Findings from the individualized management of a patient with Acyl-CoA Oxidase-1 (ACOX1) deficiency: A bedside-to-bench-to-bedside strategy.

Acyl-CoA Oxidase-1 (ACOX1) deficiency (MIM 264470) is an autosomal recessive disease characterized by impairments in the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, which is the first step in the catalysis of the ß-oxidative breakdown of very long chain fatty acids (VLCFA) occuring in peroxisomes. The deleterious accumulation of VLCFA in several organs, including the brain, is a key biochemical feature of this disease which has devastating neurological consequences. ACOX1 deficiency is ultra-rare; as such, few studies have been conducted to determine the leading causes of symptoms or uncover new therapeutics. When confronted with one such case, we decided to bring drug discovery tools to the patient's bedside in an attempt to identify a cure. A skin biopsy was performed on a young patient with ACOX1 deficiency, following which screening technologies and mass spectrometry analysis techniques were applied to design a cellular assay that enabled the direct measurement of the effect of small molecules on the patient's primary fibroblasts. This approach is particularly well adapted to inherited metabolic disorders such as ACOX1 deficiency. Through the evaluation of a proprietary library of repurposable drugs, we found that the anthelmintic drug niclosamide led to a significant reduction in VLCFA in vitro. This drug was subsequently administered to the patient for more than six years. This study outlines the screening and drug selection processes. Additionally, we present our comprehensive clinical and biochemical findings that aided in understanding the patient's natural history and analysis of the progression of the patient's symptoms throughout the treatment period. Although the patient's overall lifespan was extended compared to the average age at death in severe early onset cases of ACOX1 deficiency, we did not observe any definitive evidence of clinical or biochemical improvement during niclosamide treatment. Nonetheless, our study shows a good safety profile of long-term niclosamide administration in a child with a rare neurodegenerative disease, and illustrates the potential of individualized therapeutic strategies in the management of inherited metabolic disorders, which could benefit both patients and the broader scientific and medical communities.

Set up and validation of a sensitive method to quantify prostaglandins, prostaglandin-glycerol esters and prostaglandin-ethanolamides, as well as their respective precursors.

Arachidonic acid-derived prostaglandins are widely studied for their role in inflammation. However, besides arachidonic acid, other arachidonic moiety-containing lipids can be metabolized by COX-2. Indeed, the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) can follow the same biochemical pathways than arachidonic acid leading to the formation of prostaglandin-glycerol esters (PG-G) and prostaglandin-ethanolamides (or prostamides, PG-EA), respectively. The data reported so far support the interest of these bioactive lipids in inflammatory conditions. However, there is only a handful of methods described for their quantification in biological matrices. Moreover, given the shared biochemical pathways for arachidonic acid, 2-AG and AEA, a method allowing for the quantification of these precursors and the corresponding prostaglandin derivatives appears as largely needed. Thus, we report here the development and validation of a single run UPLC-MS/MS quantification method allowing the quantification of these endocannabinoids-derived mediators together with the classical prostaglandin. Moreover, we applied the method to the quantification of these lipids in vitro (using lipopolysaccharides-activated J774 macrophage cells) and in vivo in several tissues from DSS-induced colitis mice. This femtomole-range method should improve the understanding of the interaction between these lipid mediators and inflammation.

Genetic Polymorphisms in SLCO2B1 and ABCC1 Conjointly Modulate Atorvastatin Intracellular Accumulation in HEK293 Recombinant Cell Lines.

BACKGROUND: Although atorvastatin (ATV) is well-tolerated, patients may report muscle complaints. These are difficult to predict owing to high interindividual variability. Such side effects are linked to intramuscular accumulation of ATV. This study aimed to investigate the relative role of transporters expressed in muscle tissue in promoting or limiting drug access to cells. The impact of common single nucleotide polymorphisms (SNPs) in SLCO2B1 coding for OATP2B1 and ABCC1 coding for MRP1 on ATV transport was also evaluated. METHODS: HEK293 cells were stably transfected with plasmids containing cDNA encoding wild-type or variant SLCO2B1 and/or ABCC1 to generate single and double stable transfectant HEK293 recombinant models overexpressing variant or wild-type OATP2B1 (influx) and/or MRP1 (efflux) proteins. Variant plasmids were generated by site-directed mutagenesis. Expression analyses were performed to validate recombinant models. Accumulation and efflux experiments were performed at different concentrations. ATV was quantified by LC-MS/MS, and kinetic parameters were compared between single and double HEK transfectants expressing wild-type and variant proteins. RESULTS: The results confirm the involvement of OATP2B1 and MRP1 in ATV cellular transport because it was demonstrated that intracellular accumulation of ATV was boosted by OATP2B1 overexpression, whereas ATV accumulation was decreased by MRP1 overexpression. In double transfectants, it was observed that increased ATV intracellular accumulation driven by OATP2B1 influx was partially counteracted by MRP1 efflux. The c.935G > A SNP in SLCO2B1 was associated with decreased ATV OATP2B1-mediated influx, whereas the c.2012G > T SNP in ABCC1 seemed to increase MRP1 efflux activity against ATV. CONCLUSIONS: Intracellular ATV accumulation is regulated by OATP2B1 and MRP1 transporters, whose functionality is modulated by natural genetic variants. This is significant because it may play a role in ATV muscle side-effect susceptibility.

Lipid nanocapsules for the nose-to-brain delivery of the anti-inflammatory bioactive lipid PGD2-G.

Here, prostaglandin D2-glycerol ester (PGD2-G) was selected to target neuroinflammation. As PGD2-G is reported to have a short plasmatic half-life, we propose to use lipid nanocapsules (LNC) as vehicle to safely transport PGD2-G to the central nervous system (CNS). PGD2-G-loaded LNC (PGD2-G-LNC) reduced pro-inflammatory cytokine expression in activated microglial cells, even so after crossing a primary olfactory cell monolayer. A single nasal administration of PGD2-G-LNC in lipopolysaccharide (LPS)-treated mice reduced pro-inflammatory cytokine expression in the olfactory bulb. Coating LNC's surface with a cell-penetrating peptide, transactivator of transcription (TAT), increased its accumulation in the brain. Although TAT-coated PGD2-G-LNC modestly exerted its anti-inflammatory effect in a mouse model of multiple sclerosis similar to free PGD2-G after nasal administration, TAT-coated LNC surprisingly reduced the expression of pro-inflammatory chemokines in the CNS. These data propose LNC as an interesting drug delivery tool and TAT-coated PGD2-G-LNC remains a good candidate, in need of further work.

Dysosmobacter welbionis is a newly isolated human commensal bacterium preventing diet-induced obesity and metabolic disorders in mice.

OBJECTIVE: To investigate the abundance and the prevalence of Dysosmobacter welbionis J115T, a novel butyrate-producing bacterium isolated from the human gut both in the general population and in subjects with metabolic syndrome. To study the impact of this bacterium on host metabolism using diet-induced obese and diabetic mice. DESIGN: We analysed the presence and abundance of the bacterium in 11 984 subjects using four human cohorts (ie, Human Microbiome Project, American Gut Project, Flemish Gut Flora Project and Microbes4U). Then, we tested the effects of daily oral gavages with live D. welbionis J115T on metabolism and several hallmarks of obesity, diabetes, inflammation and lipid metabolism in obese/diabetic mice. RESULTS: This newly identified bacterium was detected in 62.7%-69.8% of the healthy population. Strikingly, in obese humans with a metabolic syndrome, the abundance of Dysosmobacter genus correlates negatively with body mass index, fasting glucose and glycated haemoglobin. In mice, supplementation with live D. welbionis J115T, but not with the pasteurised bacteria, partially counteracted diet-induced obesity development, fat mass gain, insulin resistance and white adipose tissue hypertrophy and inflammation. In addition, live D. welbionis J115T administration protected the mice from brown adipose tissue inflammation in association with increased mitochondria number and non-shivering thermogenesis. These effects occurred with minor impact on the mouse intestinal microbiota composition. CONCLUSIONS: These results suggest that D. welbionis J115T directly and beneficially influences host metabolism and is a strong candidate for the development of next-generation beneficial bacteria targeting obesity and associated metabolic diseases.

25-Hydroxycholesterol metabolism is altered by lung inflammation, and its local administration modulates lung inflammation in mice.

Inflammation is a critical component of many lung diseases including asthma and acute lung injury (ALI). Using high-performance liquid chromatography-mass spectrometry, we quantified the levels of oxysterols in two different murine models of lung diseases. These are lipid mediators derived from cholesterol and known to modulate immunity and inflammation. Interestingly, 25-hydroxycholesterol (25-OHC) was the only oxysterol with altered levels during lung inflammation, and its levels were differently affected according to the model. Therefore, we sought to assess how this oxysterol would affect lung inflammatory responses. In a model of lipopolysaccharide (LPS)-induced acute lung inflammation, 25-OHC levels were increased, and most of the hallmarks of the model (eg, leukocyte recruitment, mRNA expression, and secretion of inflammatory cytokines) were decreased following its intratracheal administration. We also found that, when administered in the lung, 25-OHC is metabolized locally into 25-hydroxycholesterol-3-sulfate and 7α,25-dihydroxycholesterol. Their administration in the lungs did not recapitulate all the effects of 25-OHC. Conversely, in a model of allergic asthma induced by intranasal administration of house dust mites (HDM), 25-OHC levels were decreased, and when intranasally administered, this oxysterol worsened the hallmarks of the model (eg, leukocyte recruitment, tissue remodeling [epithelium thickening and peribranchial fibrosis], and cytokine expression) and induced changes in leukotriene levels. Ex vivo, we found that 25-OHC decreases LPS-induced primary alveolar macrophage activation while having no effect on neutrophil activation. Its sulfated metabolite, 25-hydroxycholesterol-3-sulfate, decreased neutrophil, but not macrophage activation. Taken together, our data support a differential role of 25-OHC in ALI and allergic inflammation models.

Effects of R-flurbiprofen and the oxygenated metabolites of endocannabinoids in inflammatory pain mice models.

Pain is one of the cardinal signs accompanying inflammation. The prostaglandins (PGs), synthetized from arachidonic acid by cyclooxygenase (COX)-2, are major bioactive lipids implicated in inflammation and pain. However, COX-2 is also able to metabolize other lipids, including the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA), to give glycerol ester (PG-G) and ethanolamide (PG-EA) derivatives of the PGs. Consequently, COX-2 can be considered as a hub not only controlling PG synthesis, but also PG-G and PG-EA synthesis. As they were more recently characterized, these endocannabinoid metabolites are less studied in nociception compared to PGs. Interestingly R-profens, previously considered as inactive enantiomers of nonsteroidal anti-inflammatory drugs (NSAIDs), are substrate-selective COX inhibitors. Indeed, R-flurbiprofen can selectively block PG-G and PG-EA production, without affecting PG synthesis from COX-2. Therefore, we compared the effect of R-flurbiprofen and S-flurbiprofen in models of inflammatory pain triggered by local administration of lipopolysaccharides (LPS) and carrageenan in mice. Remarkably, the effects of flurbiprofen enantiomers on mechanical hyperalgesia seem to depend on (i) the inflammatory stimuli, (ii) the route of administration, and (iii) the timing of administration. We also assessed the effect of administration of the PG-Gs, PG-EAs, and PGs on LPS-induced mechanical hyperalgesia. Our data support the interest of studying the nonhydrolytic endocannabinoid metabolism in the context of inflammatory pain.

Identification of new enterosynes using prebiotics: roles of bioactive lipids and mu-opioid receptor signalling in humans and mice.

OBJECTIVE: The enteric nervous system (ENS) plays a key role in controlling the gut-brain axis under normal and pathological conditions, such as type 2 diabetes. The discovery of intestinal actors, such as enterosynes, able to modulate the ENS-induced duodenal contraction is considered an innovative approach. Among all the intestinal factors, the understanding of the role of gut microbes in controlling glycaemia is still developed. We studied whether the modulation of gut microbiota by prebiotics could permit the identification of novel enterosynes. DESIGN: We measured the effects of prebiotics on the production of bioactive lipids in the intestine and tested the identified lipid on ENS-induced contraction and glucose metabolism. Then, we studied the signalling pathways involved and compared the results obtained in mice to human. RESULTS: We found that modulating the gut microbiota with prebiotics modifies the actions of enteric neurons, thereby controlling duodenal contraction and subsequently attenuating hyperglycaemia in diabetic mice. We discovered that the signalling pathway involved in these effects depends on the synthesis of a bioactive lipid 12-hydroxyeicosatetraenoic acid (12-HETE) and the presence of mu-opioid receptors (MOR) on enteric neurons. Using pharmacological approaches, we demonstrated the key role of the MOR receptors and proliferator-activated receptor γ for the effects of 12-HETE. These findings are supported by human data showing a decreased expression of the proenkephalin and MOR messanger RNAs in the duodenum of patients with diabetic. CONCLUSIONS: Using a prebiotic approach, we identified enkephalin and 12-HETE as new enterosynes with potential real beneficial and safety impact in diabetic human.

Diesel exhaust particles alter the profile and function of the gut microbiota upon subchronic oral administration in mice.

BACKGROUND: Ambient air pollution by particulate matters, including diesel exhaust particles (DEP), is a major cause of cardiovascular and metabolic mortality worldwide. The mechanisms by which DEP cause these adverse outcomes are not completely understood. Because the gut microbiota controls cardiovascular and metabolic health, we hypothesized that the fraction of inhaled DEP which reach the gut after mucociliary clearance and swallowing might induce gut dysbiosis and, in turn, contribute to aggravate or induce cardiovascular and metabolic diseases. RESULTS: Female ApoE-/- mice fed a Western diet, and wild-type (C57Bl/6) mice fed standard diet were gavaged with DEP (SRM2975) doses corresponding to mucociliary clearance from inhalation exposure (200 or 1000 ng/day, 3 times a week for 3 months; and 40, 200 or 1000 ng/day, 3 times a week for 6 months, respectively). No mortality, overt systemic or digestive toxicity was observed. A dose-dependent alteration of the gut microbiota was recorded in both strains. In ApoE-/-, ß-diversity was modified by DEP, but no significant modification of the relative abundance of the phyla, families or genera was identified. In C57BL/6 mice, DEP reduced α-diversity (Shannon and Simpson indices), and modified ß-diversity, including a reduction of the Proteobacteria and Patescibacteria phyla, and an increase of the Campylobacterota phylum. In both mouse models, perturbation of the gut microbiota composition was associated with a dose-dependent reduction of bacterial short chain fatty acids (butyrate and propionate) in cecal content. However, DEP ingestion did not aggravate (ApoE-/-), or induce (C57BL/6 mice) atherosclerotic plaques, and no metabolic alteration (glucose tolerance, resistance to insulin, or lipidemia) was recorded. CONCLUSIONS: We show here that oral exposure to DEP, at doses relevant for human health, changes the composition and function of the gut microbiota. These modifications were, however, not translated into ultimate atherosclerotic or metabolic outcomes.

Systemic effects and impact on the gut microbiota upon subacute oral exposure to silver acetate in rats.

CONTEXT: The addition of silver (Ag) to food items, and its migration from food packaging and appliances results in a dietary exposure in humans, estimated to 70-90 µg Ag/day. In view of the well-known bactericidal activity of Ag ions, concerns arise about a possible impact of dietary Ag on the gut microbiota (GM), which is a master determinant of human health and diseases. Repeated oral administration of Ag acetate (AgAc) can also cause systemic toxicity in rats with reported NOAELs of 4 mg AgAc/b.w./d for impaired fertility and 0.4 mg AgAc/b.w./d for developmental toxicity. OBJECTIVE: The objective of this study was to investigate whether oral exposure to AgAc can induce GM alterations at doses causing reproductive toxicity in rats. METHODS: Male and female Wistar rats were exposed during 10 weeks to AgAc incorporated into food (0, 0.4, 4 or 40 mg/kg b.w./d), and we analyzed the composition of the GM (α- and ß-diversity). We documented bacterial function by measuring short-chain fatty acid (SCFA) production in cecal content. Ferroxidase activity, a biomarker of systemic Ag toxicity, was measured in serum. RESULTS AND CONCLUSIONS: From 4 mg/kg b.w./d onwards, we recorded systemic toxicity, as indicated by the reduction of serum ferroxidase activity, as well as serum Cu and Se concentrations. This systemic toxic response to AgAc might contribute to explain reprotoxic manifestations. We observed a dose-dependent modification of the GM composition in male rats exposed to AgAc. No impact of AgAc exposure on the production of bacterial SCFA was recorded. The limited GM changes recorded in this study do not appear related to a reprotoxicity outcome.

The α/ß-hydrolase domain 6 inhibitor WWL70 decreases endotoxin-induced lung inflammation in mice, potential contribution of 2-arachidonoylglycerol, and lysoglycerophospholipids.

Lung inflammation plays a crucial role in the pathogenesis of many respiratory diseases that are in need of new therapeutic strategies. Previously, we showed that inhibition of α/ß-hydrolase domain 6 (ABHD6) decreased macrophage activation and exerted anti-inflammatory effects. Therefore, we thought to assess the effects of ABHD6 inhibition in a mouse model of acute lung injury (ALI) induced by intratracheal administration of lipopolysaccharides. ABHD6 inhibition with N-methyl-N-{[3-(4-pyridinyl)phenyl]methyl}-carbamic acid 4'-(aminocarbonyl)(1,1'-biphenyl)-4-yl ester (WWL70) decreases most of the hallmarks of ALI, including neutrophil infiltration, cytokine secretion, and protein extravasation. mRNA expression of proinflammatory markers in the cells recovered in the bronchoalveolar lavage was also decreased. Interestingly, ABHD6 inhibition was more efficient than monoacylglycerol lipase inhibition by 4-nitrophenyl-4-[dibenzo(d)(14)dioxol-5-yl(hydroxy)methyl]piperidine-1-carboxylate. We also studied ABHD6 inhibition on primary alveolar macrophages and neutrophils to explore their potential implication in the effects of ABHD6 inhibition in vivo. Moreover, we quantified by high-performance liquid chromatography-mass spectrometry the levels of reported substrates of ABHD6 [i.e., 2-arachidonoylglycerol (2-AG) and lysophospholipids]. The potential implication of these lipid mediators in the effects of WWL70 was further investigated on primary alveolar macrophages. Taken together, these data support ABHD6 inhibition as an interesting anti-inflammatory strategy in acute lung inflammation and assess the possible contribution of 2-AG and lysophospholipids in the observed effects.-Bottemanne, P., Paquot, A., Ameraoui, H., Alhouayek, M., Muccioli, G. G. The α/ß-hydrolase domain 6 inhibitor WWL70 decreases endotoxin-induced lung inflammation in mice, potential contribution of 2-arachidonoylglycerol, and lysoglycerophospholipids.

Dysosmobacter welbionis gen. nov., sp. nov., isolated from human faeces and emended description of the genus Oscillibacter.

A strictly anaerobic, Gram-stain-negative, non-spore-forming, non-motile, non-pigmented bacterium, strain J115T, was isolated from human faeces. Cells of strain J115T were straight rods, generally 1.8-3.0 µm, but could be up to 18 µm long. Growth occurred below 2 % (w/v) NaCl and 2 % (v/v) bile. Strain J115T produced acid from myo-inositol but not from d-glucose, d-ribose or d-xylose. Butyric acid was the major end-product from myo-inositol. The genomic DNA G+C content was 58.92 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the closest cultivated neighbours of strain J115T were Oscillibacter ruminantium GH1T (95.4 % similarity) and Oscillibacter valericigenes Sjm18-20T (94.1 %). Strain J115T was also related to the not-yet-cultured bacterium Oscillospira guilliermondii(92-93 % similarity). Coherently with the 16S rRNA gene sequence results, the ANI scores don't have units of strain J115T to O. ruminantium GH1T and O. valericigenes Sjm18-20T were 73.37 and 73.24, respectively, while in silico estimations of DNA-DNA hybridization were both 20.4 %, with confidence intervals of 18.2-22.9 % and 18.2-22.8 %, respectively. The major fatty acids were iso-C15 : 0 (24.2 %), C18 : 0 DMA (18.4 %), anteiso-C15 : 0 (15.2 %) and C16 : 0 DMA (7.6 %). No respiratory quinone was detected. Based on phenotypic features and phylogenetic position, it is proposed that this isolate represents a novel species in a new genus, Dysosmobacter welbionis gen. nov., sp. nov. The type strain of Dysosmobacter welbionis is J115T (DSM 106889T=LMG 30601T).

The endogenous bioactive lipid prostaglandin D2-glycerol ester reduces murine colitis via DP1 and PPARγ receptors.

Cyclooxygenase-2 (COX-2) has long been implicated in the pathogenesis of inflammatory bowel diseases (IBDs). COX-2 is mostly known for the production of prostaglandins (PGs) from arachidonic acid. However, it also metabolizes the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide into the less well-studied bioactive lipids PG-glycerol esters (PG-Gs) and PG-ethanolamides (PG-EAs or prostamides). We previously showed that PGD2-G, a product of 2-AG oxygenation by COX-2, has anti-inflammatory effects. Therefore, we used the dextran sulfate sodium (DSS)-induced model of colitis in mice to explore the role of PGD2-G in murine models of IBD. Colon inflammation was assessed using macroscopic and histologic scores, myeloperoxidase activity, and expression of inflammatory mediators by real-time quantitative PCR and ELISA. We also compared the effects of PGD2-G with those of PGD2 and PGD2-EA. Finally, we used receptor antagonists to gain mechanistic insight into the receptors responsible for the observed effects. PGD2-G reduced DSS-induced colitis, but PGD2 and PGD2-EA did not have the same effect. Furthermore, we showed that PGD2-G is an agonist of the PGD2 receptor 1 (DP1) and that some of the effects of PGD2-G were blocked by antagonism of peroxisome proliferator-activated receptor γ and DP1. Therefore, PGD2-G could be one of the products from the COX-2/prostaglandin D synthase axis to exert beneficial effects in colitis.-Alhouayek, M., Buisseret, B., Paquot, A., Guillemot-Legris, O., Muccioli, G. G. The endogenous bioactive lipid prostaglandin D2-glycerol ester reduces murine colitis via DP1 and PPARγ receptors.

Butyricimonas faecalis sp. nov., isolated from human faeces and emended description of the genus Butyricimonas.

A Gram-negative, strictly anaerobic, non-spore forming, non-motile, non-pigmented bacterial strain, designated H184T, was isolated from human faeces. 16S rRNA gene sequence analysis showed that strain H184T represents a member of the genus Butyricimonas. Strain H184T is related to but distinct from Butyricimonasvirosa JCM 15149T and Butyricimonasparavirosa JCM 18677T, with 16S rRNA gene sequence similarities of 96.32 and 96.24 %, respectively. Strain H184T shared 90.50 % hsp60 gene sequence similarity to B. virosa JCM 15149T and B. paravirosa JCM 18677T. Growth occurs between 25 and 42 °C with an optimum at 37 °C. Bile and NaCl concentration range allowing growth are 0-3.75 % and 0-1.8 %, respectively. pH range for growth is 5.5-8. The strain produced propionate as the major end product from glucose. The major cellular fatty acids of strain H184T were iso-C15 : 0 (63.5 %) and iso-C17 : 0 3-OH (12.8%). The major menaquinone of the strain was MK-10 (86 %). DNA G+C content of the isolate H184T was 44.2 mol%. The genome-based comparison between strain H184T and B. virosa JCM 15149T by pairwise average nucleotide identity indicated a clear distinction with a score of 87.22. On the basis of these data, strain H184T represents a novel species of the genus Butyricimonas, for which the name Butyricimonas faecalis sp. nov. is proposed. The type strain of B. faecalis is H184T (DSM 106867T, LMG 30602T).

Membrane cholesterol delays cellular apoptosis induced by ginsenoside Rh2, a steroid saponin.

Saponins exhibit several biological and pharmacological activities, such as antibacterial, anti-inflammatory and anticancer effects. Many studies attribute their activities to their interactions with cholesterol. In this study, we focus on the steroid saponin ginsenoside Rh2, one of the active principles of Panax ginseng root. Some evidence suggests that lipid rafts, defined as nanodomains enriched in cholesterol and sphingolipids, could be involved in the Rh2-induced apoptosis. However, the role of membrane lipids, especially cholesterol, in this process is still poorly understood. Here, we demonstrate that (i) A549, THP-1 and U937 cells are all susceptible to the Rh2-induced apoptosis but to a differential extent and (ii) the cytotoxic effect inversely correlates with the cell membrane cholesterol content. Upon cholesterol depletion via methyl-ß-cyclodextrin, those three cells lines become more sensitive to Rh2-induced apoptosis. Then, focusing on the cholesterol-auxotroph U937 cell line, we showed that Rh2 alters plasma membrane fluidity by compacting the hydrophobic core of lipid bilayer (DPH anisotropy) and relaxing the interfacial packaging of the polar head of phospholipids (TMA-DPH anisotropy). The treatment with Rh2 conducts to the dephosphorylation of Akt and the activation of the intrinsic pathway of apoptosis (loss of mitochondrial membrane potential, caspase-9 and -3 activation). All these features are induced faster in cholesterol-depleted cells, which could be explained by faster cell accumulation of Rh2 in these conditions. This work is the first reporting that membrane cholesterol could delay the activity of ginsenoside Rh2, renewing the idea that saponin cytotoxicity is ascribed to an interaction with membrane cholesterol.

Inulin increases the beneficial effects of rhubarb supplementation on high-fat high-sugar diet-induced metabolic disorders in mice: impact on energy expenditure, brown adipose tissue activity, and microbiota.

Consumption of prebiotics and plant-based compounds have many beneficial health effects through modulation of gut microbiota composition and are considered as promising nutritional strategy for the treatment of metabolic diseases. In the present study, we assessed the separated and combined effects of inulin and rhubarb on diet-induced metabolic disease in mice. We showed that supplementation with both inulin and rhubarb abolished the total body and fat mass gain upon high-fat and high-sucrose diet (HFHS) as well as several obesity-associated metabolic disorders. These effects were associated with increased energy expenditure, lower whitening of the brown adipose tissue, higher mitochondria activity and increased expression of lipolytic markers in white adipose tissue. Despite modifications of intestinal gut microbiota and bile acid compositions by inulin or rhubarb alone, combination of both inulin and rhubarb had minor additional impact on these parameters. However, the combination of inulin and rhubarb increased the expression of several antimicrobial peptides and higher goblet cell numbers, thereby suggesting a reinforcement of the gut barrier. Together, these results suggest that the combination of inulin and rhubarb in mice potentiates beneficial effects of separated rhubarb and inulin on HFHS-related metabolic disease and could be considered as nutritional strategy for the prevention and treatment of obesity and related pathologies.

"To brain or not to brain": evaluating the possible direct effects of the satiety factor oleoylethanolamide in the central nervous system.

Introduction: Oleoylethanolamide (OEA), an endogenous N-acylethanolamine acting as a gut-to-brain signal to control food intake and metabolism, has been attracting attention as a target for novel therapies against obesity and eating disorders. Numerous observations suggested that the OEA effects might be peripherally mediated, although they involve central pathways including noradrenergic, histaminergic and oxytocinergic systems of the brainstem and the hypothalamus. Whether these pathways are activated directly by OEA or whether they are downstream of afferent nerves is still highly debated. Some early studies suggested vagal afferent fibers as the main route, but our previous observations have contradicted this idea and led us to consider the blood circulation as an alternative way for OEA's central actions. Methods: To test this hypothesis, we first investigated the impact of subdiaphragmatic vagal deafferentation (SDA) on the OEA-induced activation of selected brain nuclei. Then, we analyzed the pattern of OEA distribution in plasma and brain at different time points after intraperitoneal administration in addition to measuring food intake. Results: Confirming and extending our previous findings that subdiaphragmatic vagal afferents are not necessary for the eating-inhibitory effect of exogenous OEA, our present results demonstrate that vagal sensory fibers are also not necessary for the neurochemical effects of OEA. Rather, within a few minutes after intraperitoneal administration, we found an increased concentration of intact OEA in different brain areas, associated with the inhibition of food intake. Conclusion: Our results support that systemic OEA rapidly reaches the brain via the circulation and inhibits eating by acting directly on selected brain nuclei.

Dual-drug loaded nanomedicine hydrogel as a therapeutic platform to target both residual glioblastoma and glioma stem cells.

Glioblastoma (GBM) recurrences are inevitable, and mainly originate from residual tumor cells and the presence of glioma stem cells (GSC) around the resection cavity borders. We previously showed that the local treatment of GBM with nanomedicine-based Lauroyl-gemcitabine lipid nanocapsules (GemC12-LNC) hydrogel delayed tumor onset in various preclinical models and can be used as a scaffold to deliver multiple drugs. However, it does not inhibit tumor relapse in the long-term. In this work, we aim at encapsulating an anti-GSC molecule in the GemC12-LNC hydrogel to eliminate both GBM cells and GSC. We performed a screening on GBM cell lines (GL261 and U-87 MG) and patient-derived GSC (GBM9) to select the anti-GSC molecule that could act synergically with GemC12. Based on our results, salinomycin (Sal) and curcumin (Cur) were selected for further development. Both GemC12-Sal-LNC and GemC12-Cur LNC showed similar size (55 nm), zeta potential (- 2 mV) and viscoelastic properties compared to the GemC12-LNC hydrogel. Encapsulation efficiency was above 95 %. Moreover, the GemC12-Sal-LNC hydrogel was stable for at least 6 months and released both drugs over 30 days in vitro. Both hydrogels inhibited the growth of GL261 and U-87 MG spheroids. Flow cytometry analysis showed that Sal reduced the GSC population in GL261 and U-87 MG cells. Our results show that the co-encapsulation of Sal in the GemC12-LNC hydrogel can reduce both GBM cells and GSC, and therefore might be promising to avoid the onset of GBM recurrences.

Atorvastatin population pharmacokinetics in a real-life setting: Influence of genetic polymorphisms and association with clinical response.

The purpose of this study was to investigate the potential clinical relevance of estimating the apparent clearance (CL/F) of atorvastatin through population pharmacokinetic (PopPK) modeling with samples collected in a real-life setting in a cohort of ambulatory patients at risk of cardiovascular disease by using an opportunistic sampling strategy easily accessible in clinical routine. A total of 132 pharmacokinetic (PK) samples at a maximum of three visits were collected in the 70 included patients. The effects of demographic, genetic, and clinical covariates were also considered. With the collected data, we developed a two-compartment PopPK model that allowed estimating atorvastatin CL/F relatively precisely and considering the genotype of the patient for SLCO1B1 c.521T>C single-nucleotide polymorphism (SNP). Our results indicate that the estimation of the CL/F of atorvastatin through our PopPK model might help in identifying patients at risk of myalgia. Indeed, we showed that a patient presenting a CL/F lower than 414.67 L h-1 is at risk of suffering from muscle discomfort. We also observed that the CL/F was correlated with the efficacy outcomes, suggesting that a higher CL/F is associated with a better drug efficacy (i.e., a greater decrease in total and LDL-cholesterol levels). In conclusion, our study demonstrates that PopPK modeling can be useful in daily clinics to estimate a patient' atorvastatin clearance. Notifying the clinician with this information can help in identifying patients at risk of myalgia and gives indication about the potential responsiveness to atorvastatin therapy.

Tumor Metabolism Is Affected by Obesity in Preclinical Models of Triple-Negative Breast Cancer.

Obesity is characterized by an excessive fat mass accumulation associated with multiple disorders, including impaired glucose homeostasis, altered adipokine levels, and hyperlipidemia. Despite clear associations between tumor progression and obesity, the effects of these disorders on tumor metabolism remain largely unknown. Thus, we studied the metabolic differences between tumors of obese and lean mice in murine models of triple-negative breast cancer (E0771 and PY8819). For this purpose, a real-time hyperpolarized 1-13C-pyruvate-to-lactate conversion was studied before and after glucose administration in fasting mice. This work was completed by U-13C glucose tracing experiments using nuclear magnetic resonance (NMR) spectroscopy, as well as mass spectrometry (MS). Ex vivo analyses included immunostainings of major lipid, glucose, and monocarboxylic acids transporters. On the one hand, we discovered that tumors of obese mice yield higher lactate/pyruvate ratios after glucose administration. On the other hand, we found that the same tumors produce higher levels of lactate and alanine from glucose than tumors from lean mice, while no differences on the expression of key transporters associated with glycolysis (i.e., GLUT1, MCT1, MCT4) have been observed. In conclusion, our data suggests that breast tumor metabolism is regulated by the host's physiological status, such as obesity and diabetes.