance to cytarabine (AraC) through the activation of oxidative phosphorylation (OxPHOS) via regulation of c-MYC. Initially, we established a murine model of AML with high Prdm16 expression. We transduced c-Kit + bone marrow cells from wild-type mice with a retrovirus vector encoding MLL-AF9 (MF9) and the oncogenic short form of Prdm16 (sPrdm16). Using MF9/sPrdm16 cells, we next investigated whether AML cells with high Prdm16 expression exhibited resistance to AraC and/or daunorubicin (DNR), chemotherapeutic agents commonly used for induction chemotherapy. In the cell viability assay, MF9/sPrdm16 cells showed significantly higher IC 50 against AraC, but not against DNR, compared to MF9/control (control) cells, suggesting that high sPrdm16 expression confers relative resistance to AraC on AML cells. Colony-forming assay also confirmed resistant to AraC in MF9/sPrdm16 cells compared to control. Moreover, MF9/sPrdm16 cells were also resistant to AraC in vivo as demonstrated by the shortened survival of mice receiving MF9/sPrdm16 cells. To elucidate the mechanism of AraC resistance, we first examined cell cycle status of MF9/sPrdm16 cells since AraC exerts cytotoxicity in cell cycle dependent manner. To our surprise, MF9/sPrdm16 cells were cycling more rapidly than control cells as demonstrated by decreased G 0 phase and increased S/G 2/M phase. Intriguingly, RNA-sequencing analysis revealed that MF9/sPrdm16 cells displayed a signature of high OxPHOS by gene set enrichment analysis (GSEA). Indeed, oxygen consumption rate, superoxide production from mitochondria, and mitochondrial membrane potential were significantly elevated in MF9/sPrdm16 cells compared to control cells. The inhibition of mitochondrial respiration with metformin, the electron transport chain complex I inhibitor, or tigecycline, the mitochondrial protein synthesis inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells by inducing an energetic shift towards a lower OxPHOS status. These results clearly indicated that sPrdm16 overexpression conferred MF9 cells with AraC resistance byincreasing OxPHOS in mitochondria. Next, we explored the mechanism how sPrdm16 regulates OxPHOS. In addition to elevated OxPHOS signature, RNA-sequencing data also showed strong enrichment of Myc target-signature in MF9/sPrdm16 cells. Western blot analysis showed that sPrdm16 overexpression induced up-regulation of c-Myc and its transcriptional target, Slc1a5, a glutamine transporter. We went on to analyze metabolic changes induced by sPrdm16 overexpression by capillary electrophoresis and mass spectrometry-based metabolome analysis. We found that sPrdm16 induced the accumulation of glutamine, glutamate, α-ketoglutaric acid, succinate, fumarate, and malate in MF9 cells, possibly by activating glutaminolysis via the regulation of c-Myc and Slc1a5 expression, leading to the activation of tricarboxylic acid (TCA) cycle and OxPHOS. We also confirmed the contribution of c-Myc to AraC resistance by overexpressing c-Myc in MF9 cells. As expected, MF9/c-Myc cells mimicked the phenotype of MF9/sPrdm16 cells such as resistance to AraC and high OxPHOS status. Furthermore, 10058-F4, a c-Myc inhibitor, abrogated AraC resistance in MF9/sPrdm16 cells. Next, we asked if the observation in MF9/sPrdm16 cells could be translated into human AML cells. Human AML cell lines, MOLM-13 and THP-1, with sPRDM16 overexpression also showed significantly higher IC 50 against AraC compared to Mock expressed cells. Moreover, RNA-seq data from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 clinical trial showed that AML blasts with high PRDM16 expression demonstrated a gene expression signature associated with “OxPHOS” and “Myc Targets”. In conclusion, our findings demonstrate that PRDM16 overexpression activates mitochondrial respiration through metabolic reprogramming via c-MYC, thereby conferres AraC resistance on AML cells. These results suggest that targeting mitochondrial respiration might be a novel treatment strategy to overcome chemoresistance in AML patients with high PRDM16 expression.