bluuebunny/crossref_metadata_2025_split huggingface dataset:
Elucidating the Biosynthetic Pathway of Rhodoquinone in Caenorhabditis elegans
Authors: Babcock Shannon , Shepherd Jennifer 
Published: November 2023

Parasitic helminthes are worm‐like, obligate endoparasites that can use humans and livestock as their definitive host. The WHO reports about 24% of the world population is infected with a soil‐transmitted parasitic helminth, especially in tropical, third‐world countries where sanitation and hygiene are poor. These infections can cause economical and sociological effects along with negative or even detrimental health effects on the host. There are currently anti‐helminthic drugs available; however, the increased resistance to those drugs and lack of specificity has called for a new drug target. Parasitic helminthes utilize the fumarate reductase pathway for anaerobic respiration while inside of the host. Rhodoquinone (RQ), the final electron transporter in this pathway, is a potential new target for anti‐helminthic drugs because it is required for metabolism while in the host, and humans don't utilize or make RQ. However, the biosynthetic pathway of RQ is not currently known. This study aimed to elucidate the biosynthetic pathway of RQ in the model organism, Caenorhabditis elegans, using RNA interference (RNAi) to knockdown four potential genes involved in the pathway. C. elegans was used as the model organism because of the similar physiology and close phylogeny to parasitic helminthes. Liquid chromatography‐mass spectrometry (LC‐MS) and real time quantitative polymerase chain reaction (RT‐qPCR) were used to quantify RQ levels in C. elegans after RNAi feeding experiments.Support or Funding InformationNational Institutes of Health (1R15GM096398‐01) and Howard Hughes Medical Institute (award to Gonzaga University)
Lactotransferrin Prevents Hepatic Steatosis through Inhibition of Hepatic Dietary Lipid Uptake in Hormone Disturbance Non‐alcoholic Fatty Liver Disease Model
Authors: Lee Sungmin , Son Beomseok , Park Gaeul , Kim Wanyeon , Youn HyeSook , Youn BuHyun 
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a globally emerging metabolic disease which even could develop to hepatic cirrhosis and hepatoma. Although many studies suggested the mechanism and therapeutic targets, development of NAFLD is not completely understood. In this study, we established IR (Ionizing radiation)‐induced fatty liver mouse model which is observed to develop hepatic lipid accumulation, an initial step of the NAFLD without alteration of caloric intake. Through transcriptome analysis, we found that hepatic secretory protein lactotransferrin (Ltf) was down‐regulated in the model and expression of Ltf was modulated by growth hormone (GH). IR‐induced fatty liver mouse showed lower serum GH levels. Moreover, we investigated the role of Ltf in prevention of chylomicron uptake and lipid accumulation in the hepatocytes. Notably, we uncovered that coumestrol as a stimulant for expression of hepatic Ltf and therapeutic compound of fatty liver. Taken together, we suggest the novel fatty liver mouse model, the role of Ltf in inhibiting hepatic lipid accumulation and novel natural compound for improvement of NAFLD.Support or Funding InformationThe Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (20131610101840)
Niclosamide blocks glucagon phosphorylation of serine 552 on β‐catenin leading to decrease target genes expression and improve glucose metabolism via PKA signalling pathway
Authors: Chowdhury Md. Kamrul Hasan , Morris Margaret , Turner Nigel , Wu Lindsay , Shepherd Peter , Smith Greg 
Published: November 2023

It has been found that glucagon is able to activate the β‐catenin signalling pathway leading to increased cyclin D1 and c‐Myc expression in liver. Therefore, the main aim of this study is to determine if the effect of glucagon activating β‐catenin signalling leading to increased target gene expression is mediated through cAMP activation of protein kinase A. Primary rat hepatocytes were incubated with insulin, glucagon or epinephrine and a range of inhibitors of PI 3‐kinase, Wnt, mitochondrial uncoupler (niclosamide) or PKA inhibitors to dissection out the pathway leading to increased serine 552 phosphorylation of β‐catenin following glucagon exposure. In primary rat liver cells, we found that short exposure of glucagon or epinephrine caused a rapid increase in serine 552 phosphorylation on β‐catenin that leads to increased cyclin D1 and c‐Myc expression. Both glucose and insulin had no effect on this pathway. A range of PI 3‐kinase and Wnt inhibitors were unable to block the effect of glucagon phosphorylating β‐catenin. Interestingly, both niclosamide and the PKA inhibitor H89 blocked the glucagon effect on β‐catenin signalling leading to a reduction in the target genes expression. Our study also provide evidence that inhibition of the glucagon signalling pathway contributes to the beneficial effects of niclosamide (both forms of niclosamide) on whole body glucose metabolism. We have identified a new pathway via glucagon signalling that leads to increased β‐catenin activity that can be reversed with the antihelminthic drug niclosamide, and decrease blood glucose levels, which has recently shown promise as a potential treatment of type 2 diabetes (T2D). This novel finding could be a useful adjunctive therapeutic strategy to treat T2D, as hepatic glucose output is elevated in people with T2D and in liver cancer treatment, particularly in the context of T2D with increased β‐catenin activity.Support or Funding Information

‐ UNSW Australia, PhD scholarship
‐ Diabetes Australian Research Grant
Potential role of dietary fat‐ and obesity‐sensitive adipose PKCbeta signaling in pathophysiology of adipose dysfunction
Authors: Mehta Neil K , Huang Wei 
Published: November 2023

Excess delivery of nutrients to adipose tissue in obesity results in an increase in adipose tissue mass, followed by an increase in stress signals, and thereby an altered production of proinflammatory adipokines, ultimately leading to the progression of chronic inflammation and development of insulin‐resistance. Although the features of chronic inflammation in obese adipose tissue are clearly defined, the signals and mechanisms that trigger chronic inflammation (origin or mechanism of initiation of inflammatory signaling) and maintenance are not well understood and remains a critical area of research. There is clear evidence that the inflammatory trigger is metabolic and a strong correlation between adipocyte size and macrophage infiltration has also been reported. Importantly, specialized metabolic cells (such as adipocytes) are the cells that sustain the insult and whose expansion begins the inflammatory program. Accordingly, adipocyte hypertrophy and local hypoxia are implicated in immune cell infiltration, cytokine secretion and disruption of different signaling cascades. Our recent study suggested that protein kinase C beta (PKC beta) might be a pivotal protein regulating triglyceride storage and could be a target for the pathogenesis of adipose dysfunction in response to nutritional changes. Accordingly, a systemic PKC beta deficiency protects mice from diet‐induced adiposity and development of insulin‐resistance. To determine how PKC beta deficiency exerts its effect, we determined high‐fat diet‐PKC beta interaction and the impact of PKC beta deficiency on relevant adipose signaling pathways and metabolism. Here, we report that dietary fat strongly increased adipose PKC beta expression in a time‐dependent manner. The induction was observed at 2–6 weeks, peaked at 10 weeks, and remained elevated for at least 16 weeks (the last time point measured). We and others have reported involvement of PKC beta in regulating phosphorylation of retinoblastoma, I kappa b kinase, insulin‐receptor substrate, and p66shc, as well as dictates autophagy levels, we therefore compared changes in phosphorylation of these proteins and autophagy levels in different adipose tissues of above mice. The time‐dependent changes were also compared using adipose tissues from PKC beta‐deficient mice fed same high‐fat diet for identical periods. The available results support the possibility that adipocyte PKC beta is a key sensor to detect dangerous dietary signal, whose activation initiates adipose inflammation by sequential events consisting of PKC beta‐induced changes, thus mediating the interface between metabolic input and inflammatory output in the expanding adipose tissue. This study opens the possibility that modulation of PKC beta activity in the adipose tissue may be an effective approach to attenuate adipose dysfunction.Support or Funding InformationHL079091
Mutational analysis of the Arabidopsis thaliana lysoglycerophospholipid acyltransferase AT1g78690
Authors: Nag Saparja , Garrett Teresa A. 
Published: November 2023

Headgroup‐acylated glycerophospholipids (HAGPLs) are a class of poorly understood lipids responsible for supporting cell division and maintenance of the outer cell membrane in Escherichia coli. Overexpression of AT1g78690 in E. coli results in the accumulation of the HAGPL acylphosphatidylglycerol (acyl‐PG). However, this peripheral membrane protein, native to Arabidopsis thaliana, does not directly acylate PG but in vitro is a lysophospholipid acyltransferase that catalyzes the formation of di‐acylated glycerophospholipids, PE or PG, from mono‐acylated glycerophospholipid substrates, 1‐acyl lyso PE or 1‐acyl lyso PG respectively. AT1g78690 bears 39% and 41% sequence identity to human and Drosophila tafazzin, respectively, a monolysocardiolipin remodeling enzyme implicated in Barth Syndrome. Mutations to tafazzin are implicated in altered cardiolipin metabolism found in Barth Syndrome patients. To understand the catalytic and kinetic properties of AT1g78690, several amino acids that are 100% conserved among members of the lysophospholipid acyltransferase (LPLAT) superfamily, threonine‐63, asparagine‐66, histidine‐67, aspartate‐72, and arginine‐92 were targeted for mutation to alanine. Histidine‐67 and arginine‐92 are particularly interesting because mutation of analogous amino acids in tafazzin are associated with Barth Syndrome. Following cloning into pET28a for overexpression in E. coli, membranes will be isolated and used as the enzyme source for a thin layer chromatography based in vitro enzyme assay that follows the transfer of radiolabeled palmitate from palmitoyl Co‐A to lyso PE to form PE. Kinetic parameters, including the Michaelis‐Menten constant (Km) and catalytic efficiency (kcat) as well as substrate specificity will be examined to better understand the catalytic mechanism of AT1g78690 and its homologs.Support or Funding InformationNational Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463
Saccharomyces cerevisiae Coq10, A Putative START Domain Protein Binds Coenzyme Q And Late‐Stage Q‐Biosynthetic Intermediates
Authors: Tsui Hui , Clarke Catherine 
Published: November 2023

Coenzyme Q (ubiquinone or Q) functions as an essential redox‐active lipid in respiratory electron and proton transport in cellular energy metabolism, and is an important lipid‐soluble antioxidant in cellular membranes. In Saccharomyces cerevisiae, at least 13 nuclear‐encoded genes COQ1‐COQ11, YAH1, and ARH1 have been identified as necessary for Q biosynthesis and its function in respiration and as an antioxidant. Many of the Coq polypeptides responsible for Q biosynthesis assemble into a high molecular weight, multi‐subunit complex localized to the matrix side of the mitochondrial inner membrane. This study focuses on characterizing the Coq10 polypeptide, a putative steroidogenic acute regulatory (StAR)‐related lipid transfer (StART) domain protein in S. cerevisiae. Unlike Q‐less coq1‐coq9 deletion mutants, yeast coq10 null mutants contain near wild‐type steady‐state levels of Q6, but are nonetheless respiratory deficient. Yeast coq10 null mutants show decreased de novo Q biosynthesis and accumulate high levels of the early Q‐intermediates, especially during early log phase of growth. The structure of Caulobacter crescentus CC1736, an ortholog of yeast Coq10, contains a hydrophobic tunnel characteristic of StART domain proteins. Purified CC1736 is capable of binding Q and a farnesylated analog of a late‐stage Q biosynthetic intermediate. Overexpression of CC1736 in the coq10 null mutant is able to rescue the respiratory‐deficient phenotype and partially restore efficient Q biosynthesis, suggesting a strong functional conservation. These observations suggest the Coq10 StART domain protein is required for efficient Q biosynthesis, crucial for Q function in respiratory electron transport, and for Q to function as an antioxidant. Coq10 is speculated to be a Q‐binding protein interacting with the multi‐subunit Coq polypeptide complex and/or respiratory complexes, facilitating final delivery of Q from its site of synthesis to sites of function. In order to better understand the biological function of yeast Coq10 we describe the isolation and characterization of the high molecular weight Coq10 oligomer and are characterizing lipid ligand interactions with mature Coq10. The results will provide important clues about the physiological role of Coq10 as well as the molecular interactions required for its function. This research was supported by NSF MCB‐1330803 and Ruth L. Kirschstein National Research Service Award GM007185.Support or Funding InformationThis research was supported by NSF MCB‐1330803 and Ruth L. Kirschstein National Research Service Award GM007185.
Elevated Uptake of Glycerophosphoinositol Through the Git1 Permease Causes Cell Growth Inhibition in Saccharomyces cerevisiae
Authors: Ziegler Phillip , Nikiforov Alexiy , Robinson Benjamin , Bakalinsky Alan , O'Donnell Allyson , Patton‐Vogt Jana 
Published: November 2023

The Git1 permease is responsible for the transport of the phospholipid metabolites glycerophosphoinositol (GPI) and glycerophosphocholine (GPC) in Saccharomyces cerevisiae. GPI is produced via the deacylation of phosphatidylinositol (PI), primarily by the phospholipase Plb3. Extracellular GPI is then transported into the cell via Git1 under phosphate or inositol limiting conditions, where it is catabolized to form inositol and glycerol‐3‐phosphate. Inside the cell, the inositol can then be utilized to produce PI. Unpublished data suggests that the α‐arrestins Aly1 and Aly2 regulate the endocytosis of the Git1 permease, as Git1 is retained at the cell surface in an aly1Δaly2Δ mutant. Importantly, provision of the Git1 substrate GPI to aly1Δaly2Δ cells constitutively expressing GIT1 results in growth inhibition. This inhibition is not observed when the other Git1 substrates, GPC and glycerol‐3‐phosphate (G3P), are provided. We are identifying proteins that are involved in this growth inhibition by GPI. To this end, tools have been developed to employ the Yeast Genomic Tiling Collection (a previously constructed library containing >95% of the functional yeast genome in 1,588 plasmids) to screen for genes that restore cell growth upon overexpression. S. cerevisiae cells constitutively expressing GIT1 and containing the tiling library will be screened for growth in the presence of an inhibitory concentration of GPI. In total, our results suggest a novel role for GPI in growth regulation.Support or Funding InformationNational Institute of Health Grant NIH R15 GM104876
IDENTIFICATION OF INHIBITORS OF ACSVL3, A THERAPEUTIC TARGET IN GLIOMA
Authors: Clay Emily , Shi Xiaohai , Liu Yanqiu , DiRusso Concetta C. , Black Paul N. , Watkins Paul A. 
Published: November 2023

The objective of this study was to identify inhibitors of the enzyme Very Long Chain Acyl‐CoA Synthetase 3 (ACSVL3; SLC27A3). ACSVL3 is present in human glioma cells but not healthy glial cells, and knocking out ACSVL3 in the U87 human glioblastoma cell line has previously been shown to result in slower growth rate, fewer and smaller tumors formed in xenografts, and disrupted Akt signaling. Therefore, an inhibitor of its activity is desired for both understanding its mechanism of action in tumorigenesis and for potential chemotherapeutic usage. Several drugs and compounds that were previously shown to inhibit the homologous enzyme ACSVL1 (SLC27A2) were studied to see if they had similar inhibitory effects on ACSVL3. COS‐1 cells stably expressing ACSVL1 or ACSVL3 were harvested and cell pellets assayed for activation of [1‐14C]stearic acid (C18:0, a preferred ACSVL3 substrate) to its CoA derivative in the absence and presence of inhibitors. We also developed a fluorescence‐based assay using COS‐1 cells stably expressing a chimeric ACSVL1/3 protein containing the regulatory domain of ACSVL1 and the catalytic domain of ACSVL3. The regulatory domain of ACSVL1 conferred the ability to uptake the fluorescent fatty acid analog, C1‐BODIPY‐C12, in a process requiring functional ACSVL3 enzyme activity. Most ACSVL1 inhibitors tested also inhibited ACSVL3 using in vitro acyl‐CoA synthetase assays. These compounds inhibited BODIPY fatty acid uptake in COS‐1 cells expressing chimeric ACSVL1/3 in a dose‐dependent manner. One compound, Grassofermata/CB5, was a significantly more potent inhibitor of stearic acid activation by ACSVL3 than by ACSVL1 and was tested further. CB5 was the most potent inhibitor of stearic acid activation in U87 glioma cells. To assess effects on cell growth, CB5 was added to U87 cells in culture. At a concentration of 3 μM, CB5 drastically slowed cell growth rate but did not lead to increased cell mortality. The effects of CB5 on cell growth were reversible; when CB5 was removed from cells after several days of treatment, they reverted to a faster growth rate. A dose response curve of cell growth to CB5 concentration revealed a narrow therapeutic window. In conclusion, many known inhibitors of ACSVL1 are also potent inhibitors of ACSVL3, and the small molecule CB5 shows promise as a potential chemotherapeutic drug and tool to understand the mechanism(s) underlying the role of ACSVL3 in glioma.Support or Funding InformationKennedy Krieger institutional funds.
Dual Site Mechanism Governs Sphingosine Kinase 1 Membrane Binding and Biological Function
Authors: Pulkoski‐Gross Michael , Truman Jean‐Philip , Salama Mohamed , Clarke Christopher , Hannun Yusuf A. , Obeid Lina 
Published: November 2023

Sphingosine kinase 1 (SK1) is an enzyme at a critical junction in sphingolipid metabolism as it can regulate the balance between the pro‐apoptotic lipids ceramide and sphingosine and the pro‐survival lipid sphingosine 1‐phosphate. Here we identify a novel membrane binding motif in SK1 which is responsible for interaction with anionic phospholipids in the membrane, such as phosphatidic acid (PA). We believe that this newly identified site works in conjunction with a previously identified hydrophobic site in order to regulate the biological function of SK1. In vitro biochemical analysis shows that the anionic binding site is responsible for binding to PA. However, both sites are needed to effectively bind to liposomes containing PA. This provides evidence for SK1's binding to membranes being dictated by both sites. In cells, these sites are important for membrane binding as shown in both fractionation experiments as well as membrane localization by confocal imaging. However, this effect isn't limited to biochemical observations as these sites also affect the biology of SK1.It has been shown that SK1 is involved in regulating cell motility in cancer cells including invasion and involvement in the phosphorylation of Ezrin‐Radixin‐Moesin (ERM) proteins. We show that the mutation of either of these sites, in cells that overexpress these mutant SK1s and in which endogenous SK1 is knocked down with CRISPR technology, is sufficient to abrogate invasion of colon cancer HCT116 cells to invade through the Matrigel. Furthermore, when these mutants are combined this dual site mutant is no longer able to result in the phosphorylation of ERM proteins. Finally, membrane disruption with methyl β‐cyclodextrin, induces pit formation and co‐localizes SK1 and Endophilin‐2 as previously shown. However, when either site is mutated this co‐localization no longer happens even in the presence of Endophilin‐2 associated pits showing these sites are important for SK1s function in endocytosis. Altogether, our data suggest that SK1 uses a dual site mechanism to ensure that it can function at membranes as well as find the correct context in which it should be active.Support or Funding InformationThis work was supported by NIH grants P01CA097132 to LMO and YAH as well as a National Research Service Award F31 CA196315 to MPG.
Acyl‐CoA Thioesterase 1 regulates hepatic lipid metabolism and PPAR‐α signaling
Authors: Franklin Mallory P , Sathyanarayan Aishwarya , Mashek Douglas 
Published: November 2023

Peroxisome proliferator‐activated receptor alpha (PPAR‐α) is a major transcription factor that governs the expression of fatty acid oxidation (FAO) and anti‐inflammatory genes during fasting. Acyl‐CoA thioesterase 1 (ACOT1) hydrolyzes acyl‐CoAs to long‐chain free fatty acids and Coenzyme A during fasting. ACOT1 activity seemingly removes acyl‐CoA substrate for mitochondrial FAO and produces long‐chain free fatty acids that could serve as ligands to activate PPAR‐α. The objective of this research was to identify the role of ACOT1 in hepatic fasting lipid metabolism. To test our objectives, we injected C57Bl/6 mice with an adenovirus harboring shRNA targeted to ACOT1 or a scrambled control. Week‐long liver specific knockdown of ACOT1 increased FAO in vivo measured by serum β‐hydroxybutyrate and reduced liver triglyceride. These data correlated to results from in vitro fatty acid tracer studies in which ACOT1 knockdown increased FAO and triglyceride turnover. Despite increased FAO, ACOT1 knockdown reduced the expression of PPAR‐α target genes involved in FAO. In addition, ACOT1 knockdown led to increased expression of inflammatory and oxidative stress genes, due to reduced PPAR‐α dependent anti‐inflammatory signaling. Moreover, in vitro experiments exhibited increased PPAR‐α reporter activity in COS7 cells overexpressing ACOT1. The ability of ACOT1 to increase PPAR‐α activity was dependent on its catalytic activity, suggesting that ACOT1 produces fatty acids that serve as ligands for PPAR‐α. To confirm this theory, mice were supplemented with a synthetic PPAR‐α ligand, Wy‐14643, which rescued the expression of PPAR‐α target genes and mitigated the inflammation and oxidative stress observed in response to ACOT1 knockdown. Importantly, ACOT1 exhibits nuclear localization during prolonged fasting or under β‐adrenergic stimulation, suggesting a local pool of free fatty acids activate PPAR‐α.Together these studies suggest that ACOT1 coordinates the rate of fatty acid oxidation while simultaneously producing fatty acids that activate PPAR‐α, potentially in the nucleus, to protect the liver from inflammation and oxidative stress.Support or Funding InformationNIH/NIDDK T32 Training grant, Minnesota Obesity Training Program Predoctoral Fellowship (DK083250), awarded to Mallory Franklin.