NMR Metabolomics Analysis Research Articles

Membrane alteration, anti-virulence properties and metabolomic perturbation of a chionodracine-derived antimicrobial peptide, KHS-Cnd, on two bacteria models

Antarctic fishes, living in an extreme environment and normally exposed to pathogens, are a promising source of antimicrobial peptides (AMPs). These are emerging as next-generation drugs due to their activity against multidrug resistant (MDR) bacteria. To infect hosts, beyond intrinsic/acquired resistance, MDR species also use virulence factors such as protease secretion. Hence, AMPs targeting virulence factors could represent a novel strategy to counteract the antimicrobial resistance (AMR).In this paper, we focused on a mutant peptide, named KHS-Cnd, that was obtained from the scaffold of the chionodracine (Cnd), a natural peptide identified in the icefish Chionodraco hamatus. We studied different effects caused by the peptide interaction with the cell membrane of two model bacteria, E. coli and B. cereus. First, we investigated its membranolytic activity revealing that the peptide action is more evident on E. coli, with a 69% uptake of the used dye at 3 μM, whereas for B. cereus we found only a 65% uptake at 6 μM. Successively, we determined the impact of this lysis on total protein concentration in the medium and an increase was estimated for both bacteria (84% after 1h for E. coli and 90% for B. cereus, respectively). Moreover, we evaluated the changes in the proteolytic activity of the supernatant, that is an important aspect of bacterial resistance, showing that there was a significant reduction for both bacteria, although at higher level in the case of E. coli. The membranolytic activity was evidenced also morphologically with TEM analysis and a different alteration was evidenced for the two bacteria.Moreover, NMR metabolomics analysis showed that peptide induces changes in E. coli and B. cereus extracellular metabolites especially at the higher tested concentrations: this metabolic variation could be used as a fingerprinting of the peptide action on bacteria physiology due to its interaction with cell wall.Finally, we determined the KHS-Cnd cytotoxicity on human primary cell lines to verify its selectivity toward bacterial cell membranes and we found low toxicity until a concentration of 5 μM. Considering that the peptide exerts both membranolytic and anti-virulence activity on E. coli at 1.5 μM, we confirmed the interesting potential of this AMP as a new drug to counteract AMR.

Taurine Alleviates Ferroptosis-Induced Metabolic Impairments in C2C12 Myoblasts by Stabilizing the Labile Iron Pool and Improving Redox Homeostasis.

Ferroptosis adversely affects the viability, differentiation, and metabolic integrity of C2C12 myoblasts, contributing to the decline in skeletal muscle health. The intricate mechanisms behind this process are not fully understood. In this study, we induced ferroptosis in myoblasts using targeted inducers and found a marked decrease in specific redox metabolites, particularly taurine. Taurine supplementation effectively reversed the deleterious effects of ferroptosis, significantly increased cellular glutathione levels, reduced MDA and ROS levels, and rejuvenated impaired myogenic differentiation. Furthermore, taurine downregulated HO-1 expression and decreased intracellular Fe2+ levels, thereby stabilizing the labile iron pool. Using NMR metabolomic analysis, we observed that taurine profoundly promoted glycerophospholipid metabolism, which is critical for cell membrane repair, and enhanced mitochondrial bioenergetics, thereby increasing the energy reserves essential for muscle satellite cell regeneration. These results suggest that taurine is a potent ferroptosis inhibitor that attenuates key drivers of this process, strengthens oxidative defenses, and improves redox homeostasis. This combined effect protects cells from ferroptosis-induced damage. This study highlights the potential of taurine as a valuable ferroptosis inhibitor that protects skeletal muscle from ferroptosis-induced damage and provides a basis for therapeutic strategies to rejuvenate and facilitate the regeneration of aging skeletal muscle.

Investigating the Effectiveness of a Carb-Free Oloproteic Diet in Fibromyalgia Treatment.

Fibromyalgia (FM), a chronic disease with a high incidence in women, poses a significant challenge for diagnosis and treatment, especially due to the absence of specific biomarkers and the multifaceted nature of its symptoms, which range from neuromuscular pain to mood disorders and intestinal dysbiosis. While diagnosis currently relies on rheumatological clinical evaluations and treatment options mainly focus on symptom management, FM seems to have possible links with systemic metabolic dysfunctions with a common inflammatory root. In this context, a new therapeutic avenue emerges: could a therapeutic nutritional approach be the missing piece of the puzzle? Indeed, diet therapies employed particularly for metabolic syndromes proved recently to be efficacious for correcting systemic dysmetabolism and a high number of chronic inflammation conditions. In particular, the very-low-calorie ketogenic diet (VLCKD) demonstrated therapeutic benefits in many disorders. In the present study, we aimed to investigate the specific effects of two dietary interventions, namely the oloproteic VLCKD and the low-glycemic insulinemic (LOGI) diet, on two groups of female FM patients (FM1 and FM2) over a 45-day period. Utilizing clinical and laboratory tests, as well as non-invasive NMR metabolomic analysis of serum, urine, and saliva samples, we sought to uncover how these dietary regimens impact the metabolic dysfunctions associated with FM.

Multivariate curve resolution-based data fusion approaches applied in 1H NMR metabolomic analysis of healthy cohorts

BackgroundMetabolomics plays a critical role in deciphering metabolic alterations within individuals, demanding the use of sophisticated analytical methodologies to navigate its intricate complexity. While many studies focus on single biofluid types, simultaneous analysis of multiple matrices enhances understanding of complex biological mechanisms. Consequently, the development of data fusion methods enabling multiblock analysis becomes essential for comprehensive insights into metabolic dynamics. ResultsThis study introduces a novel guideline for jointly analyzing diverse metabolomic datasets (serum, urine, metadata) with a focus on metabolic differences between groups within a healthy cohort. The guideline presents two fusion strategies, ‘Low-Level data fusion’ (LLDF) and ‘Mid-Level data fusion’ (MLDF), employing a sequential application of Multivariate Curve Resolution with Alternating Least Squares (MCR-ALS), linking the outcomes of successive analyses. MCR-ALS is a versatile method for analyzing mixed data, adaptable at various stages of data processing—encompassing resonance integration, data compression, and exploratory analysis. The LLDF and MLDF strategies were applied to 1H NMR spectral data extracted from urine and serum samples, coupled with biochemical metadata sourced from 145 healthy volunteers. SignificanceBoth methodologies effectively integrated and analysed multiblock datasets, unveiling the inherent data structure and variables associated with discernible factors among healthy cohorts. While both approaches successfully detected sex-related differences, the MLDF strategy uniquely revealed components linked to age. By applying this analysis, we aim to enhance the interpretation of intricate biological mechanisms and uncover variations that may not be easily discernible through individual data analysis.

Application of 1H NMR metabolomics analysis to sparkling wines aged with different types of sugar added to the liqueur de dosage

Over the past decade, 1H NMR metabolomic studies have become increasingly popular in wine research since they can provide a chemical fingerprint with information regarding authenticity, geographical origin, vintage, and wine composition. Comparatively few 1H NMR-based studies have evaluated sparkling wines. In the present study, quantitative 1H NMR metabolomics was applied to assess the composition of finished traditional method (bottle-fermented) sparkling wines aged for 18-months in cellar conditions (14 °C, 70% relative humidity) following liqueur d′expedition (dosage) additions with six sugar types (glucose, fructose, sucrose derived from sugar beets and sugar cane, maltose, and MCR Sucraisin®) and a zero-dosage control. No systematic trends were observed when comparing the metabolite profile across sugar types, nor between the zero-dosage control and treatments with sugar additions. Multivariate statistical approaches using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) did not effectively discriminate between dosage treatments nor wine age, while orthogonal projections to latent structures discriminant analysis (OPLS-DA) successfully discriminated between wines by age and was validated through permutation tests. This study presents the first application of 1H NMR metabolomics coupled with multivariate analysis to monitor the aging of sparkling wines.

Physiological and metabolomic responses of the ethylene insensitive squash mutant etr2b to drought

The squash gain-of-function mutant etr2b disrupts the ethylene-binding domain of ethylene receptor CpETR2B, conferring partial ethylene insensitivity, changes in flower and fruit development, and enhanced salt tolerance. In this paper, we found that etr2b also confers a growth advantage as well as a physiological and metabolomic response that make the mutant better adapted to drought. Mutant plants had a higher root and leaf biomass than WT under both well-watered and drought conditions, but the reduction in growth parameters in response to drought was similar in WT and etr2b. Water deficit reduced all gas-exchange parameters in both WT and etr2b, but under moderate drought the mutant increased photosynthesis rate in comparison with control conditions, and showed a higher leaf CO2 concentration, transpiration rate, and stomata conductance than WT. The response of etr2b to drought indicates that ethylene is a negative regulator of plant growth under both control and drought. Since etr2b increased ABA content in well-watered plant, but prevented the induction of ABA production in response to drought, it is likely that the etr2b response under drought is not mediated by ABA. A 1H NMR metabolomic analysis revealed that etr2b enhances the accumulation of osmolytes (soluble sugars and trigonelline), unsaturated and polyunsaturated fatty acids, and phenolic compounds under drought, concomitantly with a reduction of malic- and fumaric-acid. The role of CpETR2B and ethylene in the regulation of these drought-protective metabolites is discussed.

Assessing the Acute Toxicological Effects of Annona muricata Leaf Ethanol Extract on Rats: Biochemical, Histopathological, and Metabolomics Analyses.

Annona muricata is a common plant used in Africa and South America to manage various types of disease. However, there is insufficient toxicological information or published standard available regarding repeated dose animal toxicity data. As part of the safety assessment, we exposed Sprague Dawley rats to an acute oral toxicity of A. muricata. The intent of the current study was to use advanced proton nuclear magnetic resonance (1H NMR) in serum and urinary metabolomics evaluation techniques to provide the in vivo acute toxicological profile of A. muricata leaf ethanol extract in accordance with the Organization for Economic Co-operation and Development's (OECD) 423 guidelines. A single 2000 mg/kg dose of A. muricata leaf ethanol extract was administered to Sprague Dawley rats over an observational period of 14 days. The toxicity evaluation (physical and behavior observation, body weight, renal function test, liver function test and 1H NMR analysis) showed no abnormal toxicity. Histopathological analysis manifested mild changes, i.e., the treated kidney manifested mild hypercellularity of mesangial cells and mild red blood cell congestion. In addition, there was mild hemorrhage into tissue with scattered inflammatory cells and mild dilated central vein with fibrosis in the liver. However, the changes were very mild and not significant which correlate with other analyses conducted in this study (biochemical test and 1H NMR metabolomic analysis). On the other hand, urinary 1H NMR analysis collected on day 15 revealed high similarity on the metabolite variations for both untreated and treated groups. Importantly, the outcomes suggest that A. muricata leaf ethanol extract can be safely consumed at a dose of 2000 mg/kg and the LD50 must be more than 2000 mg/kg.

Study on the acute toxicity of sodium taurocholate via zebrafish mortality, behavioral response, and NMR-metabolomics analysis

Sodium taurocholate (NaT) is a hydrophobic bile salt that exhibits varying toxicity and antimicrobial activity. The accumulation of BSs during their entero-hepatic cycle causes cytotoxicity in the liver and intestine and could also alter the intestinal microbiome leading to various diseases. In this research, the acute toxicity of sodium taurocholate in different concentrations (3000 mg/L, 1500 mg/L, 750 mg/L, 375 mg/L, and 0 mg/L) was investigated on four months old zebrafish by immersion in water for 96 h. The results were determined based on the fish mortality, behavioral response, and NMR metabolomics analysis which revealed LC50 of 1760.32 mg/L and 1050.42 mg/L after 72 and 96 h treatment, respectively. However, the non-lethal NaT concentrations of 750 mg/L and 375 mg/L at 96 h exposure significantly (p ≤ 0.05) decreased the total distance traveled and the activity duration, also caused surface respiration on the zebrafish. Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) revealed that the metabolome of the fish treated with 750 mg/L was discriminated from that of the control by PC1. Major significantly downregulated metabolites by NaT-induction include valine, isoleucine, 2-hydroxyvalerate, glycine, glycerol, choline, glucose, pyruvate, anserine, threonine, carnitine and homoserine. On the contrary, taurine, creatine, lactate, acetate and 3-hydroxybutyrate were upregulated suggesting cellular consumption of lipids, glucose and amino acids for adenosine triphosphate (ATP) generation during immune and inflammatory response. whereby these metabolites were released in the process. In conclusion, the research revealed the toxic effect of NaT and its potential to trigger changes in zebrafish metabolism.

Quantitative plasma profiling by 1H NMR-based metabolomics: impact of sample treatment.

Introduction: There is evidence that sample treatment of blood-based biosamples may affect integral signals in nuclear magnetic resonance-based metabolomics. The presence of macromolecules in plasma/serum samples makes investigating low-molecular-weight metabolites challenging. It is particularly relevant in the targeted approach, in which absolute concentrations of selected metabolites are often quantified based on the area of integral signals. Since there are a few treatments of plasma/serum samples for quantitative analysis without a universally accepted method, this topic remains of interest for future research. Methods: In this work, targeted metabolomic profiling of 43 metabolites was performed on pooled plasma to compare four methodologies consisting of Carr-Purcell-Meiboom-Gill (CPMG) editing, ultrafiltration, protein precipitation with methanol, and glycerophospholipid solid-phase extraction (g-SPE) for phospholipid removal; prior to NMR metabolomics analysis. The effect of the sample treatments on the metabolite concentrations was evaluated using a permutation test of multiclass and pairwise Fisher scores. Results: Results showed that methanol precipitation and ultrafiltration had a higher number of metabolites with coefficient of variation (CV) values above 20%. G-SPE and CPMG editing demonstrated better precision for most of the metabolites analyzed. However, differential quantification performance between procedures were metabolite-dependent. For example, pairwise comparisons showed that methanol precipitation and CPMG editing were suitable for quantifying citrate, while g-SPE showed better results for 2-hydroxybutyrate and tryptophan. Discussion: There are alterations in the absolute concentration of various metabolites that are dependent on the procedure. Considering these alterations is essential before proceeding with the quantification of treatment-sensitive metabolites in biological samples for improving biomarker discovery and biological interpretations. The study demonstrated that g-SPE and CPMG editing are effective methods for removing proteins and phospholipids from plasma samples for quantitative NMR analysis of metabolites. However, careful consideration should be given to the specific metabolites of interest and their susceptibility to the sample treatment procedures. These findings contribute to the development of optimized sample preparation protocols for metabolomics studies using NMR spectroscopy.

A serum NMR metabolomic analysis of the Corynebacterium pseudotuberculosis infection in goats.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants, is highly prevalent worldwide. Economic losses have already been associated with the disease, and little is known about the host-pathogen relationship associated with the disease. The present study aimed to perform a metabolomic study of the C. pseudotuberculosis infection in goats. Serum samples were collected from a herd of 173 goats. The animals were classified as controls (not infected), asymptomatic (seropositives but without detectable CLA clinical signs), and symptomatic (seropositive animals presenting CLA lesions), according to microbiological isolation and immunodiagnosis. The serum samples were analyzed using nuclear magnetic resonance (1H-NMR), nuclear Overhauser effect spectroscopy (NOESY), and Carr-Purcell-Meiboom-Gill (CPMG) sequences. The NMR data were analyzed using chemometrics, and principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were performed to discover specific biomarkers responsible for discrimination between the groups. A high dissemination of the infection by C. pseudotuberculosis was observed, being 74.57% asymptomatic and 11.56% symptomatic. In the evaluation of 62 serum samples by NMR, the techniques were satisfactory in the discrimination of the groups, being also complementary and mutually confirming, demonstrating possible biomarkers for the infection by the bacterium. Twenty metabolites of interest were identified by NOESY and 29 by CPMG, such as tryptophan, polyunsaturated fatty acids, formic acid, NAD+, and 3-hydroxybutyrate, opening promising possibilities for the use of these results in new therapeutic, immunodiagnosis, and immunoprophylactic tools, as well as for studies of the immune response against C. pseudotuberculosis. KEY POINTS: • Sixty-two samples from healthy, CLA asymptomatic, and symptomatic goats were screened • Twenty metabolites of interest were identified by NOESY and 29 by CPMG • 1H-NMR NOESY and CPMG were complementary and mutually confirming.

Urinary 1H NMR Metabolomic Analysis of Prenatal Maternal Stress Due to a Natural Disaster Reveals Metabolic Risk Factors for Non-Communicable Diseases: The QF2011 Queensland Flood Study.

Prenatal stress alters fetal programming, potentially predisposing the ensuing offspring to long-term adverse health outcomes. To gain insight into environmental influences on fetal development, this QF2011 study evaluated the urinary metabolomes of 4-year-old children (n = 89) who were exposed to the 2011 Queensland flood in utero. Proton nuclear magnetic resonance spectroscopy was used to analyze urinary metabolic fingerprints based on maternal levels of objective hardship and subjective distress resulting from the natural disaster. In both males and females, differences were observed between high and low levels of maternal objective hardship and maternal subjective distress groups. Greater prenatal stress exposure was associated with alterations in metabolites associated with protein synthesis, energy metabolism, and carbohydrate metabolism. These alterations suggest profound changes in oxidative and antioxidative pathways that may indicate a higher risk for chronic non-communicable diseases such obesity, insulin resistance, and diabetes, as well as mental illnesses, including depression and schizophrenia. Thus, prenatal stress-associated metabolic biomarkers may provide early predictors of lifetime health trajectories, and potentially serve as prognostic markers for therapeutic strategies in mitigating adverse health outcomes.

Antibiofilm activity and NMR-based metabolomic characterization of cell-free supernatant of Limosilactobacillus reuteri DSM 17938.

The microbial biofilm has been defined as a "key virulence factor" for a multitude of microorganisms associated with chronic infections. Its multifactorial nature and variability, as well as an increase in antimicrobial resistance, suggest the need to identify new compounds as alternatives to the commonly used antimicrobials. The aim of this study was to assess the antibiofilm activity of cell-free supernatant (CFS) and its sub-fractions (SurE 10 K with a molecular weight <10 kDa and SurE with a molecular weight <30 kDa), produced by Limosilactobacillus reuteri DSM 17938, vs. biofilm-producing bacterial species. The minimum inhibitory biofilm concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) were determined via three different methods and an NMR metabolomic analysis of CFS and SurE 10K was performed to identify and quantify several compounds. Finally, the storage stability of these postbiotics was evaluated by a colorimetric assay by analyzing changes in the CIEL*a*b parameters. The CFS showed a promising antibiofilm activity against the biofilm developed by clinically relevant microorganisms. The NMR of CFS and SurE 10K identifies and quantifies several compounds, mainly organic acids and amino acids, with lactate being the most abundant metabolite in all the analyzed samples. The CFS and SurE 10 K were characterized by a similar qualitative profile, with the exception of formate and glycine detected only in the CFS. Finally, the CIEL*a*b parameters assess the better conditions to analyze and use these matrices for the correct preservation of bioactive compounds.

Abstract 1476: 1H NMR Metabolomic Multivariate Analysis of Rat Plasma Reveals the Correlation of HIV-1 Genotype with Age and Substance Use

Abstract 1476: 1H NMR Metabolomic Multivariate Analysis of Rat Plasma Reveals the Correlation of HIV-1 Genotype with Age and Substance Use

1H NMR metabolomics analysis of oil palm stem tissue infected by Ganoderma boninense based on field severity Indices

Basal stem rot disease (BSR) caused by G. boninense affects most oil palm plants in Southeast Asia. This disease can be fatal to palm oil production. BSR shows no signs on the tree in the early stages of infection. Therefore, it is essential to find an approach that can detect BSR disease in oil palm, especially at any level of disease severity in the field. This study aims to identify biomarkers of BSR disease in oil palm stem tissue based on various disease severity indices in the field using 1H NMR-based metabolomics analysis. The crude extract of oil palm stem tissue with four disease severity indices was analyzed by 1H NMR metabolomics. Approximately 90 metabolites from oil palm stem tissue were identified.Twenty of these were identified as metabolites that significantly differentiated the four disease severity indices. These metabolites include the organic acid group, the carbohydrate group, the organoheterocyclic compound group, and the benzoid group. In addition, different tentative biomarkers for different disease severity indices were also identified. These tentative biomarkers consist of groups of organic acids, carbohydrates, organoheterocyclic compounds, nitrogenous organic compounds, and benzene. There are five pathways in oil palm that are potentially affected by BSR disease.

Effect of Different Climatic Regions and Seasonal Variation on the Antibacterial and Antifungal Activity, and Chemical Profile of Helichrysum aureonitens Sch. Bip.

Native South Africans make use of Helichrysum aureonitens Sch. Bip. extracts for the treatment of a variety of infections and they are important in traditional medicinal preparations. This study investigated the effect of seasonal variation and geographical location on the antibacterial and antifungal activities of H. aureonitens. Material was collected in two different seasons: early spring, with high rainfall and high temperatures (October), and late autumn, with low rainfall and lower temperatures (May). Further analysis was carried out using 1H-NMR based metabolomics to analyse and compare the chemical profiles of the plants in both seasons and locations. Plant materials were collected from two sites for each season, at Wakefield farm (KwaZulu-Natal), representing a colder, wetter environment, and Telperion (Mpumalanga), representing a drier and warmer environment. Leaves of H. aureonitens were tested against bacteria (Proteus vulgaris (P. vulgaris) and Bacillus subtilis (B. subtilis)) as well as fungi (Aspergillus flavus (A. flavus), Aspergillus nomius (A. nomius), Cladosporium cladosporioides (C. cladosporioides), Fusarium oxysporum (F. oxysporum) and Penicillum halotolerans (P. halotolerans)). Extracts from the October harvest showed significant activities against the Gram-negative bacterium P. vulgaris compared to the May harvest, with an MIC value of 62.5 µg/mL. Similar activity was observed between the extracts from the wet season across the two geographically different locations. There was generally very good antifungal activity observed for all the species, with the exception of A. nomius, which had MIC values ranging from 0.39–1.56 µg/mL. Extracts of plant materials harvested in the wetter region had a significantly higher activity against A. flavus and F. oxysporum in both seasons than those from plants harvested in the drier region. Telperion-harvested plants exhibited better activity against F. oxysporum in the autumn. Hydrogen-1 NMR metabolomic analysis confirmed the significant effects of the seasons and the peculiar climates of different localities on the secondary metabolite profile of H. aureonitens.

Urine 1H-NMR Metabolomics to Discriminate Neurocysticercosis Patients from Healthy Controls: An Exploratory Study.

The diagnosis of neurocysticercosis (NCC) is principally based on neuroimaging (magnetic resonance imaging or computed tomography), instrumentation that is scarcely available in the rural regions where Taenia solium transmission, primarily occurs due to poor sanitation conditions. Immunological assays for antigen or antibody detection complement the neuroimaging approach. However, no field-applicable assays to diagnose viable NCC or to guide the referral of cases for neuroimaging or for appropriate management are available. We performed an exploratory study on urine and serum samples using 1H-nuclear magnetic resonance (NMR)-based metabolomics to discriminate NCC patients (n = 14) from healthy control subjects (n = 22). Metabolic profiles demonstrated a discrimination between the urines of NCC patients and noninfected control subjects with a moderate predictive accuracy (R2 = 0.999, Q2 = 0.434). NMR metabolomics analysis has been proven useful in depicting biomarkers linked to other infectious diseases, various types of cancer, and other disorders. Our results, albeit preliminary, open a door to the development of better methods for detecting NCC through the identification of biomarkers participating in disturbed metabolic pathways.

Abstract 1065: The potential chemo-modulatory effect of the marine-derived secondary metabolite terrein on the anticancer properties of gemcitabine in colorectal cancer cells

Abstract Terrein is a bioactive marine secondary metabolite isolated from the fungal strain of Penicillium species SF-7181 and Aspergillus terreus. Previous studies showed that terrein possesses cytotoxic properties attributed to interrupting various molecular pathways, such as nuclear factor kappa B (NF-kB). Gemcitabine (GCB) is an anticancer drug commonly used to treat colorectal cancer; however, it suffers from tumor cell resistance and subsequently treatment failure. In this study, the potential anticancer properties of terrein as well as its chemomodulatory effect on GCB are assessed against various colorectal cancer cell lines (SW620, HT29 and HCT116) under normoxic and hypoxic (pO2 ≤ 1%) conditions. The antiproliferative effects of terrein, GCB and their equitoxic combination were evaluated using sulforhodamine-B assay. In SW620, terrein had IC50’s of 66.89±8.5 µM and 73.4±1.3 µM as single treatment, and the IC50’s of GCB in combination treatment were 0.27± 0.01 µM and 0.24±0.04 µM, under hypoxic and normoxic conditions, respectively. In HT29, terrein had IC50’s of 85.5±4.4 µM and 49.6±11.4 µM, and the IC50’s of GCB in combination treatment were 3.2±0.08 µM and 0.27±0.005 µM, under hypoxic and normoxic conditions, respectively. In HCT116, terrein had IC50’s of 21.6±2.891 µM and 74.76±0.97 µM, and the IC50’s of GCB in combination treatment were 0.23±0.03 µM and 0.23±0.005 µM, under hypoxic and normoxic conditions, respectively. In both SW620 and HCT116, terrein exhibited a preferential antitumor effect in hypoxic conditions; however, the opposite was observed in HT29. Upon further analysis via flow cytometry after annexin-V/FITC and PI staining, GCB and its combination treatment with terrein were found to induce necrosis in all cell lines under investigation. In addition, terrein was found to significantly induce significant S-phase cell arrest compared to GCB treatment alone using DNA content cytometry assay coupled with PI staining. Currently, we are studying the expression of multiple cyclins/CDK’s involved in the different cell cycle phases for further understanding of the detailed mechanism of action. Using flowcytometry assessment after staining with acridine orange, terrein suppressed autophagy compared to control cells as well as GCB treatment. NMR metabolomic analysis revealed that the change in oxygen levels significantly affected extracellular amino acid metabolite profiling such as leucin, which increased significantly due to hypoxia; while tyrosine decreased significantly in response to hypoxic environment. Currently, the gene expression profiling corresponding to these amino acids metabolomic changes are undergoing. Therefore, oxygen concentration plays a vital role in the intratumoral amino acid metabolism in response to terrein, GCB and their combination within colorectal cancer cells. Citation Format: Reham K. Abu Hijjleh, Dalia Y. Al Saeedy, Naglaa S. Ashmawy, Ahmed Gouda, Sameh S. Elhady, Ahmed M. Al-Abd. The potential chemo-modulatory effect of the marine-derived secondary metabolite terrein on the anticancer properties of gemcitabine in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1065.

Alterations in Microbial-Associated Fecal Metabolites in Relation to Arsenic Exposure Among Infants.

In utero and early life exposure to inorganic arsenic (iAs) alters immune response in experimental animals and is associated with an increased risk of infant infections. iAs exposure is related to differences in the gut microbiota diversity, community structure, and the relative abundance of individual microbial taxa both in laboratory and human studies. Metabolomics permits a direct measure of molecular products of microbial and host metabolic processes. We conducted NMR metabolomics analysis on infant stool samples and quantified the relative concentrations of 34 known microbial-related metabolites. We examined these metabolites in relation to both in utero and infant log2 urinary total arsenic concentrations (utAs, the sum of iAs and iAs metabolites) collected at approximately 6 weeks of age using linear regression models, adjusted for infant sex, age at sample collection, type of delivery (vaginal vs. cesarean section), feeding mode (breast milk vs. any formula), and specific gravity. Increased fecal butyrate (b = 214.24), propionate (b = 518.33), cholate (b = 8.79), tryptophan (b= 14.23), asparagine (b = 28.80), isoleucine (b = 65.58), leucine (b = 95.91), malonate (b = 50.43), and uracil (b = 36.13), concentrations were associated with a doubling of infant utAs concentrations (p< 0.05). These associations were largely among infants who were formula fed. No clear associations were observed with maternal utAs and infant fecal metabolites. Metabolomic analyses of infant stool samples lend further evidence that the infant gut microbiota is sensitive to As exposure, and these effects may have functional consequences.

Saffron extract (Safr'Inside™) improves anxiety related behaviour in a mouse model of low-grade inflammation through the modulation of the microbiota and gut derived metabolites.

Treatment of anxiety and depression predominantly centres around pharmacological interventions, which have faced criticism for their associated side effects, lack of efficacy and low tolerability. Saffron, which is reportedly well tolerated in humans, has been recognised for its antidepressant and anti-anxiety properties. Indeed, we previously reported upon the efficacy of saffron extract supplementation in healthy adults with subclinical anxiety. However, the molecular aetiology remains unclear. In a rodent model of low-grade chronic inflammation, we explored the impact of a saffron extract (Safr'Inside™) supplemented at a physiological dose, which equated to 22 ± 1.2 mg per day human equivalent dose for a person of 60 kg. Behavioural tests (Open Field task, Y maze, Novel object recognition), caecal 16S rRNA microbial sequencing, caecal 1H NMR metabolomic analysis and 2DE brain proteomic analyses were completed to probe gut-brain axis interactions. Time occupying the centre of the Open Field maze (OF) was increased by 62% in saffron supplemented animals. This improvement in anxiety-related behaviour coincided with gut microbial shifts, notably Akkermansia, Muribaculaceae, Christensenellacae and Alloprevotella which significantly increased in response to saffron supplementation. Akkermansia and Muribaculaceae abundance negatively correlated with the neurotoxic metabolite dimethylamine which was reduced in saffron supplemented animals. Brain proteomic analysis highlighted several significantly altered proteins including ketimine reductase mu-crystallin which also correlated with dimethylamine concentration. Both dimethylamine and ketimine reductase mu-crystallin were associated with OF performance. This may be indicative of a novel interaction across the gut-brain axis which contributes to anxiety-related disorders.

Metabolic Rewiring Is Essential for AML Cell Survival to Overcome Autophagy Inhibition by Loss of ATG3.

Simple SummaryThe importance of autophagy in leukemia progression and survival has been studied previously. However, little is known about the development of resistance mechanisms to autophagy inhibition in leukemia. Here, we present data on the mechanisms by which leukemia cells maintain their cell survival after inhibition of autophagy by the loss of ATG3. After the loss of ATG3, leukemia cells upregulated their energy metabolism by increasing glycolysis and mitochondrial metabolism, in particular oxidative phosphorylation, which resulted in higher ATP levels. Moreover, inhibition of mitochondrial function strongly impaired cell survival in ATG3 deficiency, thus demonstrating the importance of ATG3 in the regulation of metabolism and survival of leukemic cells. Therefore, our data provide a rationale for combining autophagy inhibitors with inhibitors targeting mitochondrial metabolism for the development of leukemia therapy to overcome the potential obstacle of emerging resistance to autophagy inhibition.Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.