bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipid droplet degradation through a novel and direct engulfment by endo‐lysosomes
Authors: Schott Micah B. , Mehall Bridget , Weller Shaun , Schulze Ryan J. , McNiven Mark 
Published: April 2020

Lipid droplets (LDs) are fat‐storage organelles that play critical roles in energy homeostasis, but can excessively accumulate in metabolic diseases such as obesity and fatty liver where hepatocytes become grossly engorged with excess lipid. The catabolism of LDs aids in disease resolution and occurs in part by lipophagy, a form of selective autophagy, but the molecular and vesicle trafficking events that facilitate this process are not fully understood. During lipophagy, autophagosomes are thought to target LDs prior to fusion with acidic lysosomes, where neutral lipids are terminally degraded by lysosomal acid lipase. Ultrastructural viewing of hepatocytes showing LDs contained within electron‐dense acidic vesicles is consistent with this concept. In addition to canonical autophagy, we find that most LDs undergoing degradation are encased within single‐membrane endo‐lysosomes, and few LDs are observed within traditional double‐membrane autophagosomes. Further, studies of LD catabolism in yeast have shown that LDs can be directly internalized by the large acidic vacuole through a process known as microlipophagy. Thus, the GOAL of this study was to test if similar microlipophagy mechanisms occur in mammalian cells such as hepatocytes, and whether this process involves autophagy.METHODSTo test this, we used microscopic fatty acid tracing studies and time‐lapse confocal microscopy to determine the role of autophagosomes versus endo‐lysosomes in LD catabolism. To trace the fate of LDs into terminal degradative vesicles, cells were first loaded with the fluorescent lipid marker Bodipy C12, then treated for 24h with LAListat, an inhibitor of lysosomal acid lipase.RESULTSWe observed a ~5‐fold higher incidence of LDs within endo‐lysosomes (marked with the tetraspanin CD63), compared with autophagosomes (marked by the autophagy protein LC3). Furthermore, siRNA knockdown of macroautophagy proteins (Atg5, Atg7, and Fip200) did not inhibit LD internalization by endo‐lysosomes, suggesting that autophagy is not a prerequisite for this process. In addition, time‐lapse microscopy revealed a direct internalization of cytosolic LDs by endo‐lysosomes positive for lysotracker or CD63‐mCherry.CONCLUSIONThese findings suggest that hepatocytes utilize a distinct form of microautophagy that entails the direct envelopment of cytoplasmic LDs by endocytic vesicles such as multivesicular bodies and lysosomes/late endosomes. In addition to lipolysis and canonical macroautophagy, this process plays an important role in the catabolic utilization of LDs by the hepatocyte.Support or Funding InformationThis study was supported by NIH R01DK044650 (to M.A.M.), RO1AA020735 (to M.A.M.), and the K99AA020735 (to M.B.S.).
Coevolution of secreted proteins and members of the secretory pathway reveals interactions and motifs used for proper secretion
Authors: Shamie Isaac , Lewis Nathan E. 
Published: April 2020

The secretory pathway consists of hundreds of macromolecules that function by physically interacting with and modifying secreted proteins to bring them to their final form and location. These include include chaperones, translocases, and enzymes for post‐translational modifications. Some of these are generic proteins for maintaining homeostasis in the secretory pathway, while others are specific for processing groups or individual secreted proteins. Additionally, many of these machinery come from common families that are conserved across eukaryotes. Understanding how these proteins are able to process thousands of secreted proteins across organisms and cell types is important and still lack fine‐grained details. In this study, we take an evolutionary approach to systematically characterize interactions between secreted proteins and the machinery proteins. Using mutual information and directed coupling analysis, we discover amino acids between pairs of proteins that have coevolved. We use this to predict interactions between proteins and compare them to the literature as well as computationally predicted database PrePPI. Additionally, we discover functional amino acid motifs within this pathway seen across organisms. By examining regions not found in the most conserved regions, this technique can capture previously unknown functional residues. This approach adds to work done in prokaryotes, here showing that interactions can be inferred between proteins in the secretory pathway by studying their amino acid content.Support or Funding InformationNovo Nordisk Foundation
CHANGES IN THE METABOLIC PROFILE OF MDA‐MB231 BREAST CANCER DUE TO LDHA AND LDHB DOUBLE KNOCKOUT
Authors: Mack Nzinga , Soliman Karam , Mazzio Elizabeth 
Published: April 2020

PURPOSETriple Negative Breast Cancer (TNBC) is an aggressive form of cancer that disproportionately affects African Americans and has a high mortality rate with no known cure. All solid tumors display an elevated glycolytic flux accounting for increased levels of lactic acid production, which is believed to be driven by the various isoforms of the enzyme Lactate Dehydrogenase (LDH). LDHA and LDHB are highly expressed in MDA‐MB‐231 cells. Increased lactic acid secretion contributes to significant pro oncogenic malignant and metastatic processes.METHODSEdited MDA‐MB 231 TNBC cells with LDHA and LDHB double gene knockouts were provided by Snynthego from their engineered cell product, knockout cell pool. The effects of LDHA and LDHB double knockouts on genetic parameters in MDA‐MB‐231 cells were evaluated by examining whole transcriptomic influence on mRNAs and long intergenic non‐coding RNA transcripts (lincRNA) using Affymetrix human 2.1‐ST microarrays. Genome studies were complemented by metabolic analysis of mitochondrial function, lactic acid production, glucose consumption and ATP production.RESULTSThe data provide evidence to suggest that there are alternative pathways to the production of lactic acid outside of LDH conversion of pyruvate to lactic acid. The transcriptome analysis shows specific genes that could be involved with an alternative route of lactic acid production in cancer cells.DISCUSSION/CONCLUSIONThere is clearly enough evidence to suggest that acidity drives malignant processes using lactic acid as a means to control pH. Prevention of lactic acid production would be a formidable detriment to survival and metastasis of human malignancies, including TNBC.Support or Funding InformationNIH 2U54MD00758234A1
Mitochondrial function after bisphenol S exposure: a safe substitute for BPA?
Authors: Singh Radha Dutt , Easson Sarah , Thompson Jennifer A. 
Published: April 2020

ObjectiveBisphenol A (BPA) is one of the most common industrial chemicals, detected in the urine of 93 percent of US adults. BPA was declared a toxic substance in 2010 and has been linked to metabolic and cardiovascular disease. Amid growing consumer concern, manufacturers increasingly turned to BPA substitutes, the most common of which is bisphenol S (BPS); however, its safety was not determined before introduction to market. Thus, exposure to BPS is on the rise; but its health effects are unclear. Our aim was to determine the impact of BPS exposure on mitochondrial dynamics in vascular smooth muscle cells.MethodsVascular smooth muscle cells (VSMC) were isolated by enzymatic digestion from the thoracic aorta of juvenile (6–7 week old) male C56BL/6 mice. Cells were cultured in DMEM/F12 media supplemented with 10% fetal bovine serum and 1% penicillin‐streptomycin. At 70–80% confluency, cells were treated with vehicle (DMSO) or variable doses of BPS [2.5nM (levels in drinking water); 250nM (permissible limit) and 25μM]. Reactive oxygen species (ROS) levels from cellular and mitochondrial sources were determined by 2′,7′‐dichlorofluorescin diacetate (DCFDA) and MitoSOX staining, respectively. Membrane integrity was assessed by evaluating mitochondrial membrane potential (MMP) by JC1 staining. Mitochondrial fragmentation was measured by MitoTracker Red CMXROS staining and confocal microscopy. Real‐time PCR will be used to measure the expression of genes involved in mito‐fission and fussion and mitochondrial DNA copy number.ResultsAfter 30 min and 1 hr of BPS exposure, cellular and mitochondrial ROS levels were increased in a dose‐dependent manner, with a more prominent increase at 1 hr (all doses p<0.001). At 1, 12 and 24 hrs after exposure, mitochondrial depolarization was observed at all doses. At 24 hours, the reduction in mitochondrial membrane potential was greatest at the lowest dose (3‐fold vs. vehicle, p<0.0001). MitoTracker Red CMXROS revealed significant mitochondrial fragmentation at 24 hrs after exposure to 25μM BPS.ConclusionMitochondrial function is disrupted in VSMCs exposed to BPS at doses lower than the permissible limit and equivalent to human exposure. Studies are underway to determine the mechanistic basis of BPS‐induced mitochondrial dysfunction and long‐term effects on vascular health.Support or Funding InformationThis study was supported the CIHR, Heart and Stroke Foundation of Canada, and a Kertland Fellowship.
Asperuloside reduces food intake and body weight via downregulation of orexigenic hypothalamic signalling in a mouse model of metabolic syndrome.
Authors: Caruso Vanni , Ishaq Muhammad , Tran Duyen , Yang Cheng , Vicenzi Silvia , Guven Nuri , Deans Bianca , Randall Cameron , Smith Jason , Jacobson Glenn , Ronci Maurizio , Eri Raj , Canales Juan , Iglesias Miguel 
Published: April 2020

Controlling the urge to eat requires adequate integration of hypothalamic neuropeptides regulating energy balance processes with dopaminergic circuits in the mesolimbic pathway. Disruption of this signalling alters the hedonic control of reward‐driven food intake contributing to obesity. Our study aimed to understand the mechanism of action of the novel anti‐obesity compound, asperuloside (ASP), and whether ASP treatment could be used as safe weight loss treatment.Mice were fed with a 45% high fat diet (HFD) and concurrently treated with ASP for 12 weeks. ASP reduced significantly (10.50%, p<0.05) final body weight of HFD mice compared to their control group. The effects of ASP reached significance after 5 weeks of treatment and were maintained throughout the experiment. Over the experimental period, the compound induced a significant daily energy intake reduction (12.8%, p<0.05) in mice consuming HFD compared to their control group. When standardised by body weight, visceral adipose mass was increased by the effect of HFD and significantly reduced by ASP treatment (35%, p<0.05). Orexigenic hypothalamic genes including NPY and ARGP, as well as important receptors involved in the control of food intake such as ghrelin, leptin and cannabinoid receptors, were significantly (p<0.05) downregulated by ASP treatment. These results suggest that ASP might induce weight loss plausibly via downregulation of hypothalamic signalling and support ASP candidacy as a potential and safe drug for the treatment of obesity.
Stepwise autophosphorylation regulates biased agonism of the insulin receptor
Authors: Yunn Na-Oh , Park Mangeun , Ryu Sung Ho 
Published: April 2020

Insulin is a key regulator of energy metabolism in peripheral tissues but also functions as a growth factor. Insulin binding to the insulin receptor (IR) leads to autophosphorylation of intracellular tyrosine residues, which simultaneously initiates a multitude of signals and functions. In contrast, some artificial (non‐insulin) ligands for the IR result in biased agonism, selectively activating the PI3K/AKT pathway and metabolic effects without activating mitogen‐activated protein kinase (MAPK) pathway and mitogenic effects. However, the precise mechanism of biased agonism at the receptor level remains unclear. The biased agonist IR‐A48 aptamer selectively induces mono‐phosphorylation of Tyr1150 residue (m‐pY1150) in the kinase domain of IR. Hence, we hypothesized that IR autophosphorylation is a stepwise process in which formation of m‐pY1150 represents an intermediate step. To explore this idea, we used hybrid receptors in which insulin can bind only one of the two binding sites of the dimeric receptor. Asymmetric insulin binding selectively induced symmetric m‐pY1150 in both kinase domains. Moreover, the juxtamembrane domain, which interacts with the kinase domain, restricted the full activation of IR, and symmetric m‐pY1150 played a crucial role in the rearrangement of intracellular domains to release this restriction. Our findings demonstrate that the symmetry of insulin binding to a dimeric receptor determines the stepwise autophosphorylation of IR. Furthermore, considering that the degree of ligand symmetry on a receptor depends mainly on ligand concentration, our results suggest that IR may play metabolic‐biased roles in peripheral tissues dependent on local insulin concentrations.Support or Funding InformationThis research was supported by the Global Research Laboratory (GRL) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. NRF‐2016K1A1A2912722).
Cytochrome B5 Reductase 3 Can Be Approached As A Contemporary Therapeutic Target to Restrain Hypertension
Authors: Kumar Gaurav , Dey Sanjay Kumar , Kundu Suman 
Published: April 2020

Hypertension or high blood pressure is attributed as a critical health problem. Currently available therapeutics are indeed effective in combating hypertension, but are not always bereft of complications. Hence, to pursue alternate anti‐hypertensive therapeutics, the present study investigated human Cytochrome B5 Reducatse3 (hCYB5R3) enzyme as a “contemporary” therapeutic target, since this enzyme regulates the bioavailability of nitric oxide, a vasodilator involved in governing vascular tone; and hence hypertension. In the present investigation, small molecule databases were virtually screened against the crystal structure of the enzyme to identify potential inhibitors as well as to obtain binding energies and inhibitory constants. The enzyme was purified to homogeneity from recombinant sources and the inhibitors so obtained were tested in vitro using potassium ferricyanide assay in order to calculate IC50 values as a measure of inhibitor efficacy of these compounds against hCYB5R3. Further, the effect of these inhibitors was tested using various biophysical techniques including fluorescence and CD spectroscopy so as to obtain binding affinity, number of binding sites and effect on secondary structure, respectively. Fluorescence analysis has revealed promising binding constants (in micro molar ranges) and stoichiometric ratios (≈1:1) for some of the inhibitors. CD analysis revealed that these inhibitors do not alter the secondary structure of the FAD containing enzyme. Further, selected inhibitors were able to curb hypertension in vivo in spontaneously hypertensive rats. These encouraging findings have poised us to investigate the inhibitors further since overall, the identified and screened leads showed promising potential to be developed as novel anti‐hypertensive drugs against hCYB5R3.Support or Funding InformationThe funding support to conduct this study was provided by Department of Biotechnology (DBT) and Council of Scientific and Industrial Research (CSIR), Government of India. For experiments in rats, ethical clearance was obtained from Rajiv Gandhi Centre for Biotechnology (RGCB), India (IAEC/2015/23/CCKartha/RGCB/20.05.2015 (Protocol No. IAEC/570/CCK/2016)
Identification of Microglial Cell Molecular Markers During Chick Embryo Brain Development and Human Xenograft Tumor Formation
Authors: Lin Amy 
Published: April 2020

Glioblastoma (GBM) is the most aggressive and invasive human brain tumor, which despite advancement in surgery and immunology, only 10% of patients survive 18 months after diagnosis. The Galileo lab studies GBM, focusing on the role of the protein L1CAM on increasing the motility, proliferation, and invasiveness of GBM cells using in vitro cell tracking and a novel xenograft chick embryo brain tumor model. The work here focused on microglial cells, which are resident brain immune system cells that are not well understood. Studies in rodents and GBM patients showed that microglia play a critical role in promoting GBM growth via an activation of complex network of cytokines, chemokines and other metabolic factors that occur in the extracellular matrix. In the context of brain tumors, microglia promote neuroinflammation, which induces the BBB breakdown and inhibits antitumor immune responses. The first aim for my project was to find biomarkers that specifically identify microglial cells in normal chick embryo brain during development. The second aim was to use those markers in xenograft tumors in the chick embryo brain to look for any proinflammatory changes in microglial cells. Chick embryo brains were fixed, frozen, and cut into serial cryostat sections or fixed and vibratome sectioned, followed by immunofluorescent staining using antibodies or lectins that specifically identified microglial cells and endothelial cells in other model organisms. The optimal staining protocol required overnight primary antibody/lectin with 0.1% Triton X‐100 detergent and 5% normal goat serum in phosphate buffered saline. The antibodies and lectins investigated included Isolectin B4, Tomato (Lycopersicon esculentum) lectin, anti‐ferritin, anti‐laminin and CD45. Immunofluorescence analysis revealed CD45 to stain microglial cells with the most specificity. CD45 staining reveal microglial cell morphology to progressively change from a compact ameboid shape (E5) into ramified branched shape (E12 and E15). In addition to the increased quantity of ramified microglial cells in chick brain with xenograft human tumors, the microglia also appeared to be localized near endothelia. Further investigations are needed to verify the increased presence of reactive microglial cells and their interaction with tumor cells and angiogenesis.Support or Funding InformationThis work was supported by the Delaware INBRE program, with a grant from the National Institute of General Medical Sciences‐NIGMS (8 P20 GM103446‐16) from the National Institutes of Health, a grant to DSG from the National Cancer Institute, and a generous gift from Pamela S. Simpson ‘97 Translational Cancer Research Fund. I would like to thank Tyler Hellmig and Emily Kollenbroich for their guidance.
Anatomy of the round window: visibility prediction from preoperative CT scanners in cochlear implantation: literature criteria study and three‐dimensional reconstruction evaluation
Authors: Labrousse Marc , Veleine Yohanan , Dubernard Xavier , Brenet Esteban , Braun Marc , Renard Yohann 
Published: April 2020

IntroductionDuring cochlear implantation, round window membrane visibility through posterior tympanotomy is associated with type of cochleostomy used. Limiting bone drilling helps preserve residual hearing. Studies of preoperative scanner sections were proposed to anticipate its visibility with variable results. Based on preoperative scanner three‐dimensional reconstruction the purpose of this study is to evaluate the predictive capacity of the round window membrane.Materials and methodsProspective monocentric observational study conducted at Reims University Hospital Center on all patients who were candidates for cochlear implantation from December 2018 to May 2019. The exposure of the round window membrane is classified as “good exposure” and “bad exposure” according to the St Thomas’ Hospital classification, on three‐dimensional (3D) reconstruction and on the basis of intraoperative vision, blind to each other. The criteria in the literature measured on the CT scan sections were also studied. Their correlation is then evaluated, as well as the diagnostic performance of the 3D analysis to predict the intraoperative exposure of the round window membrane.Results25 procedures were carried out in 10 children and 15 adults. Among literature criteria only the distance from the facial nerve to the membrane of the oblique sagittal round window is statistically significantly associated with intraoperative exposure of the membrane of the round window (good exposure: 5.5 ± 0.6[4.7–6.7] mm versus poor exposure: 4.8 ± 0.6[3.9–5.3] mm ; p=0.05). Exposure of the round window membrane in 3D reconstruction is associated with intraoperative exposure of the round window membrane in a statistically significant manner (p=0.0005).DiscussionIn our study, among literature criteria only distance from facial nerve to round window membrane in oblique sagittal plane is statistically significantly associated with intraoperative exposure of the round window membrane. For a safe cochlear implantation, we propose a new criterion using petrous 3D CT scanner reconstructions which is strongly associated with intraoperative exposure of the round window membrane.
Retrotransposition Bursts in the Adult Brain: Stress, Running, and Instrumental Learning Increase the Number of L1 Retrotransposon DNA Copies in the Mouse Nervous System
Authors: Anokhin Konstantin , Ivashkina Olga 
Published: April 2020

Retrotransposons, the DNA elements which proliferate by reverse transcription of their RNA intermediates, comprise a major fraction of mammalian genomes. In the human genome a particularly abundant retrotransposon ‐ long interspersed nuclear element‐1 (LINE‐1 or L1) has 5x105 copies 80–100 of which are active and competent for retrotransposition. Recent findings indicate that LINE‐1 is active in neuronal precursor cells during differentiation, and its de novo insertions can change expression of neuron‐specific genes and cause somatic mosaicism in the adult brain. However, there has been limited understanding whether such retrotranspositions happen in the adult brain, when and how they occur, and what is their importance for the CNS function. In our study we tested whether prolonged stress, wheel running, or operant learning can lead to changes in L1 DNA copy number in the mouse brain. For this purpose, we subjected mice to immobilization stress (2 hours a day, 10 days), wheel running (unlimited access, 10 days) or nose‐poke operant training (1 session a day, 10 days). We collected the samples of hippocampus, frontal cortex, cerebellum, muscle and testicle tissues from experimental and home cage control mice. We performed qPCR for analysis of 3′‐UTR and ORF‐1 copy number. We observed significant increase in the 3′‐UTR and ORF‐1 copy number in all brain structures of the stress group compared to the home cage group. Nose‐poke training was associated with specific increase in 3′‐UTR copy number in the hippocampus. We also observed a small increase in 3′‐UTR and ORF‐1 copy number in all investigated brain structures in wheel running group. Altogether our results show that retrotranspositions is a common event in the adult brain under different conditions of novel experience.Support or Funding InformationRFBR # 19‐015‐00550