Energy Resolved Mass Spectrometry for Interoperable Non-resonant Collisional Spectra in Metabolomics.

In untargeted metabolomics, the unambiguous identification of metabolites remains a major challenge. This requires high-quality spectral libraries for reliable metabolite identification, which is essential for translating metabolomics data into meaningful biological information. Several attempts have been made to generate reproducible product ion spectra (PIS) under a low collision energy (ELab) regime and nonresonant collisional conditions but have not fully succeeded. We examined the ERMS (energy-resolved mass spectrometry) breakdown curves of two lipo-amino acids and showed the possibility to highlight "singular points", called descriptors hereafter (linked to respective ELab depending on the instrument), for each of the monomodal product ion profiles. Using several instruments based on different technologies, the PIS recorded at these specific ELab sites shows remarkable similarities. The descriptors appeared as being independent of the fragmentation mechanisms and can be used to overcome the main instrumental effects that limit the interoperability of spectral libraries. This proof-of-concept study, performed on two particular lipo-amino acids, demonstrates the high potential of ERMS-derived information to determine the instrument-specific ELab at which PIS recorded in nonresonant conditions become highly similar and instrument-independent, thus comparable across platforms. This innovative but straightforward approach could help remove some of the obstacles to metabolite identification in nontargeted metabolomics, putting an end to a challenging chimera.

Alterations of Prostanoid Expression and Intestinal Epithelial Barrier Functions in Ileus.

INTRODUCTION: Intestinal manipulation (IM)-induced inflammation could contribute to postoperative ileus (POI) pathophysiology via the modulation of prostanoid pathways. To identify the prostanoids involved, we aimed to characterize the profile of prostanoids and their synthesis enzyme expression in a murine model of POI and to determine whether the altered prostanoids could contribute to POI. METHODS: Four or 14 h after IM in mice, gastrointestinal (GI) motility and intestinal epithelial barrier (IEB) permeability were assessed in vivo and ex vivo in Ussing chambers. Using high sensitivity liquid chromatography-tandem mass spectrometry, we characterized the tissue profile of polyunsaturated fatty acid metabolites in our experimental model. Finally, we evaluated in vivo the effects of the prostanoids studied upon IM-induced gut dysfunctions. RESULTS: We first showed that 14 h after IM was significantly faster than jejunal transit at 4 h post-IM, although it remained significantly increased compared to the control. In contrast, we showed that IM-induced inflammation increase in jejunum permeability was similar after four and 14 h. We next showed that expression of prostacyclin synthase and hemopoietic prostaglandin-D synthase mRNA and their products were significantly reduced 14 h after IM as compared to controls. Furthermore, 15-deoxy-delta 12,14-Prostaglandin J2 reduced the IM-induced inflammation increase in IEB permeability but had no effect on GI motility. In contrast, PGI2 increased IM-induced IEB permeability and motility dysfunctions. CONCLUSIONS: Arachidonic acid derivative contributes differentially to GI dysfunction in POI. The decrease of 15-deoxy-delta 12,14-Prostaglandin J2 levels induced by IM could contribute to impaired GI dysfunctions in POI and could be considered as putative therapeutic targets to restore barrier dysfunctions associated with POI.

Proenkephalin deletion in hematopoietic cells induces intestinal barrier failure resulting in clinical feature similarities with irritable bowel syndrome in mice.

Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammation-induced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteria-epithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer's patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients.

Dysosmobacter welbionis effects on glucose, lipid, and energy metabolism are associated with specific bioactive lipids.

The newly identified bacterium Dysosmobacter welbionis J115T improves host metabolism in high-fat diet (HFD)-fed mice. To investigate mechanisms, we used targeted lipidomics to identify and quantify bioactive lipids produced by the bacterium in the culture medium, the colon, the brown adipose tissue (BAT), and the blood of mice. In vitro, we compared the bioactive lipids produced by D. welbionis J115T versus the probiotic strain Escherichia coli Nissle 1917. D. welbionis J115T administration reduced body weight, fat mass gain, and improved glucose tolerance and insulin resistance in HFD-fed mice. In vitro, 19 bioactive lipids were highly produced by D. welbionis J115T as compared to Escherichia coli Nissle 1917. In the plasma, 13 lipids were significantly changed by the bacteria. C18-3OH was highly present at the level of the bacteria, but decreased by HFD treatment in the plasma and normalized in D. welbionis J115T-treated mice. The metabolic effects were associated with a lower whitening of the BAT. In the BAT, HFD decreased the 15-deoxy-Δ12,14-prostaglandin J2, a peroxisome proliferator-activated receptor (PPAR-γ) agonist increased by 700% in treated mice as compared to HFD-fed mice. Several genes controlled by PPAR-γ were upregulated in the BAT. In the colon, HFD-fed mice had a 60% decrease of resolvin D5, whereas D. welbionis J115T-treated mice exhibited a 660% increase as compared to HFD-fed mice. In a preliminary experiment, we found that D. welbionis J115T improves colitis. In conclusion, D. welbionis J115T influences host metabolism together with several bioactive lipids known as PPAR-γ agonists.

Identification of bacterial lipopeptides as key players in IBS.

OBJECTIVES: Clinical studies revealed that early-life adverse events contribute to the development of IBS in adulthood. The aim of our study was to investigate the relationship between prenatal stress (PS), gut microbiota and visceral hypersensitivity with a focus on bacterial lipopeptides containing γ-aminobutyric acid (GABA). DESIGN: We developed a model of PS in mice and evaluated, in adult offspring, visceral hypersensitivity to colorectal distension (CRD), colon inflammation, barrier function and gut microbiota taxonomy. We quantified the production of lipopeptides containing GABA by mass spectrometry in a specific strain of bacteria decreased in PS, in PS mouse colons, and in faeces of patients with IBS and healthy volunteers (HVs). Finally, we assessed their effect on PS-induced visceral hypersensitivity. RESULTS: Prenatally stressed mice of both sexes presented visceral hypersensitivity, no overt colon inflammation or barrier dysfunction but a gut microbiota dysbiosis. The dysbiosis was distinguished by a decreased abundance of Ligilactobacillus murinus, in both sexes, inversely correlated with visceral hypersensitivity to CRD in mice. An isolate from this bacterial species produced several lipopeptides containing GABA including C14AsnGABA. Interestingly, intracolonic treatment with C14AsnGABA decreased the visceral sensitivity of PS mice to CRD. The concentration of C16LeuGABA, a lipopeptide which inhibited sensory neurons activation, was decreased in faeces of patients with IBS compared with HVs. CONCLUSION: PS impacts the gut microbiota composition and metabolic function in adulthood. The reduced capacity of the gut microbiota to produce GABA lipopeptides could be one of the mechanisms linking PS and visceral hypersensitivity in adulthood.

Energy-Resolved Ion Mobility Spectrometry: Composite Breakdown Curves for Distinguishing Isomeric Product Ions.

Identification of lipopeptides (LpAA) synthesized from bacteria involves the study of structural characterization. Twenty LpAA have been characterized using commercial tandem high-resolution mass spectrometers in negative electrospray, employing nonresonant excitation in "RF only" collision cells and generally behave identically. However, [LpAA-H]- (AA = Asp or Glu) shows surprising fragmentation pathways, yielding a complementary fatty acid carboxylate and dehydrated amino acid fragment anions. In this study, the dissociation mechanisms of [C12Glu-H]- were determinate using energy-resolved mass spectrometry (ERMS). Product ion breakdown profiles are, generally, unimodal with full width at half-maximum (fwhm) increasing as product ion m/z ratios decrease, except for the two product ions of interest (fatty acid carboxylate and dehydrated glutamate) characterized by broad and composite profiles. Such behavior was already shown for other ions using a custom-built guided ion beam mass spectrometer. In this study, we investigate the meaning of these particular profiles from an ERMS breakdown, using fragmentation mechanisms depending on the collision energy. ERMS on line with ion mobility spectrometry (IMS), here called ER-IMS, provides a way to probe such questions. Broad or composite profiles imply that the corresponding product ions may be generated by two (or more) pathways, resulting in common or isomeric product ion structures. ER-IMS analysis indicates that the fatty acid carboxylate product ion is produced with a common structure through different pathways, while dehydrated glutamate has two isomeric forms depending on the mechanism involved. Drift time values correlate with the calculated collision cross section that confirms the product ion structures and fragmentation mechanisms.

Prenatal stress induces changes in PAR2- and M3-dependent regulation of colon primitive cells.

Prenatal stress is associated with a high risk of developing adult intestinal pathologies, such as irritable bowel syndrome, chronic inflammation, and cancer. Although epithelial stem cells and progenitors have been implicated in intestinal pathophysiology, how prenatal stress could impact their functions is still unknown. We have investigated the proliferative and differentiation capacities of primitive cells using epithelial crypts isolated from colons of adult male and female mice whose mothers have been stressed during late gestation. Our results show that stem cell/progenitor proliferation and differentiation in vitro are negatively impacted by prenatal stress in male progeny. This is promoted by a reinforcement of the negative proliferative/differentiation control by the protease-activated receptor 2 (PAR2) and the muscarinic receptor 3 (M3), two G protein-coupled receptors present in the crypt. Conversely, prenatal stress does not change in vitro proliferation of colon primitive cells in female progeny. Importantly, this maintenance is associated with a functional switch in the M3 negative control of colonoid growth, becoming proliferative after prenatal stress. In addition, the proliferative role of PAR2 specific to females is maintained under prenatal stress, even though PAR2-targeted stress signals Dusp6 and activated GSK3ß are increased, reaching the levels of males. An epithelial serine protease could play a critical role in the activation of the survival kinase GSK3ß in colonoids from prenatally stressed female progeny. Altogether, our results show that following prenatal stress, colon primitive cells cope with stress through sexually dimorphic mechanisms that could pave the way to dysregulated crypt regeneration and intestinal pathologies.NEW & NOTEWORTHY Primitive cells isolated from mouse colon following prenatal stress and exposed to additional stress conditions such as in vitro culture, present sexually dimorphic mechanisms based on PAR2- and M3-dependent regulation of proliferation and differentiation. Whereas prenatal stress reinforces the physiological negative control exerted by PAR2 and M3 in crypts from males, in females, it induces a switch in M3- and PAR2-dependent regulation leading to a resistant and proliferative phenotype of progenitor.

Altered Subpopulations of Red Blood Cells and Post-treatment Anemia in Malaria.

In acute malaria, the bulk of erythrocyte loss occurs after therapy, with a nadir of hemoglobin generally observed 3-7 days after treatment. The fine mechanisms leading to this early post-treatment anemia are still elusive. We explored pathological changes in RBC subpopulations by quantifying biochemical and mechanical alterations during severe malaria treated with artemisinin derivatives, a drug family that induce "pitting" in the spleen. In this study, the hemoglobin concentration dropped by 1.93 G/dl during therapy. During the same period, iRBC accounting for 6.12% of all RBC before therapy (BT) were replaced by pitted-RBC, accounting for 5.33% of RBC after therapy (AT). RBC loss was thus of 15.9%, of which only a minor part was due to the loss of iRBC or pitted-RBC. When comparing RBC BT and AT to normal controls, lipidomics revealed an increase in the cholesterol/phosphatidylethanolamine ratio (0.17 versus 0.24, p < 0.001) and cholesterol/phosphatidylinositol ratio (0.36 versus 0.67, p = 0.001). Using ektacytometry, we observed a reduced deformability of circulating RBC, similar BT and AT, compared to health control donors. The mean Elongation Index at 1.69Pa was 0.24 BT and 0.23 AT vs. 0.28 in controls (p < 0.0001). At 30Pa EI was 0.56 BT and 0.56 AT vs. 0.60 in controls (p < 0.001). The retention rate (rr) of RBC subpopulations in spleen-mimetic microsphere layers was higher for iRBC (rr = 20% p = 0.0033) and pitted-RBC (rr = 19%, p = 0.0031) than for healthy RBC (0.12%). Somewhat surprisingly, the post-treatment anemia in malaria results from the elimination of RBC that were never infected.

Delta opioid receptors on nociceptive sensory neurons mediate peripheral endogenous analgesia in colitis.

BACKGROUND: Inflammatory visceral pain is endogenously controlled by enkephalins locally released by mucosal CD4+ T lymphocytes in mice. The present study aimed at identifying opioid receptor(s) expressed on nociceptive sensory nerves involved in this peripheral opioid-mediated analgesia. METHODS: The peripheral analgesia associated with the accumulation of CD4+ T lymphocytes within the inflamed colonic mucosa was assessed in conditional knockout mice specifically deleted for either of the two opioid receptors for enkephalins (i.e., µ (MOR) and δ (DOR) receptors) in Nav1.8-expressing sensory neurons in the dextran sulfate sodium (DSS)-induced colitis model. RESULTS: Endogenous analgesia is lost in conditional knockout mice for DOR, but not MOR at the later phase of the DSS-induced colitis. The absence of either of the opioid receptors on sensory nerves had no impact on both the colitis severity and the rate of T lymphocytes infiltrating the inflamed colonic mucosa. CONCLUSION: The key role of DOR on primary afferents in relieving intestinal inflammatory pain opens new therapeutic opportunities for peripherally restricted DOR analgesics to avoid most of the side effects associated with MOR-targeting drugs used in intestinal disorders.

Identification of bacterial lipo-amino acids: origin of regenerated fatty acid carboxylate from dissociation of lipo-glutamate anion.

The identification of bacterial metabolites produced by the microbiota is a key point to understand its role in human health. Among them, lipo-amino acids (LpAA), which are able to cross the epithelial barrier and to act on the host, are poorly identified. Structural elucidation of few of them was performed by high-resolution tandem mass spectrometry based on electrospray combined with selective ion dissociations reach by collision-induced dissociation (CID). The negative ions were used for their advantages of yielding only few fragment ions sufficient to specify each part of LpAA with sensitivity. To find specific processes that help structural assignment, the negative ion dissociations have been scrutinized for an LpAA: the N-palmitoyl acyl group linked to glutamic acid (C16Glu). The singular behavior of [C16Glu-H]¯ towards CID showed tenth product ions, eight were described by expected fragment ions. In contrast, instead of the expected product ions due to CONH-CH bond cleavage, an abundant complementary dehydrated glutamic acid and fatty acid anion pair were observed. Specific to glutamic moiety, they were formed by a stepwise dissociation via molecular isomerization through ion-dipole formation prior to dissociation. This complex dissociated by partner splitting either directly or after inter-partner proton transfer. By this pathway, surprising regeneration of deprotonated fatty acid takes place. Such regeneration is comparable to that occurred from dissociation to peptides containing acid amino-acid. Modeling allow to confirm the proposed mechanisms explaining the unexpected behavior of this glutamate conjugate.

Discovery and quantification of lipoamino acids in bacteria.

Improving knowledge about metabolites produced by the microbiota is a key point to understand its role in human health and disease. Among them, lipoamino acid (LpAA) containing asparagine and their derivatives are bacterial metabolites which could have an impact on the host. In this study, our aim was to extend the characterization of this family. We developed a semi-targeted workflow to identify and quantify new candidates. First, the sample preparation and analytical conditions using liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) were optimized. Using a theoretical homemade database, HRMS raw data were manually queried. This strategy allowed us to find 25 new LpAA conjugated to Asn, Gln, Asp, Glu, His, Leu, Ile, Lys, Phe, Trp and Val amino acids. These metabolites were then fully characterized by MS2, and compared to the pure synthesized standards to validate annotation. Finally, a quantitative method was developed by LC coupled to a triple quadrupole instrument, and linearity and limit of quantification were determined. 14 new LpAA were quantified in gram positive bacteria, Lactobacilus animalis, and 12 LpAA in Escherichia coli strain Nissle 1917.

Maternal prebiotic supplementation impacts colitis development in offspring mice.

Background and aims: Maternal diet plays a key role in preventing or contributing to the development of chronic diseases, such as obesity, allergy, and brain disorders. Supplementation of maternal diet with prebiotics has been shown to reduce the risk of food allergies and affect the intestinal permeability in offspring later in life. However, its role in modulating the development of other intestinal disorders, such as colitis, remains unknown. Therefore, we investigated the effects of prebiotic supplementation in pregnant mice on the occurrence of colitis in their offspring. Materials and methods: Offspring from mothers, who were administered prebiotic galacto-oligosaccharides and inulin during gestation or fed a control diet, were subjected to three cycles of dextran sulphate sodium (DSS) treatment to induce chronic colitis, and their intestinal function and disease activity were evaluated. Colonic remodelling, gut microbiota composition, and lipidomic and transcriptomic profiles were also assessed. Results: DSS-treated offspring from prebiotic-fed mothers presented a higher disease score, increased weight loss, and increased faecal humidity than those from standard diet-fed mothers. DSS-treated offspring from prebiotic-fed mothers also showed increased number of colonic mucosal lymphocytes and macrophages than the control group, associated with the increased colonic concentrations of resolvin D5, protectin DX, and 14-hydroxydocosahexaenoic acid, and modulation of colonic gene expression. In addition, maternal prebiotic supplementation induced an overabundance of eight bacterial families and a decrease in the butyrate caecal concentration in DSS-treated offspring. Conclusion: Maternal prebiotic exposure modified the microbiota composition and function, lipid content, and transcriptome of the colon of the offspring. These modifications did not protect against colitis, but rather sensitised the mice to colitis development.

Peripheral Opioid Receptor Blockade Enhances Epithelial Damage in Piroxicam-Accelerated Colitis in IL-10-Deficient Mice.

Mucosal CD4+ T lymphocytes display a potent opioid-mediated analgesic activity in interleukin (IL)-10 knockout mouse model of inflammatory bowel diseases (IBD). Considering that endogenous opioids may also exhibit anti-inflammatory activities in the periphery, we examined the consequences of a peripheral opioid receptor blockade by naloxone-methiodide, a general opioid receptor antagonist unable to cross the blood-brain barrier, on the development of piroxicam-accelerated colitis in IL-10-deficient (IL-10-/-) mice. Here, we show that IL-10-deficient mice treated with piroxicam exhibited significant alterations of the intestinal barrier function, including permeability, inflammation-related bioactive lipid mediators, and mucosal CD4+ T lymphocyte subsets. Opioid receptor antagonization in the periphery had virtually no effect on colitis severity but significantly worsened epithelial cell apoptosis and intestinal permeability. Thus, although the endogenous opioid tone is not sufficient to reduce the severity of colitis significantly, it substantially contributes to the protection of the physical integrity of the epithelial barrier.

PGI2 Inhibits Intestinal Epithelial Permeability and Apoptosis to Alleviate Colitis.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBDs) that encompass both ulcerative colitis and Crohn's disease are a major public health problem with an etiology that has not been fully elucidated. There is a need to improve disease outcomes and preventive measures by developing new effective and lasting treatments. Although polyunsaturated fatty acid metabolites play an important role in the pathogenesis of several disorders, their contribution to IBD is yet to be understood. METHODS: Polyunsaturated fatty acids metabolite profiles were established from biopsy samples obtained from Crohn's disease, ulcerative colitis, or control patients. The impact of a prostaglandin I2 (PGI2) analog on intestinal epithelial permeability was tested in vitro using Caco-2 cells and ex vivo using human or mouse explants. In addition, mice were treated with PGI2 to observe dextran sulfate sodium (DSS)-induced colitis. Tight junction protein expression, subcellular location, and apoptosis were measured in the different models by immunohistochemistry and Western blotting. RESULTS: A significant reduction of PGI2 in IBD patient biopsies was identified. PGI2 treatment reduced colonic inflammation, increased occludin expression, decreased caspase-3 cleavage and intestinal permeability, and prevented colitis development in DSS-induced mice. Using colonic explants from mouse and human control subjects, the staurosporine-induced increase in paracellular permeability was prevented by PGI2. PGI2 also induced the membrane location of occludin and reduced the permeability observed in colonic biopsies from IBD patients. CONCLUSIONS: The present study identified a PGI2 defect in the intestinal mucosa of IBD patients and demonstrated its protective role during colitis.

Uropathogenic E. coli induces DNA damage in the bladder.

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells.

Mas-related G protein-coupled receptors (Mrgprs) - Key regulators of neuroimmune interactions.

Interplay between physiological systems in the body plays a prominent role in health and disease. At the cellular level, such interplay is orchestrated through the binding of specific ligands to their receptors expressed on cell surface. G protein-coupled receptors (GPCR) are seven-transmembrane domain receptors that initiate various cellular responses and regulate homeostasis. In this review, we focus on particular GPCRs named Mas-related G protein-coupled receptors (Mrgprs) mainly expressed by sensory neurons and specialized immune cells. We describe the different subfamilies of Mrgprs and their specific ligands, as well as recent advances in the field that illustrate the role played by these receptors in neuro-immune biological processes, including itch, pain and inflammation in diverse organs.

Endothelin-1 Exhibiting Pro-Nociceptive and Pro-Peristaltic Activities Is Increased in Peritoneal Carcinomatosis.

Background: Peritoneal carcinomatosis often results in alterations in intestinal peristalsis and recurrent abdominal pain. Pain management in these patients is often unsatisfactory. This study aimed to investigate whether endothelin-1 (EDN1) was involved in pain mediation in peritoneal carcinomatosis, and thus whether the EDN1 pathway could be a new therapeutic target for peritoneal carcinomatosis-associated pain. Methods: EDN1 plasma levels and abdominal pain severity were assessed in patients with abdominal tumors, with or without peritoneal carcinomatosis, and in healthy donors. The effects of EDN1 on the visceromotor response to colorectal distension, and on colonic contractions were then examined in mice, and the mechanism of action of EDN1 was then investigated by measuring the impact of EDN1 exposure on calcium mobilization in cultured neurons. Inhibition studies were also performed to determine if the effects of EDN1 exposure could be reversed by EDN1-specific receptor antagonists. Results: A positive correlation between EDN1 plasma levels and abdominal pain was identified in patients with peritoneal carcinomatosis. EDN1 exposure increased visceral sensitivity and the amplitude of colonic contractions in mice and induced calcium mobilization by direct binding to its receptors on sensory neurons. The effects of EDN1 were inhibited by antagonists of the EDN1 receptors. Conclusions: This preliminary study, using data from patients with peritoneal carcinomatosis combined with data from experiments performed in mice, suggests that EDN1 may play a key role mediating pain in peritoneal carcinomatosis. Our findings suggest that antagonists of the EDN1 receptors might be beneficial in the management of pain in patients with peritoneal carcinomatosis.

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.

Cholesterol-rich naked mole-rat brain lipid membranes are susceptible to amyloid beta-induced damage in vitro.

Naked mole-rats are extraordinarily long-lived rodents that offer unique opportunities to study the molecular origins of age-related neurodegenerative diseases. Remarkably, they do not accumulate amyloid plaques, even though their brains contain high concentrations of amyloid beta (Aß) peptide from a young age. Therefore, they represent a particularly favourable organism to study the mechanisms of resistance against Aß neurotoxicity. Here we examine the composition, phase behaviour, and Aß interactions of naked mole-rat brain lipids. Relative to mouse, naked mole-rat brain lipids are rich in cholesterol and contain sphingomyelin in lower amounts and of shorter chain lengths. Proteins associated with the metabolism of ceramides, sphingomyelins and sphingosine-1-phosphate receptor 1 were also found to be decreased in naked mole-rat brain lysates. Correspondingly, we find that naked mole-rat brain lipid membranes exhibit a high degree of phase separation, with the liquid ordered phase extending to 80% of the supported lipid bilayer. These observations are consistent with the 'membrane pacemaker' hypothesis of ageing, according to which long-living species have lipid membranes particularly resistant to oxidative damage. We also found that exposure to Aß disrupts naked mole-rat brain lipid membranes significantly, breaking the membrane into pieces while mouse brain derived lipids remain largely intact upon Aß exposure.

Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets.

BACKGROUND: The hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process. METHODS: Peripheral blood neutrophils were isolated from newborn CFTR+/+ and CFTR-/- piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated. RESULTS: Polyunsaturated fatty acid metabolites analysis did not show any difference between CFTR+/+ and CFTR-/- neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR-/- neutrophils to kill pseudomonas aeruginosa in vitro was observed. CONCLUSIONS: ICM-MS demonstrated that CFTR-/- neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.