FAME4-associating YEATS2 knockdown impairs dopaminergic synaptic integrity and leads to seizure-like behaviours in Drosophila melanogaster.

Familial adult myoclonus epilepsy (FAME) is a neurological disorder caused by a TTTTA/TTTCA intronic repeat expansion. FAME4 is one of the six types of FAME that results from the repeat expansion in the first intron of the gene YEATS2. Although the RNA toxicity is believed to be the primary mechanism underlying FAME, the role of genes where repeat expansions reside is still unclear, particularly in the case of YEATS2 in neurons. This study used Drosophila to explore the effects of reducing YEATS2 expression. Two pan-neuronally driven dsDNA were used for knockdown of Drosophila YEATS2 (dYEATS2), and the resulting molecular and behavioural outcomes were evaluated. Drosophila with reduced dYEATS2 expression exhibited decreased tolerance to acute stress, disturbed locomotion, abnormal social behaviour, and decreased motivated activity. Additionally, reducing dYEATS2 expression negatively affected tyrosine hydroxylase (TH) gene expression, resulting in decreased dopamine biosynthesis. Remarkably, seizure-like behaviours induced by knocking down dYEATS2 were rescued by the administration of L-DOPA. This study reveals a novel role of YEATS2 in neurons in regulating acute stress responses, locomotion, and complex behaviours, and suggests that haploinsufficiency of YEATS2 may play a role in FAME4.

Metabolomic analyses uncover an inhibitory effect of niclosamide on mitochondrial membrane potential in cholangiocarcinoma cells.

Background: Niclosamide is an oral anthelminthic drug that has been used for treating tapeworm infections. Its mechanism involves the disturbance of mitochondrial membrane potential that in turn inhibits oxidative phosphorylation leading to ATP depletion. To date, niclosamide has been validated as the potent anti-cancer agent against several cancers. However, the molecular mechanisms underlying the effects of niclosamide on the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cell functions remain to be elucidated. The aims of this study were to investigate the effects of niclosamide on CCA cell proliferation and on metabolic phenoconversion through the alteration of metabolites associated with mitochondrial function in CCA cell lines. Materials and Methods: The inhibitory effect of niclosamide on CCA cells was determined using SRB assay. A mitochondrial membrane potential using tetramethylrhodamine, ethyl ester-mitochondrial membrane potential (TMRE-MMP) assay was conducted. Liquid chromatography-mass spectrometry-based metabolomics was employed to investigate the global metabolic changes upon niclosamide treatment. ATP levels were measured using CellTiter-Glo® luminescent cell viability assay. NAD metabolism was examined by the NAD+/NADH ratio. Results: Niclosamide strongly inhibited CCA cell growth and reduced the MMP of CCA cells. An orthogonal partial-least square regression analysis revealed that the effects of niclosamide on suppressing cell viability and MMP of CCA cells were significantly associated with an increase in niacinamide, a precursor in NAD synthesis that may disrupt the electron transport system leading to suppression of NAD+/NADH ratio and ATP depletion. Conclusion: Our findings unravel the mode of action of niclosamide in the energy depletion that could potentially serve as the promising therapeutic strategy for CCA treatment.

1H NMR fecal metabolic phenotyping of periductal fibrosis- and cholangiocarcinoma-specific metabotypes defining perturbation in gut microbial-host co-metabolism.

Background: The liver fluke Opisthorchis viverrini (OV), which subsequently inhabits the biliary system and results in periductal fibrosis (PDF), is one of the primarily causes of cholangiocarcinoma (CCA), a bile duct cancer with an exceptionally high incidence in the northeast of Thailand and other Greater Mekong Subregion (GMS) countries. Insights in fecal metabolic changes associated with PDF and CCA are required for further molecular research related to gut health and potential diagnostic biological marker development. Methods: In this study, nuclear magnetic resonance (NMR) metabolomics was applied for fecal metabolic phenotyping from 55 fecal water samples across different study groups including normal bile duct, PDF and CCA groups. Results: By using NMR spectroscopy-based metabolomics, fecal metabolic profiles of patients with CCA or PDF and of individuals with normal bile duct have been established with a total of 40 identified metabolites. Further multivariate statistical analysis and hierarchical clustering heat map have demonstrated the PDF- and CCA-specific metabotypes through various altered metabolite groups including amino acids, alcohols, amines, anaerobic glycolytic metabolites, fatty acids, microbial metabolites, sugar, TCA cycle intermediates, tryptophan catabolism substrates, and pyrimidine metabolites. Compared to the normal bile duct group, PDF individuals showed the significantly elevated relative concentrations of fecal ethanol, glycine, tyrosine, and N-acetylglucosamine whereas CCA patients exhibited the remarkable fecal metabolic changes that can be evident through the increased relative concentrations of fecal uracil, succinate, and 5-aminopentanoate. The prominent fecal metabolic alterations between CCA and PDF were displayed by the reduction of relative concentration of methanol observed in CCA. The metabolic alterations associated with PDF and CCA progression have been proposed with the involvement of various metabolic pathways including TCA cycle, ethanol biogenesis, hexamine pathway, methanol biogenesis, pyrimidine metabolism, and lysine metabolism. Among them, ethanol, methanol, and lysine metabolism strongly reflect the association of gut-microbial host metabolic crosstalk in PDF and/or CCA patients. Conclusion: The PDF- and CCA-associated metabotypes have been investigated displaying their distinct fecal metabolic patterns compared to that of normal bile duct group. Our study also demonstrated that the perturbation in co-metabolism of host and gut bacteria has been involved from the early step since OV infection to CCA tumorigenesis.

The impact of hypoxia and oxidative stress on proteo-metabolomic alterations of 3D cholangiocarcinoma models.

The three-dimensional multicellular spheroid (3D MCS) model has been employed in cholangiocarcinoma research as it generates 3D architecture and includes more physiological relevance with the multicellular arrangement. However, it is also essential to explain the molecular signature in this microenvironment and its structural complexity. The results indicated that poorly differentiated CCA cell lines were unable to form 3D MCS due to the lack of cell adhesion molecules with more mesenchymal marker expression. The well-differentiated CCA and cholangiocyte cell lines were able to develop 3D MCSs with round shapes, smooth perimeter, and cell adhesion molecules that led to the hypoxic and oxidative microenvironment detected. For MMNK-1, KKU-213C, and KKU-213A MCSs, the proteo-metabolomic analysis showed proteins and metabolic products altered compared to 2D cultures, including cell-cell adhesion molecules, energy metabolism-related enzymes and metabolites, and oxidative-related metabolites. Therefore, the 3D MCSs provide different physiological states with different phenotypic signatures compared to 2D cultures. Considering the 3D model mimics more physiological relevance, it might lead to an alternate biochemical pathway, targeting to improve drug sensitivity for CCA treatment.

Bacterial challenge-associated metabolic phenotypes in Hermetia illucens defining nutritional and functional benefits.

Black soldier fly (BSF, Hermetia illucens) is popular for its applications in animal feed, waste management and antimicrobial peptide source. The major advantages of BSF larva include their robust immune system and high nutritional content that can be further developed into more potential agricultural and medical applications. Several strategies are now being developed to exploit their fullest capabilities and one of these is the immunity modulation using bacterial challenges. The mechanism underlying metabolic responses of BSF to different bacteria has, however, remained unclear. In the current study, entometabolomics was employed to investigate the metabolic phenoconversion in response to either Escherichia coli, Staphylococcus aureus, or combined challenges in BSF larva. We have, thus far, characterised 37 metabolites in BSF larva challenged with different bacteria with the major biochemical groups consisting of amino acids, organic acids, and sugars. The distinct defense mechanism-specific metabolic phenotypes were clearly observed. The combined challenge contributed to the most significant metabolic phenoconversion in BSF larva with the dominant metabolic phenotypes induced by S. aureus. Our study suggested that the accumulation of energy-related metabolites provided by amino acid catabolism is the principal metabolic pathway regulating the defense mechanism. Therefore, combined challenge is strongly recommended for raising BSF immunity as it remarkably triggered amino acid metabolisms including arginine and proline metabolism and alanine, aspartate and glutamate metabolism along with purine metabolism and pyruvate metabolism that potentially result in the production of various nutritional and functional metabolites.

Lipidomic Analyses Uncover Apoptotic and Inhibitory Effects of Pyrvinium Pamoate on Cholangiocarcinoma Cells via Mitochondrial Membrane Potential Dysfunction.

Pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, has been validated as a highly potent anti-cancer agent and patented recently as a potential chemotherapeutic drug for various cancers. The aims of this study were, therefore, to investigate the ability of PP in anti-proliferative activity and focused on the lipid profiles revealing the alteration of specific lipid species in the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cells. PP inhibited CCA cell viability through suppressing mitochondrial membrane potential (MMP) and ATP productions, leading to apoptotic cell death. Liquid chromatography-mass spectrometry combined with chemometrics was performed to investigate lipid alteration during PP-induced apoptosis. The lipidomic analyses showed the altered lipid signatures of CCA cell types including S-acetyldihydrolipoamide, methylselenopyruvate, and triglycerides that were increased in PP-treated CCA cells. In contrast, the levels of sphinganine and phosphatidylinositol were lower in the PP-treated group compared with its counterpart. The orthogonal partial-least squares regression analysis revealed that PP-induced MMP dysfunction, leading to remarkably reduced ATP level, was significantly associated with triglyceride (TG) accumulation observed in PP-treated CCA cells. Our findings indicate that PP could suppress the MMP function, which causes inhibition of CCA cell viability through lipid production, resulting in apoptotic induction in CCA cells. These findings provide an anti-cancer mechanism of PP under apoptotic induction ability that may serve as the alternative approach for CCA treatment.

1H NMR metabolic phenotyping of Dipterocarpus alatus as a novel tool for age and growth determination.

Dipterocarpus alatus belongs to Family Dipterocarpaceae that can be commonly found in Southeast Asian countries. It is a perennial plant with oval-shaped leaves and oleoresin-rich wood. It has been considered as a multipurpose plant since all parts can be practically utilized. One of the major problems for utilizing Dipterocarpus alatus is the difficulty knowing the exact age as this kind of plant is ready for multipurpose use after 20 years of age. At present, the most commonly used method for determining age of Dipterocarpus alatus is the annual ring estimation. However, this conventional method is unable to provide the high precision and accuracy of age determination due to its limitation including blurry annual rings caused by enriched oleoresin in the wood. The current study aimed to investigate the differences of 1H -NMR spectroscopy-based metabolic profiles from bark and leaf of Dipterocarpus alatus at different ages including 2, 7, 15 and 25 years. Our findings demonstrated that there is a total of 56 metabolites shared between bark and leaf. It is noticeable that bark at different ages exhibited the strongest variation and sugar or sugar derivatives that were found in higher concentrations in bark compared with those in leaf. We found that decreasing levels of certain metabolites including tagatose, 1'kestose and 2'-fucosyllactose exhibited the promising patterns. In conclusion, panel metabolites involved in the sucrose biosynthesis can precisely determine the age and growth of Dipterocarpus alatus.