Metabolomic changes in adults with status epilepticus: A human case-control study.

OBJECTIVE: Status epilepticus (SE) is a life-threatening prolonged epileptic seizure that affects ~40 per 100 000 people yearly worldwide. The persistence of seizures may lead to excitotoxic processes, neuronal loss, and neuroinflammation, resulting in long-term neurocognitive and functional disabilities. A better understanding of the pathophysiological mechanisms underlying SE consequences is crucial for improving SE management and preventing secondary neuronal injury. METHODS: We conducted a comprehensive untargeted metabolomic analysis, using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), on plasma and cerebrospinal fluid (CSF) samples from 78 adult patients with SE and 107 control patients without SE, including 29 with CSF for both groups. The metabolomic fingerprints were compared between patients with SE and controls. Metabolites with differences in relative abundances that could not be attributed to treatment or nutrition provided in the intensive care unit were isolated. Enrichment analysis was performed on these metabolites to identify the most affected pathways. RESULTS: We identified 76 metabolites in the plasma and 37 in the CSF that exhibited differential expression in patients with SE compared to controls. The enrichment analysis revealed that metabolic dysregulations in patients with SE affected primarily amino acid metabolism (including glutamate, alanine, tryptophan, glycine, and serine metabolism), pyrimidine metabolism, and lipid homeostasis. Specifically, patients with SE had elevated levels of pyruvate, quinolinic acid, and keto butyric acid levels, along with lower levels of arginine, N-acetylaspartylglutamate (NAAG), tryptophan, uracil, and uridine. The tryptophan kynurenine pathway was identified as the most significantly altered in SE, resulting in the overproduction of quinolinic acid, an N-methyl-d-aspartate (NMDA) receptor agonist with pro-inflammatory properties. SIGNIFICANCE: This study has identified several pathways that may play pivotal roles in SE consequences, such as the tryptophan kynurenine pathway. These findings offer novel perspectives for the development of neuroprotective therapeutics.

Efficient Access to Deuterated and Tritiated Nucleobase Pharmaceuticals and Oligonucleotides using Hydrogen-Isotope Exchange.

A general approach for the efficient hydrogen-isotope exchange of nucleobase derivatives is described. Catalyzed by ruthenium nanoparticles, using mild reaction conditions, and involving either D2 or T2 as isotopic sources, this reaction possesses a wide substrate scope and a high solvent tolerability. This novel method facilitates the access to essential diagnostic tools in drug discovery and development: tritiated pharmaceuticals with high specific activities and deuterated oligonucleotides suitable for use as internal standards during LC-MS quantification.

In-depth characterization of a selection of gut commensal bacteria reveals their functional capacities to metabolize dietary carbohydrates with prebiotic potential.

The microbial utilization of dietary carbohydrates is closely linked to the pivotal role of the gut microbiome in human health. Inherent to the modulation of complex microbial communities, a prebiotic implies the selective utilization of a specific substrate, relying on the metabolic capacities of targeted microbes. In this study, we investigated the metabolic capacities of 17 commensal bacteria of the human gut microbiome toward dietary carbohydrates with prebiotic potential. First, in vitro experiments allowed the classification of bacterial growth and fermentation profiles in response to various carbon sources, including agave inulin, corn fiber, polydextrose, and citrus pectin. The influence of phylogenetic affiliation appeared to statistically outweigh carbon sources in determining the degree of carbohydrate utilization. Second, we narrowed our focus on six commensal bacteria representative of the Bacteroidetes and Firmicutes phyla to perform an untargeted high-resolution liquid chromatography-mass spectrometry metabolomic analysis: Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides intestinalis, Subdoligranulum variabile, Roseburia intestinalis, and Eubacterium rectale exhibited distinct metabolomic profiles in response to different carbon sources. The relative abundance of bacterial metabolites was significantly influenced by dietary carbohydrates, with these effects being strain-specific and/or carbohydrate-specific. Particularly, the findings indicated an elevation in short-chain fatty acids and other metabolites, including succinate, gamma-aminobutyric acid, and nicotinic acid. These metabolites were associated with putative health benefits. Finally, an RNA-Seq transcriptomic approach provided deeper insights into the underlying mechanisms of carbohydrate metabolization. Restricting our focus on four commensal bacteria, including B. xylanisolvens, B. thetaiotaomicron, S. variabile, and R. intestinalis, carbon sources did significantly modulate the level of bacterial genes related to the enzymatic machinery involved in the metabolization of dietary carbohydrates. This study provides a holistic view of the molecular strategies induced during the dynamic interplay between dietary carbohydrates with prebiotic potential and gut commensal bacteria. IMPORTANCE: This study explores at a molecular level the interactions between commensal health-relevant bacteria and dietary carbohydrates holding prebiotic potential. We showed that prebiotic breakdown involves the specific activation of gene expression related to carbohydrate metabolism. We also identified metabolites produced by each bacteria that are potentially related to our digestive health. The characterization of the functional activities of health-relevant bacteria toward prebiotic substances can yield a better application of prebiotics in clinical interventions and personalized nutrition. Overall, this study highlights the importance of identifying the impact of prebiotics at a low resolution of the gut microbiota to characterize the activities of targeted bacteria that can play a crucial role in our health.

An orally active carbon monoxide-releasing molecule enhances beneficial gut microbial species to combat obesity in mice.

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, ß-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction of inflammatory markers in colon and cecum and restoration of metabolic profiles in these tissues. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

Despite similar clinical features metabolomics reveals distinct signatures in insulin resistant and progressively obese minipigs.

Obesity is a major contributor to the silent and progressive development of type 2 diabetes (T2D) whose prevention could be improved if individuals at risk were identified earlier. Our aim is to identify early phenotypes that precede T2D in diet-induced obese minipigs. We fed four groups of minipigs (n = 5-10) either normal-fat or high-fat high-sugar diet during 2, 4, or 6 months. Morphometric features were recorded, and metabolomics and clinical parameters were assessed on fasting plasma samples. Multivariate statistical analysis on 46 morphometrical and clinical parameters allowed to differentiate 4 distinct phenotypes: NFC (control group) and three others (HF2M, HF4M, HF6M) corresponding to the different stages of the obesity progression. Compared to NFC, we observed a rapid progression of body weight and fat mass (4-, 7-, and tenfold) in obese phenotypes. Insulin resistance (IR; 2.5-fold increase of HOMA-IR) and mild dyslipidemia (1.2- and twofold increase in total cholesterol and HDL) were already present in the HF2M and remained stable in HF4M and HF6M. Plasma metabolome revealed subtle changes of 23 metabolites among the obese groups, including a progressive switch in energy metabolism from amino acids to lipids, and a transient increase in de novo lipogenesis and TCA-related metabolites in HF2M. Low anti-oxidative capacities and anti-inflammatory response metabolites were found in the HF4M, and a perturbed hexose metabolism was observed in HF6M. Overall, we show that IR and progressively obese minipigs reveal phenotype-specific metabolomic signatures for which some of the identified metabolites could be considered as potential biomarkers of early progression to TD2.

Development of a Mass Spectrometry Imaging Method for Detecting and Mapping Graphene Oxide Nanoparticles in Rodent Tissues.

Graphene-based nanoparticles are continuously being developed for biomedical applications, and their use raises concerns about their environmental and biological impact. In the literature, some imaging techniques based on fluorescence and radioimaging have been used to explore their fate in vivo. Here, we report on the use of label-free mass spectrometry and mass spectrometry imaging (MSI) for graphene oxide (GO) and reduced graphene oxide (rGO) analyses in rodent tissues. Thereby, we extend previous work by focusing on practical questions to obtain reliable and meaningful images. Specific radical anionic carbon clusters ranging from C2-• to C9-• were observed for both GO and rGO species, with a base peak at m/z 72 under negative laser desorption ionization mass spectrometry (LDI-MS) conditions. Extension to an LDI-MSI method was then performed, thus enabling the efficient detection of GO nanoparticles in lung tissue sections of previously exposed mice. The possibility of quantifying those nanoparticles on tissue sections has also been investigated. Two different ways of building calibration curves (i.e., GO suspensions spotted on tissue sections, or added to lung tissue homogenates) were evaluated and returned similar results, with linear dynamic concentration ranges over at least 2 orders of magnitude. Moreover, intra- and inter-day precision studies have been assessed, with relative standard deviation below 25% for each concentration point of a calibration curve. In conclusion, our study confirms that LDI-MSI is a relevant approach for biodistribution studies of carbon-based nanoparticles, as quantification can be achieved, provided that nanoparticle suspension and manufacturing are carefully controlled.

Supercritical fluid chromatography coupled to mass spectrometry for lipidomics.

Supercritical fluid chromatography (SFC) has experienced a particular revival in recent years thanks to the development of robust and efficient commercial systems. Because of its physico-chemical properties, supercritical carbon dioxide (CO2 ) mixed with cosolvents and additives is particularly suitable for SFC to allow the elution of compounds of different polarities and more particularly complex lipids. Hyphenation with mass spectrometry (MS) is increasingly described in the literature but still requires many further developments in order to be as user-friendly as coupling with liquid chromatography. The basic concepts of SFC and MS hyphenation will be first considered. Then a representative example of method development in lipidomics will be introduced. In conclusion, the challenges and future needs in this field of research will be discussed.

Palladium-Mediated Site-Selective C-H Radio-iodination.

The palladium-mediated C-H radio-iodination of arenes using sodium iodide as the primary isotopic source is reported and performed without chemical know-how in 30 min and applied to the synthesis of complex radio-iodinated compounds of biological interest.

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA.

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

Ruthenium-catalyzed hydrogen isotope exchange of C(sp3)-H bonds directed by a sulfur atom.

We present here the first example of C(sp3)-H activation directed by a sulfur atom. Based on this transformation catalyzed by Ru/C, we have developed a hydrogen isotope exchange reaction for the deuterium and tritium labelling of thioether substructures in complex molecules.

Despite similar clinical features metabolomics reveals distinct signatures in insulin resistant and progressively obese minipigs

Obesity is a major contributor to the silent and progressive development of type 2 diabetes (T2D) whose prevention could be improved if individuals at risk were identified earlier. Our aim is to identify early phenotypes that precede T2D in diet-induced obese minipigs. We fed four groups of minipigs (n = 5–10) either normal-fat or high-fat high-sugar diet during 2, 4, or 6 months. Morphometric features were recorded, and metabolomics and clinical parameters were assessed on fasting plasma samples. Multivariate statistical analysis on 46 morphometrical and clinical parameters allowed to differentiate 4 distinct phenotypes: NFC (control group) and three others (HF2M, HF4M, HF6M) corresponding to the different stages of the obesity progression. Compared to NFC, we observed a rapid progression of body weight and fat mass (4-, 7-, and tenfold) in obese phenotypes. Insulin resistance (IR; 2.5-fold increase of HOMA-IR) and mild dyslipidemia (1.2- and twofold increase in total cholesterol and HDL) were already present in the HF2M and remained stable in HF4M and HF6M. Plasma metabolome revealed subtle changes of 23 metabolites among the obese groups, including a progressive switch in energy metabolism from amino acids to lipids, and a transient increase in de novo lipogenesis and TCA-related metabolites in HF2M. Low anti-oxidative capacities and anti-inflammatory response metabolites were found in the HF4M, and a perturbed hexose metabolism was observed in HF6M. Overall, we show that IR and progressively obese minipigs reveal phenotype-specific metabolomic signatures for which some of the identified metabolites could be considered as potential biomarkers of early progression to TD2. (AU)

Impact of peptide micropatterning on endothelial cell actin remodeling for cell alignment under shear stress.

HSVEC behavior under physiological shear stress in vitro is investigated on PET surfaces micropatterned with both RGDS and WQPPRARI peptides. This technique allows (i) creating geometries on surface to guide cell orientation under shear stress and (ii) controlling surface chemical composition in order to modulate cell behavior. Under shear stress, endothelial cells adhere on patterned PET surfaces and present a more rapid orientation in flow direction in comparison to cells cultured on homogeneous surfaces. Micropatterned surfaces presenting a large surface area ratio of RGDS/WQPPRARI peptides induce fibrillar adhesion, while surfaces presenting an equal RGDS/WQPPRARI peptides surface area ratio preferentially induce focal adhesion.

Differentiation of pre-osteoblast cells on poly(ethylene terephthalate) grafted with RGD and/or BMPs mimetic peptides.

The bone morphogenetic proteins (BMPs) are cytokines of the transforming growth factor beta family. Some BMPs such as BMP-2, BMP-7 and BMP-9 play a major role in the bone and cartilage formation. The BMP peptides corresponding to residues 73-92, 89-117, and 68-87 of BMP-2, BMP-7 and BMP-9 respectively as well as adhesion peptides (GRGDSPC) were grafted onto polyethylene terephthatalate (PET) surfaces. We evaluated the state of differentiation of pre-osteoblastic cells. The behavior of these cells on various functionalized surfaces highlighted the activity of the mimetic peptides immobilized on surfaces. The induced cells (observed in the case of surfaces grafted with BMP-2, 7 or 9 mimetic peptides and GRGDSPC peptides) were characterized on several levels. First of all, we focused on the evaluation of the osteoblastic markers such as the transcriptional factor Runx2, which is a critical regulator of osteoblastic differentiation. Secondly, the results obtained showed that these induced cells take a different morphology compared to the cells in a state of proliferation or in a state of extracellular matrix production. Induced cells were characterized by an increased thickness compared to non-induced cells. Thus, our studies prove a direct correlation between cell morphology and state of induction. Thereafter, we focused on characterizing the extracellular matrix formed by the cells on various surfaces. The extracellular matrix thickness was more significant in the case of surfaces grafted with mimetic peptides of the BMP-2, 7 or 9 and GRGDSPC peptides which once again proves their activity when immobilized on material surface. These results demonstrate that GRGDSPC and BMPs peptides, grafted to PET surface, act to enhance osteogenic differentiation and mineralization of pre-osteoblastic cells. These findings are potentially useful in developing engineered biomaterials for bone regeneration.

The effect of RGD density on osteoblast and endothelial cell behavior on RGD-grafted polyethylene terephthalate surfaces.

Hybrid materials combining polyethylene terephthalate and different types of cells (endothelial and osteoblastic cells) have been developed thanks to the covalent grafting of different densities of RGD containing peptides onto the polymer surface. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions, coupling agent grafting and the immobilization of the RGDC peptides. High resolution mu-imager was used to evaluate RGD densities (varying between 0.6 and 2.4 pmol/mm(2)) and has exhibited the stability of the surface grafted peptides when treated in harsh conditions. The efficiency of this route for biomimetic modification of a PET surface was demonstrated by measuring the adhesion of MC3T3 and HSVEC cells and by focal adhesion observation. Results obtained prove that a minimal RGDC density of 1 pmol/mm(2) is required to improve MC3T3 and HSVEC cells responses. Indeed, cells seeded onto a RGDC-modified PET with a density higher than 1 pmol/mm(2) were able to establish focal adhesion as visualized by fluorescence microscope compared to cells immobilized onto unmodified PET and RGDC-modified PET with densities lower than 1 pmol/mm(2). Moreover, the number of focal contacts was enhanced by the increase of RGDC peptide densities grafted onto the material surface. With this study we proved that the density of peptides immobilized on the surface is a very important parameter influencing osteoblast or endothelial cell adhesion and focal contact formation.