Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders.

Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)-collectively Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a "pathological package" capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein-ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.

Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism.

The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213* in svf1 and 167* in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167* in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.

The Glycine Lipids of Bacteroides thetaiotaomicron Are Important for Fitness during Growth In Vivo and In Vitro.

Acylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by the N-acylation of the amino acid, and this is followed by subsequent O-acylation of the acylated molecule, resulting in the production of the mature diacylated amino acid lipid. In this study, we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of a diacylated glycine lipid (GL) species produced in Bacteroides thetaiotaomicron We, and others, have previously reported the identification of a gene, named glsB in this study, that encodes an N-acyltransferase activity responsible for the production of a monoacylated glycine called N-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of the Bacteroidales genomes sequenced so far, the glsB gene is located immediately downstream from a gene, named glsA, that is also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that the coexpression of glsB and glsA results in the production of GL in Escherichia coli We constructed a deletion mutant of the glsB gene in B. thetaiotaomicron, and we confirm that glsB is required for the production of GL in B. thetaiotaomicron Moreover, we show that glsB is important for the ability of B. thetaiotaomicron to adapt to stress and colonize the mammalian gut. Therefore, this report describes the genetic requirements for the biosynthesis of GL, a diacylated amino acid species that contributes to fitness in the human gut bacterium B. thetaiotaomicronIMPORTANCE The gut microbiome has an important role in both health and disease of the host. The mammalian gut microbiome is often dominated by bacteria from the Bacteroidales, an order that includes Bacteroides and Prevotella In this study, we have identified an acylated amino acid, called glycine lipid, produced by Bacteroides thetaiotaomicron, a beneficial bacterium originally isolated from the human gut. In addition to identifying the genes required for the production of glycine lipids, we show that glycine lipids have an important role during the adaptation of B. thetaiotaomicron to a number of environmental stresses, including exposure to either bile or air. We also show that glycine lipids are important for the normal colonization of the murine gut by B. thetaiotaomicron This work identifies glycine lipids as an important fitness determinant in B. thetaiotaomicron and therefore increases our understanding of the molecular mechanisms underpinning colonization of the mammalian gut by beneficial bacteria.

First-in-human controlled inhalation of thin graphene oxide nanosheets to study acute cardiorespiratory responses.

Graphene oxide nanomaterials are being developed for wide-ranging applications but are associated with potential safety concerns for human health. We conducted a double-blind randomized controlled study to determine how the inhalation of graphene oxide nanosheets affects acute pulmonary and cardiovascular function. Small and ultrasmall graphene oxide nanosheets at a concentration of 200 µg m-3 or filtered air were inhaled for 2 h by 14 young healthy volunteers in repeated visits. Overall, graphene oxide nanosheet exposure was well tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected irrespective of graphene oxide particle size. Highly enriched blood proteomics analysis revealed very few differential plasma proteins and thrombus formation was mildly increased in an ex vivo model of arterial injury. Overall, acute inhalation of highly purified and thin nanometre-sized graphene oxide nanosheets was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures at a clinical setting for risk assessment of graphene oxide, and lay the foundations for investigating the effects of other two-dimensional nanomaterials in humans. Clinicaltrials.gov ref: NCT03659864.

Surviving Starvation: Proteomic and Lipidomic Profiling of Nutrient Deprivation in the Smallest Known Free-Living Eukaryote.

Marine phytoplankton, comprising cyanobacteria, micro- and pico-algae are key to photosynthesis, oxygen production and carbon assimilation on Earth. The unicellular green picoalga Ostreococcus tauri holds a key position at the base of the green lineage of plants, which makes it an interesting model organism. O. tauri has adapted to survive in low levels of nitrogen and phosphorus in the open ocean and also during rapid changes in the levels of these nutrients in coastal waters. In this study, we have employed untargeted proteomic and lipidomic strategies to investigate the molecular responses of O. tauri to low-nitrogen and low-phosphorus environments. In the absence of external nitrogen, there was an elevation in the expression of ammonia and urea transporter proteins together with an accumulation of triglycerides. In phosphate-limiting conditions, the expression levels of phosphokinases and phosphate transporters were increased, indicating an attempt to maximise scavenging opportunities as opposed to energy conservation conditions. The production of betaine lipids was also elevated, highlighting a shift away from phospholipid metabolism. This finding was supported by the putative identification of betaine synthase in O. tauri. This work offers additional perspectives on the complex strategies that underpin the adaptive processes of the smallest known free-living eukaryote to alterations in environmental conditions.

Elevated Fibroblast growth factor 21 (FGF21) in obese, insulin resistant states is normalised by the synthetic retinoid Fenretinide in mice.

Fibroblast growth factor 21 (FGF21) has emerged as an important beneficial regulator of glucose and lipid homeostasis but its levels are also abnormally increased in insulin-resistant states in rodents and humans. The synthetic retinoid Fenretinide inhibits obesity and improves glucose homeostasis in mice and has pleotropic effects on cellular pathways. To identify Fenretinide target genes, we performed unbiased RNA-seq analysis in liver from mice fed high-fat diet ± Fenretinide. Strikingly, Fgf21 was the most downregulated hepatic gene. Fenretinide normalised elevated levels of FGF21 in both high-fat diet-induced obese mice and in genetically obese-diabetic Leprdbmice. Moreover, Fenretinide-mediated suppression of FGF21 was independent of body weight loss or improved hepatic insulin sensitivity and importantly does not induce unhealthy metabolic complications. In mice which have substantially decreased endogenous retinoic acid biosynthesis, Fgf21 expression was increased, whereas acute pharmacological retinoid treatment decreased FGF21 levels. The repression of FGF21 levels by Fenretinide occurs by reduced binding of RARα and Pol-II at the Fgf21 promoter. We therefore establish Fgf21 as a novel gene target of Fenretinide signalling via a retinoid-dependent mechanism. These results may be of nutritional and therapeutic importance for the treatment of obesity and type-2 diabetes.

Fenretinide mediated retinoic acid receptor signalling and inhibition of ceramide biosynthesis regulates adipogenesis, lipid accumulation, mitochondrial function and nutrient stress signalling in adipocytes and adipose tissue.

Fenretinide (FEN) is a synthetic retinoid that inhibits obesity and insulin resistance in high-fat diet (HFD)-fed mice and completely prevents 3T3-L1 pre-adipocyte differentiation. The aim of this study was to determine the mechanism(s) of FEN action in 3T3-L1 adipocytes and in mice. We used the 3T3-L1 model of adipogenesis, fully differentiated 3T3-L1 adipocytes and adipose tissue from HFD-induced obese mice to investigate the mechanisms of FEN action. We measured expression of adipogenic and retinoid genes by qPCR and activation of nutrient-signalling pathways by western blotting. Global lipid and metabolite analysis was performed and specific ceramide lipid species measured by liquid chromatography-mass spectrometry. We provide direct evidence that FEN inhibits 3T3-L1 adipogenesis via RA-receptor (RAR)-dependent signaling. However, RARα antagonism did not prevent FEN-induced decreases in lipid levels in mature 3T3-L1 adipocytes, suggesting an RAR-independent mechanism. Lipidomics analysis revealed that FEN increased dihydroceramide lipid species 5- to 16-fold in adipocytes, indicating an inhibition of the final step of ceramide biosynthesis. A similar blockade in adipose tissue from FEN-treated obese mice was associated with a complete normalisation of impaired mitochondrial ß-oxidation and tricarboxylic acid cycle flux. The FEN catabolite, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-OXO), also decreased lipid accumulation without affecting adipogenesis. FEN and 4-OXO (but not RA) treatment additionally led to the activation of p38-MAPK, peIF2α and autophagy markers in adipocytes. Overall our data reveals FEN utilises both RAR-dependent and -independent pathways to regulate adipocyte biology, both of which may be required for FEN to prevent obesity and insulin resistance in vivo.