Saliva remains underexplored in metabolomics compared with widely used biofluids such as blood and urine. However, its noninvasive, rapid, and cost-effective collection, together with its suitability for self-sampling, makes it attractive for clinical and personalized medicine. Moreover, saliva is expected to provide complementary metabolic information on other biofluids. In this context, this study aims to identify an optimal sample collection and preparation protocol for maximizing informative metabolomic data from salivary nuclear magnetic resonance (NMR) profiles. Four preparation protocols, selected and adapted from the literature, were systematically compared using the number of identified metabolites, their concentrations, and intraday repeatability as evaluation criteria. Among them, an in-house-adapted method combining centrifugation, freeze-drying, and ultrafiltration proved most effective. This approach provided the broadest metabolome coverage, with 42 metabolites quantified. This method was further assessed using analysis of variance to determine intra- and interday precision. Most metabolites demonstrated excellent repeatability (coefficients of variation below 10%), confirming the protocol reliability for quantitative metabolomics. Overall, the optimized approach yields high-quality spectra, wide metabolic coverage, and strong analytical precision, supporting reproducible salivary NMR metabolomics. Beyond its methodological contribution, this work highlights saliva as a promising biofluid for diagnostics, disease monitoring, and personalized medicine, provided that collection and preparation procedures are appropriately standardized.

R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (BPDZ 711, 4) initially designed as a KATP channel opener, was found to exhibit diverse biological activities. The compound inhibited insulin release from rat pancreatic islets, indicating a potential effect on glucose metabolism. Oxygraphy measurements on chronic myeloid leukemia (CML) K-562 cells revealed an impact on cellular respiration. Additionally, the compound demonstrated inhibitory activity on histone deacetylase class III enzymes (sirtuins), linking metabolic and epigenetic regulation. This was corroborated by its effect on protein acetylation and modulation of the extracellular pH of treated CML cells. Alterations in CML cells' nuclear morphology and the release of high-mobility group box 1 (HMGB1) protein confirmed mechanisms related to cellular stress and immunogenic cell death. BPDZ 711 preserved the viability of peripheral blood mononuclear cells, thus demonstrating excellent differential toxicity.Since BPDZ 711 is a racemate, the present study focused on the preparation of the two enantiomers and examined the possibility that each isomer could display a distinct pharmacological profile. Our data revealed that the R-enantiomer (5) of BPDZ 711 was consistently the most biologically active compound (eutomer), making it the reference compound for future drug discovery and development.

The present study investigates the AMPA receptor potentiation and cognitive-enhancing effects of novel halosubstituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. Monohalosubstituted 4-cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides were initially identified for their promising activity. In this article, a new pharmacomodulation explored the addition of a second halogen atom on the benzene nucleus and the variation of the cycloalkyl group at the 4-position. Compound 14o (the 6,7-dichloro-4-cyclopropyl-substituted derivative) was selected based on potency and safety. It selectively potentiated AMPA receptors in human and rat models, enhanced AMPA-mediated excitatory postsynaptic responses in rat hippocampal slices, and increased brain-derived neurotrophic factor (BDNF) expression in rat cortical neurons. In vivo, 14o in animal models significantly improved long-term potentiation (LTP) and enhanced cognitive performance in memory-related behavioral tasks at low doses. Notably, 14o also exhibited neuroprotective effects in rats, delaying hippocampal neurodegeneration following transient ischemia, without proconvulsant activity. These findings support 14o as a promising candidate for the treatment of cognitive disorders.

Regular consumption of pomegranate, a polyphenol-rich fruit, is associated with multiple health benefits. As polyphenols reach the colon, they interact with the gut microbiota, influencing both its composition and metabolic activity. This study investigated the impact of a one-week supplementation with two doses of the commercial pomegranate extract Oxylent® (1.3 and 2.6 g/day) on gut microbiota and metabolite production using the SHIME® system. Bacterial metabolite production, including short-chain fatty acids (SCFA), urolithins, succinate, and lactate, was assessed using chromatographic and enzymatic assays. The bacterial composition across colonic sections, represented by different fermenters in the SHIME, was investigated using 16 S rRNA amplicon sequencing. Pomegranate extract did not significantly alter SCFA or succinate levels, but reduced L- and D-lactate in the transverse colon; the higher dose (2.6 g/day) also decreased D-lactate in the ascending colon. Microbiota profiling revealed a higher bacterial diversity following pomegranate extract supplementation. However, Prevotella abundance decreased in the ascending and transverse colonic sections, potentially explaining the reduced propionate levels observed in the transverse colon with 2.6 g/day of pomegranate extract. Interestingly, contrasting effects were noted for Mitsuokella genus, which decreased in the descending colon at 1.3 g/day but increased at 2.6 g/day in the transverse and descending colons. Furthermore, the higher dose reduced Enterocloster abundance in the descending colon. Overall, Oxylent® pomegranate extract influenced both microbial composition and metabolite production, particularly taxa associated with health-related metabolites. These results highlight the potential of pomegranate compounds to beneficially influence the gut microbiota, supporting their role in promoting intestinal health.

Glucose measurement is a critical investigation in metabolic disease management, especially in diabetes and inherited disorders. However, both laboratory-based and handheld point-of-care (HPOC) (glucometers) glucose testing face significant preanalytical and analytical challenges. In central laboratories, glycolysis in uncentrifuged samples leads to glucose consumption, which may compromise diagnostic accuracy. Although sodium fluoride (NaF) is commonly used as a glycolysis inhibitor, it has a delayed effect, requiring several hours to stabilize glucose concentrations. Recently, citrate-buffered NaF-EDTA (FCE) tubes have been introduced to inhibit glycolysis more effectively, yet they remain underused. Preanalytical variables, including sample collection, transport, and processing delays, further impact glucose stability and the diagnosis of diabetes, including gestational diabetes mellitus (GDM). HPOC devices provide an alternative by delivering rapid results and minimizing preanalytical errors, but glucose meters are prone to physiological and analytical interferences, such as hematocrit variations, environmental conditions, presence of redox-active drugs, and enzymatic specificity issues. These interferences may lead to inaccurate glucose readings, impairing clinical decision-making, especially in intensive care and emergency settings. Moreover, discrepancies between capillary and venous glucose concentrations can contribute to misdiagnosis and inappropriate glycemic management. This review provides a comprehensive analysis of glucose measurement methodologies, their limitations, and potential improvements, emphasizing the need for preanalytical harmonization in laboratory testing and a better understanding of interferences in HPOC testing. Standardization of blood sample handling and adoption of optimized collection tubes could enhance glucose measurement reliability, ultimately improving diabetes diagnosis and patient outcomes.

Background Artemisia spp. have been used for millennia in traditional medicine to treat a variety of ailments, including malaria. Extracts of Artemisia afra and A. annua remain widely used throughout Africa for healthcare purposes, notably to prevent and/or treat malaria. However, the modes of action of these plant extracts remain unclear, with contradictory reports regarding the presence and role of artemisinin in both plants. Purpose The aim of this study was to identify differences in the antimalarial mode of action of A. afra and A. annua by measuring their phenolic profiles and comparing their effect on parasite metabolism in vitro. Methods In this work, we analyzed the phenolic profile of A. afra and A. annua extracts through high-performance liquid chromatography (HPLC), detected and quantified artemisinin through HPLC and mass spectrometry (MS), and performed comparative HPLC-MS metabolomic analysis on in vitro-cultured Plasmodium falciparum trophozoites to elucidate the potential modes of action of these plant extracts. Results A. afra contained only trace amounts of artemisinin and elicited a different parasite metabolic response compared to A. annua, which contained significantly more artemisinin and correlated closely with the parasite response profile elicited by purified artemisinin. A. annua impacted parasite glutathione metabolism in agreement with the established redox activity of artemisinin, while A. afra had an effect on lipid precursors. Conclusions This study reveals that A. afra and A. annua have divergent effects on Plasmodium falciparum metabolism and provides support for ongoing efforts exploring the use of A. afra for the treatment of malaria.

The aim of this study was to explore how a metabolomic approach could provide valuable information on changes in the athletes' metabolome during a mountain ultramarathon race. To achieve this goal, we established a longitudinal cohort of athletes enrolled in the TOR des Géants, a 330 km mountain ultramarathon with 24,000 m of elevation gain. Sixteen healthy male athletes (43.9 ± 10.1 years) were recruited, and blood samples were collected at four time points: pre-race, mid-race, post-race and after 72 h recovery. Using a 1H-NMR-based metabolomic approach, we evaluated metabolic changes that occur during both race effort and recovery, and correlated them with functional muscle, cardiac, inflammatory, and renal biomarkers already used in the clinic. The processed data were analyzed using multivariate analysis tools specific to longitudinal study design, and innovative pathway analysis was used for data interpretation. Mountain ultramarathon running significantly affected the metabolism and physiology of athletes. Multivariate analysis highlighted specific metabolites and functional biomarkers associated with prolonged exercise. Neither metabolite levels nor biomarker concentrations returned to baseline after 3 days of recovery. Finally, innovative pathway analysis shed light on specific metabolic changes resulting from mountain ultramarathon exercise. In this study, we propose an NMR-based metabolomics strategy to assess exercise-associated metabolic changes during and after events such as the Tor des Géants. Using state-of-the-art data representation methods specific to metabolomics analysis, we demonstrated that such a methodology can provide a unique view of the biology associated with such extreme conditions. As this approach provides unique insights into the biology of extreme exercise, it holds promise for the development of new tools for athlete management.

Study question Do environmental chemicals play a role on deep endometriosis through metabolic disruption? Summary answer The present study support that women with deep endometriosis exhibit a specific metabolic profile associated to the internal exposure of persistent organic pollutants. What is known already A growing list of pollutants seems to be able to interact with and metabolic signaling pathways involved in the onset and progression of endometriosis. Up to date, most of research on environmental pollutants have focused on endocrine disrupting mechanisms, whereas their involvement on the metabolic shift observed on endometriosis lesions have not been studied yet. Persistent organic pollutants (POPs) such as organochlorine pesticides (OCPs) or per-/polyfluorinated substances (PFAS), have shown the capacity to alter the normal metabolic function through different mechanisms, including mitochondrial dysfunction or energy homeostatic imbalance. Previous metabolomic studies have failed to identify robust predictive biomarkers of endometriosis. Study design, size, duration We conducted an observational metabolomic-wide case-control study with French women undergoing surgery for endometriosis or In vitro fertilization, with and without surgically confirmed endometriosis (n = 137). A “meet-in-the-middle” systematic review of observational and experimental studies was conducted to identify metabolic pathways overlapping POPs and endometriosis in order to support the biological plausibility. Participants/materials, setting, methods Women’s serum was analyzed using gas and liquid chromatography coupled to high-resolution mass spectrometry (HRMS) to measure the levels of 14 polychlorinated biphenyls (PCB), six OCPs and six PFAS. A comprehensive targeted metabolomic profiling was conducted using HRMS and 1H nuclear magnetic resonance (1H NMR). An ultra-targeted study including inflammatory mediators oxylipins and twenty-six free fatty acids was conducted by liquid and gas chromatography coupled to tandem mass spectrometry, respectively. Main results and the role of chance The PCB180, PCB167 and the pesticide trans-nonachlor were associated with a higher risk of deep endometriosis. Women with endometriosis exhibited a distinctive metabolic profile, with elevated serum levels of lactate, ketone bodies and multiple amino acids and lower levels of bile acids, phosphatidylcholines (PCs), cortisol and hippuric acid. The pesticide c was positively associated with deep endometriosis risk and the alteration of 2-hydroxybutyrate pathway. In turn, negative associations were found between the fluorinated industrial pollutant, perfluoroundecanoic acid (PFUnA) and levels of prostaglandin E2, 15-hydroxyeicosatetraenoic acid (15HETE) and 5-HETE. Levels of 5-HETE and 8,9-epoxyeicosatrienoic acid were found to be at lower levels in women with endometriosis-related infertility. Limitations, reasons for caution This study has a limited sample size, and the cross-sectional design does not prevent the reverse causation. The results may not be generalized to mild or superficial forms of endometriosis and general population. Further observational and experimental studies will be required to confirm these findings. Wider implications of the findings The present study showed that women with deep endometriosis had higher levels of POPs compared to women with benign conditions. A targeted high-throughput metabolomic analysis allowed gaining insight into metabolic pathways potentially linking the pesticide trans-nonachlor with endometriosis. Trial registration number No

The present study aims to highlight the impact on biological activity of the application of the isosteric concept to 1,2,4-benzothiadiazine 1,1-dioxides (BTDs) reported as AMPA receptor positive allosteric modulators (AMPAR PAMs). In a previous work, thiochroman 1,1-dioxides were designed as AMPAR PAMs by removing the two nitrogen atoms of the thiadiazine ring, a first pharmacomodulation process that led to encouraging results. In this study, another pharmacomodulation approach was employed to assess the impact of removing only one of the two nitrogen atoms of the thiadiazine ring providing two new series of candidates: 1,2-benzothiazine 1,1-dioxides and 1,4-benzothiazine 1,1-dioxides. Moreover, the isosteric concept between the carboxamide and the sulfonamide function was also explored leading to quinazolinone analogues of BTDs. The biological data revealed that 1,4-benzothiazine 1,1-dioxides appeared to be the most promising isosteres of BTDs since a significant AMPAR potentiation activity was observed with representative compounds. Among them, the chloro-substituted compound 25b demonstrated the highest activity, being the closest structural analogue of the well-known BTD AMPAR potentiator BPAM121. On the other hand, none of the 1,2-benzothiazine 1,1-dioxides and the quinazolinones studied were found to exert a significant AMPAR potentiation activity. In conclusion, activity on AMPARs can be retained with compounds where the nitrogen atoms at the 2-position (1,4-benzothiazine 1,1-dioxides) or at the 2,4-positions (thiochroman 1,1-dioxides) of BTDs was replaced by one or two carbon atoms. Further investigations are required to explore additional structural modifications that could improve biological activity.

Background The metabolomics approach, aiming to link variation of metabolic profile to pathologic conditions, is a vital tool for personalized medicine and biomarker discovery. In the context of athletes' monitoring and injury prevention, measuring the dynamic metabolic changes resulting from pathologic conditions is crucial. Objective We aimed to develop an NMR-based metabolomic workflow to spot specific metabolic signature of athletes' fitness and recovery. Our purpose was to provide valuable information about the metabolic status of athletes by comparing metabolomic data coming from two group of individuals: Ultra-trail athletes (UT) and, sedentary individuals. Design Longitudinal, cohort study Setting UT individuals were participating to the UltraTour, a 67 km ultra-trail running, 1500 D+ in Liège (Belgium). Sedentary individuals ran 1 hour on a treadmill at the maximum of their capacity. Participants 20 UT participants, 15 sedentary. Intervention (or Assessment of risk factors) Blood samples were collected at three time points: before (TO), immediately after (T1), and three hours after the start of their effort (T3). NMR-based metabolomic analysis was employed to analyze intragroup and intergroup metabolic variations. Main outcome measurements 1H-NMR spectra were recorded and processed through an in-house pipeline to generate metabolic profiles. Intragroup and intergroup variations were analyzed using non-discriminant PCA models dedicated to multivariate analysis. Results Intragroup metabolic profile changes from T0 to T3 were observed for both groups. Indeed, three hours of recovery were insufficient for athletes to return to their baseline metabolic status. Significant differences between athletes and sedentary individuals were also highlighted. Conclusion This proof-of-concept study spotted the importance of metabolomics approach in the field of athletes' follow-up, performance monitoring and injury prevention. Indeed, by following the metabolic changes over the time, we can assess the fitness of the studied individuals and gather information about their recovery and avoid situation of high-risk of muscle injury.

The synthesis and biological evaluation on AMPA and kainate receptors of new examples of 3,4-dihydro-2 H -1,2,4-thieno[3,2- e ]-1,2,4-thiadiazine 1,1-dioxides is described. The introduction of a cyclopropyl chain instead of an ethyl chain at the 4-position of the thiadiazine ring was found to dramatically improve the potentiator activity on AMPA receptors, with compound 32 (BPAM395) expressing in vitro activity on AMPARs (EC2x = 0.24 µM) close to that of the reference 4-cyclopropyl-substituted benzothiadiazine dioxide 10 (BPAM344). Interestingly, the 4-allyl-substituted thienothiadiazine dioxide 27 (BPAM307) emerged as the most promising compound on kainate receptors being a more effective potentiator than the 4-cyclopropyl-substituted thienothiadiazine dioxide 32 and supporting the view that the 4-allyl substitution of the thiadiazine ring could be more favorable than the 4-cyclopropyl substitution to induce marked activity on kainate receptors versus AMPA receptors. The thieno-analogue 36 (BPAM279) of the clinically tested S18986 ( 11 ) was selected for in vivo evaluation in mice as a cognitive enhancer due to a safer profile than 32 after massive per os drug administration. Compound 36 was found to increase the cognition performance in mice at low doses (1 mg/kg) per os suggesting that the compound was well absorbed after oral administration and able to reach the central nervous system. Finally, compound 32 was selected for co-crystallization with the GluA2-LBD (L504Y,N775S) and glutamate to examine the binding mode of thienothiadiazine dioxides within the allosteric binding site of the AMPA receptor. At the allosteric site, this compound established similar interactions as the previously reported BTD-type AMPA receptor modulators. Highlights The study explored AMPA/kainate receptor PAMs belonging to thienothiadiazine dioxides The 4-cyclopropyl-substituted compound 32 was the most potent AMPA receptor modulator 4-Allyl substitution improved activity and selectivity for kainate receptors The tricyclic compound 36 expressed cognitive improvement in vivo in mice Compound 32 was co-crystallized with GluA2-LBD to examine receptor binding mode Graphical abstract

In order to improve our healthcare system, it is undeniable that the future of modern medicine must focus on a more preventive and personalized approach, notably based on the individual characteristics specific to each patient. In this perspective, clinical metabolomics, which focuses on metabolites, emerges as a particularly interesting and promising approach. Indeed, this science reflects the internal and external stimuli received by an individual, thus capturing their physiological and/or pathological state. Close to the phenotype, it represents the interface between the patient, their genes, and their environment in the broadest sense. Its translational nature requires the conjunction of several expertise areas, both in analytical, biostatistical, and clinical levels. Combined with other data, it allows the generation of predictive or diagnostic models useful for early detection and monitoring of pathologies, taking into account notably the individual characteristics of patients. There are, of course, many obstacles and challenges to overcome for metabolomics to transition into clinical practice, but it is evident that this innovative approach will, in the years to come, find its place among the tools available to clinicians in a more personalized vision of patient care.

R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea (BPDZ 711, 4) initially designed as a KATP channel opener, was found to exhibit diverse biological activities. The compound inhibited insulin release from rat pancreatic islets, indicating a potential effect on glucose metabolism. Oxygraphy measurements on chronic myeloid leukemia (CML) K-562 cells revealed an impact on cellular respiration. Additionally, the compound demonstrated inhibitory activity on histone deacetylase class III enzymes (sirtuins), linking metabolic and epigenetic regulation. This was corroborated by its effect on protein acetylation and modulation of the extracellular pH of treated CML cells. Alterations in CML cells’ nuclear morphology and the release of high-mobility group box 1 (HMGB1) protein confirmed mechanisms related to cellular stress and immunogenic cell death. BPDZ 711 preserved the viability of peripheral blood mononuclear cells, thus demonstrating excellent differential toxicity.Since BPDZ 711 is a racemate, the present study focused on the preparation of the two enantiomers and examined the possibility that each isomer could display a distinct pharmacological profile. Our data revealed that the R-enantiomer (5) of BPDZ 711 was consistently the most biologically active compound (eutomer), making it the reference compound for future drug discovery and development.

Malaria is a parasitic disease that remains a global concern and the subject of many studies. Metabolomics has emerged as an approach to better comprehend complex pathogens and discover possible drug targets, thus giving new insights that can aid in the development of antimalarial therapies. However, there is no standardized method to extract metabolites from in vitro Plasmodium falciparum intraerythrocytic parasites, the stage that causes malaria. Additionally, most methods are developed with either LC-MS or NMR analysis in mind, and have rarely been evaluated with both tools. In this work, three extraction methods frequently found in the literature were reproduced and samples were analyzed through both LC-MS and 1H NMR, and evaluated in order to reveal which is the most repeatable and consistent through an array of different tools, including chemometrics, peak detection and annotation. The most reliable method in this study proved to be a double extraction with methanol and methanol/water (80:20, v/v). Metabolomic studies in the field should move towards standardization of methodologies and the use of both LC-MS and 1H NMR in order to make data more comparable between studies and facilitate the achievement of biologically interpretable information.

Trypanosoma brucei (Tb) is the causative agent of human African trypanosomiasis (HAT), also known as sleeping sickness, which can be fatal if left untreated. An understanding of the parasite's cellular metabolism is vital for the discovery of new antitrypanosomal drugs and for disease eradication. Metabolomics can be used to analyze numerous metabolic pathways described as essential to Tb. brucei but has some limitations linked to the metabolites' physicochemical properties and the extraction process. To develop an optimized method for extracting and analyzing Tb. brucei metabolites, we tested the three most commonly used extraction methods, analyzed the extracts by hydrophilic interaction liquid chromatography high-resolution mass spectrometry (HILIC LC-HRMS), and further evaluated the results using quantitative criteria including the number, intensity, reproducibility, and variability of features, as well as qualitative criteria such as the specific coverage of relevant metabolites. Here, we present the resulting protocols for untargeted metabolomic analysis of Tb. brucei using (HILIC LC-HRMS). (c) 2024 Wiley Periodicals LLC. Basic Protocol 1: Culture of Trypanosoma brucei brucei parasites Basic Protocol 2: Preparation of samples for metabolomic analysis of Trypanosoma brucei brucei Basic Protocol 3: LC-HRMS-based metabolomic data analysis of Trypanosoma brucei brucei

Background: Exposure to persistent organic pollutants (POPs) has been related to the risk of endometriosis however the mechanisms remain unclear. The objective of the present study was to characterize the metabolic profiles underpinning the associations between POPs and endometriosis risk. Methodology: A hospital-based case-control study was conducted in France to recruit women with and without surgically confirmed deep endometriosis. Women's serum was analyzed using gas and liquid chromatography coupled to high -resolution mass spectrometry (HRMS) to measure the levels of polychlorinated biphenyls (PCBs), organochlorinated pesticides (OCPs) and per-/polyfluoroalkyl substances (PFAS). A comprehensive metabolomic profiling was conducted using targeted HRMS and 1H nuclear magnetic resonance (1H NMR) to cover polar and non -polar fractions. A "meet -in -the -middle" statistical framework was applied to identify the metabolites related to endometriosis and POP levels, using multivariate linear and logistic regressions adjusting for confounding variables. Results: Fourteen PCBs, six OCPs and six PFAS were widely found in almost all serum samples. The pesticide trans-nonachlor was the POP most strongly and positively associated with deep endometriosis risk, with odds ratio (95 % confidence interval) of 2.42 (1.49; 4.12), followed by PCB180 and 167. Women with endometriosis exhibited a distinctive metabolic profile, with elevated serum levels of lactate, ketone bodies and multiple amino acids and lower levels of bile acids, phosphatidylcholines (PCs), cortisol and hippuric acid. The metabolite 2hydroxybutyrate was simultaneously associated to endometriosis risk and exposure to trans-nonachlor. Conclusions: To the best of our knowledge, this is the first comprehensive metabolome-wide association study of endometriosis, integrating ultra -trace profiling of POPs. The results confirmed a metabolic alteration among women with deep endometriosis that could be also associated to the exposure to POPs. Further observational and experimental studies will be required to delineate the causal ordering of those associations and gain insight on the underlying mechanisms.

INTRODUCTION:Human African trypanosomiasis, commonly known as sleeping sickness, is a vector-borne parasitic disease prevalent in sub-Saharan Africa and transmitted by the tsetse fly. Suramin, a medication with a long history of clinical use, has demonstrated varied modes of action against Trypanosoma brucei. This study employs a comprehensive workflow to investigate the metabolic effects of suramin on T. brucei, utilizing a multimodal metabolomics approach.OBJECTIVES:The primary aim of this study is to comprehensively analyze the metabolic impact of suramin on T. brucei using a combined liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) approach. Statistical analyses, encompassing multivariate analysis and pathway enrichment analysis, are applied to elucidate significant variations and metabolic changes resulting from suramin treatment.METHODS:A detailed methodology involving the integration of high-resolution data from LC-MS and NMR techniques is presented. The study conducts a thorough analysis of metabolite profiles in both suramin-treated and control T. brucei brucei samples. Statistical techniques, including ANOVA-simultaneous component analysis (ASCA), principal component analysis (PCA), ANOVA 2 analysis, and bootstrap tests, are employed to discern the effects of suramin treatment on the metabolomics outcomes.RESULTS:Our investigation reveals substantial differences in metabolic profiles between the control and suramin-treated groups. ASCA and PCA analysis confirm distinct separation between these groups in both MS-negative and NMR analyses. Furthermore, ANOVA 2 analysis and bootstrap tests confirmed the significance of treatment, time, and interaction effects on the metabolomics outcomes. Functional analysis of the data from LC-MS highlighted the impact of treatment on amino-acid, and amino-sugar and nucleotide-sugar metabolism, while time effects were observed on carbon intermediary metabolism (notably glycolysis and di- and tricarboxylic acids of the succinate production pathway and tricarboxylic acid (TCA) cycle).CONCLUSION:Through the integration of LC-MS and NMR techniques coupled with advanced statistical analyses, this study identifies distinctive metabolic signatures and pathways associated with suramin treatment in T. brucei. These findings contribute to a deeper understanding of the pharmacological impact of suramin and have the potential to inform the development of more efficacious therapeutic strategies against African trypanosomiasis.

Supplementary Table S1: patient characteristics including age, sex, tumor type, and treatment

Supplementary Figure 1-7 on irradiation of CAF; effect of irradiated CAF on cancer cells; glutamine metabolism; proteome analysis of CM CAF; effect of recombinant IGF1 on cancer cells; effect of recombinant IGF1 on metabolism of cancer cells; schematic of in vivo set-up and Figure legends or respective figures