27Oenocytes are an insect cell type having diverse physiological functions ranging 28 from cuticular hydrocarbon (CHC) production to insecticide detoxification that may 29 impact their capacity to transmit pathogens. To develop functional genetic tools to 30 study Anopheles gambiae oenocytes, we have trapped an oenocyte enhancer to 31 create a transgenic mosquito Gal4 driver line that mediates tissue-specific 32 expression. After crossing with UAS-reporter lines, An. gambiae oenocytes are 33 fluorescently tagged through all life stages and demonstrate clearly the two 34 characteristic oenocyte cell-types arising during development. The driver was then 35 used to characterise the function of two oenocyte expressed An. gambiae cyp4g 36 genes through tissue-specific expression of UAS-RNAi constructs. Silencing of 37 cyp4g16 or cyp4g17 caused lethality in pupae of differing timing and penetrance. 38 Surviving cyp4g16 knockdown adults showed increased sensitivity to desiccation. 39 Total cuticular hydrocarbon levels were reduced by approximately 80% or 50% in 40 both single gene knockdowns when assayed in young pupa or surviving adults 41 respectively, indicating both genes are required for complete CHC production in An. 42 gambiae and demonstrate synergistic activity in young pupae. Comparative CHC 43 profiles were very similar for the two knockdowns, indicating overlapping substrate 44 specificities of the two enzymes. Differences were observed for example with 45 reduced abundance of shorter chain CHCs in CYP4G16 knockdowns, and reduction 46 in longer, branched chained CHCs in CYP4G17 knockdown adults. This is the first 47 time that two cyp4gs have both been shown to be required for complete CHC 48 production in an insect. Moreover, the generation of tagged cells and identification of 49 an enhancer region can expediate oenocyte specific transcriptomics. The novel 50 4 driver line can also be used to explore oenocyte roles in pheromone production, 51 mating behaviour and longevity in the malaria mosquito. 52 53 54
IntroductionThe pathogenicity at differing points along the aggregation pathway of many fibril-forming proteins associated with neurodegenerative diseases is unclear. Understanding the effect of different aggregation states of these proteins on cellular processes is essential to enhance understanding of diseases and provide future options for diagnosis and therapeutic intervention.ObjectivesTo establish a robust method to probe the metabolic changes of neuronal cells and use it to monitor cellular response to challenge with three amyloidogenic proteins associated with neurodegenerative diseases in different aggregation states.MethodNeuroblastoma SH-SY5Y cells were employed to design a robust routine system to perform a statistically rigorous NMR metabolomics study into cellular effects of sub-toxic levels of alpha-synuclein, amyloid-beta 40 and amyloid-beta 42 in monomeric, oligomeric and fibrillar conformations.ResultsThis investigation developed a rigorous model to monitor intracellular metabolic profiles of neuronal cells through combination of existing methods. This model revealed eight key metabolites that are altered when neuroblastoma cells are challenged with proteins in different aggregation states. Metabolic pathways associated with lipid metabolism, neurotransmission and adaptation to oxidative stress and inflammation are the predominant contributors to the cellular variance and intracellular metabolite levels. The observed metabolite changes for monomer and oligomer challenge may represent cellular effort to counteract the pathogenicity of the challenge, whereas fibrillar challenge is indicative of system shutdown. This implies that although markers of stress are more prevalent under oligomeric challenge the fibrillar response suggests a more toxic environment.ConclusionThis approach is applicable to any cell type that can be cultured in a laboratory (primary or cell line) as a method of investigating how protein challenge affects signalling pathways, providing additional understanding as to the role of protein aggregation in neurodegenerative disease initiation and progression.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-017-1289-5) contains supplementary material, which is available to authorized users.
The nuclear deubiquitylase BRCA1 associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline BAP1 mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (w-) and a promoter trap (KO) into human mesothelial cells. BAP1w-/KO MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. SILAC-mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1-deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid (TCA) cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in BAP1w-/KO MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in the cancer genome atlas (TCGA) MESO dataset. Elevated ASS1 is also evident by immunohistochemical staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival amongst patients with BAP1-negative/ASS1-expressing tumours. Alterations in arginine metabolism may sensitise cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, BAP1w-/KO MeT5A become desensitised to arginine-deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines. Implications: Our data reveal an inter-relationship between BAP1 and arginine metabolism, providing a potential means of identifying epithelioid MPM patients likely to benefit from ADI-PEG20.
Neutrophils play a key role in the pathophysiology of rheumatoid arthritis (RA) where release of ROS and proteases directly causes damage to joints and tissues. Neutrophil function can be modulated by Janus Kinase (JAK) inhibitor drugs, including tofacitinib and baricitinib, which are clinically effective treatments for RA. However, clinical trials have reported increased infection rates and transient neutropenia during therapy. The subtle differences in the mode of action, efficacy and safety of JAK inhibitors have been the primary research topic of many clinical trials and systematic reviews, to provide a more precise and targeted treatment to patients. The aim of this study was to determine both the differences in the metabolome of neutrophils from healthy controls and people with RA, and the effect of different JAK inhibitors on the metabolome of healthy and RA neutrophils. Isolated neutrophils from healthy controls (HC) (n = 6) and people with RA (n = 7) were incubated with baricitinib, tofacitinib or a pan-JAK inhibitor (all 200 ng/mL) for 2 h. Metabolites were extracted, and 1H nuclear magnetic resonance (NMR) was applied to study the metabolic changes. Multivariate analyses and machine learning models showed a divergent metabolic pattern in RA neutrophils compared to HC at 0 h (F1 score = 86.7%) driven by energy metabolites (ATP, ADP, GTP and glucose). No difference was observed in the neutrophil metabolome when treated with JAK inhibitors. However, JAK inhibitors significantly inhibited ROS production and baricitinib decreased NET production (p < 0.05). Bacterial killing was not impaired by JAK inhibitors, indicating that the effect of JAK inhibitors on neutrophils can inhibit joint damage in RA without impairing host defence. This study highlights altered energy metabolism in RA neutrophils which may explain the cause of their dysregulation in inflammatory disease.
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