bluuebunny/crossref_metadata_2025_split huggingface dataset:
CB1 BUT NOT CB2 CANNABINOID RECEPTOR INCREASES NEURITE EXTENSION IN HUMAN NEUROBLASTOMA
Authors: Lyons Erica Lynn , Kabler Sandra , Howlett Allyn , Kovach Alexander L. , Thomas Brian 
Published: June 2021

Despite neurodegenerative disease affecting more than five million Americans, there is not yet sufficient understanding of the underlying mechanism of disease to cure or even halt progression. Transplanted neurons may represent a potential treatment if they could successfully integrate with host brain tissue. Both CB1 and CB2 receptors are located in neuronal growth cone membranes, implying that cannabinoid signaling may play a role in neurite extension or guidance (Harkany et al. 2007). CB1 cannabinoid signaling has been shown to be active in neuronal extension, yet the differences between CB1R and CB2R signaling remain unclear (Berghuis et al. 2007, Duff et al. 2013). We have created a novel stable overexpression model to investigate the role of cannabinoid signaling in human neuronal extension. We quantified the endogenous production of cannabinoid ligand 2‐Arachidonoyl Glycerol (2‐AG) and its isomer 1‐AG in SHSY5Y human neuroblastoma at an abundance of 4,000 nanograms Total AG per million cells. This AG was likely 2‐AG produced by DAGL enzyme activity because calcium stimulation by Oxotremorine M increased Total AG abundance to 6,000 nanograms per million cells while inhibition of DAGL by THL decreased Total AG abundance to less than 100 nanograms per million cells. We transfected SHSY5Y cells with either CB1 or CB2 receptors, purified and maintained the transfection using geneticin selection, and isolated clonal cell lines that stably overexpress either CB1R (CB1XS) or CB2R (CB2XS). Stable overexpression of cannabinoid receptors did not alter mRNA abundance of the enzymes that synthesize and degrade the endogenous cannabinoids 2‐AG and AEA. Stable overexpression of CB1R but not CB2R increased the extension length from 30 to 200 micrometers per neurite. Our research suggests a role for CB1R but not CB2R mediated cannabinoid signaling in neurite development that may provide insights in development of therapies that support functional integration of transplanted neurons into host tissue.Support or Funding InformationSupport from NIDA grant DA03690 and T32 grant HL091797 is appreciated.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Vacuolar H+‐ATPase Inhibition on Activation of Cathepsin B and Cathepsin L Secreted from MDA‐MB231 Breast Cancer Cells
Authors: Hinton Ayana M. , Uhlman Andrew , Folkers Kelly , Liston Jared , Pancholi Harshida 
Published: June 2021

Studies indicate secreted cathepsins are involved in metastasis. V‐ATPases, which are necessary for activating intracellular cathepsins, also play a role in metastasis and are targeted to the plasma membrane of metastatic breast cancer cells. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. We investigated whether V‐ATPase inhibition would reduce the activity of secreted cathepsin B and cathepsin L. Using cell lysates and conditioned media, we measured cathepsin B and L activity within and outside of the cells. We found different forms of cathepsin B and L were secreted representing the pre‐pro, pro and active forms of the proteases. Cathepsin B activity was higher than cathepsin L in conditioned media and in cell lysates. V‐ATPase inhibition by concanamycin A decreased cathepsin B activity in conditioned media and significantly decreased cathepsin B activity in cell lysates. Cathepsin L activity showed a slight decrease in cell lysates. Changes in the activity of secreted and intracellular cathepsins following V‐ATPase inhibition were supported by changes in the amounts of pro and active forms of cathepsins B in conditioned media and cathepsin B and L in cell lysates. Overall, our data shows that inactive forms of cathepsins B and L are secreted from the MB231 cells and V‐ATPase activity is important for the activation of secreted cathepsin B. This indicates a connection between cell surface V‐ATPases in metastatic breast cancer cells and the function of secreted cathepsin B.Support or Funding InformationDenison University Anderson Fellowship, Denison University Research FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nanodiscs: A Novel Approach to Studying the Methionine ABC Transporter System
Authors: Winslow Michael G. , Yang Janet G. 
Published: June 2021

The cell membrane is a formidable yet flexible boundary that separates the interior from the exterior of the cell. The tightly regulated, selective movement of molecules across the membrane is crucial to maintain appropriate intracellular levels of metabolites. Concentrations gradients across lipid bilayers are generated and maintained by specialized integral membrane proteins known as transporters. The long‐term goal of the Yang Group is to understand how transporters catalyze substrate transport across cellular membranes.Toward this goal, we are conducting quantitative analyses of a bacterial methionine transport system. The E.coli MetNI transporter uses the energy of ATP binding and hydrolysis to drive methionine import against its concentration gradient. Unlike the majority of in vitro transporter studies conducted using detergent‐solubilized proteins, we aim to examine the MetNI reaction cycle in a native membrane environment. Here we present our preliminary work on preparing methionine transporters in nanodiscs, a discoidal phospholipid bilayer encircled by a protein helical belt.Support or Funding InformationNo external funding.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomechanical control of lysosomal secretion via the VAMP7 hub: a tug‐of‐war mechanism between VARP and LRRK1
Authors: Galli Thierry , Wang Guan , Nola Sébastien , Bovio Simone , Coppey‐Moisan Maïtté , Lafont Frank 
Published: June 2021

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we found that cells deficient in the secretory lysosome v‐SNARE VAMP7 were impaired in adapting to substrate rigidity. Conversely VAMP7‐mediated secretion was stimulated by more rigid substrate and this regulation depended on the Longin domain of VAMP7. We further found that the Longin domain bound the kinase and retrograde trafficking adaptor LRRK1 and LRRK1 negatively regulated VAMP7‐mediated exocytosis. Conversely, VARP, a VAMP7‐ and kinesin 1‐interacting protein, further controlled the availability for secretion of peripheral VAMP7 vesicles and response of cells to mechanical constraints. We propose a mechanism whereby biomechanical constraints regulate VAMP7‐dependent lysosomal secretion via LRRK1 and VARP tug‐of‐war control of the peripheral readily‐releasable pool of secretory lysosomes.Support or Funding InformationWork in our group was funded by grants from INSERM, CNRS, Association Française contre les Myopathies (Research Grant 16612), the French National Research Agency (NeuroImmunoSynapse ANR‐13‐BSV2‐0018‐02), the Ecole des Neurosciences de Paris (ENP), the Fondation pour la Recherche Médicale (FRM), Who am I? Labex (Idex ANR‐11‐IDEX‐0005‐01), awards of the Association Robert Debré pour la Recherche Médicale to T.G. and fellowship from the Region Ile‐de‐France in the framework of DIM Cerveau&Pensée and from FRM (FDT20150532766) to G.W. We acknowledge the ImagoSeine facility, and the France BioImaging infrastructure supported by ANR (10‐EQPX‐04‐01) and the EU‐FEDER (12,001,407) as part of “Investments of the future” program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AP‐3‐dependent Mechanisms Regulate the Trafficking of ATP8a1 to Lamellar Bodies in Alveolar Type 2 Cells
Authors: Kook Seunghyi , Wang Ping , Meng Shufang , Hanby Hayley A. , Jaume Alexa , Goetzl Laura , Marks Michael S. , Guttentag Susan H. 
Published: June 2021

Alveolar type 2 cells (AT2) are involved in the maintenance of epithelial homeostasis and repair after injury. Lamellar bodies (LB) in AT2 play a critical role in lung health and disease as the site of surfactant synthesis, packaging and secretion. LB biogenesis is poorly understood, especially mechanisms that foster membrane and protein trafficking to LB. In this study, we found that ATP8a1, a member of the P4 subfamily of P‐;type ATPases (P4‐;ATPases), was highly enriched in the ABCA3‐positive LB fraction derived from human fetal lung explants. In AT2 from mice lacking the adaptor protein 3 complex (AP‐3), the pearl mouse model of Hermansky Pudlak syndrome type 2 (HPS2), ATP8a1 was largely depleted from ABCA3‐positive LB and accumulated in Rab11‐positive recycling endosomes (REs). Using targeted mutagenesis, we identified a di‐leucine‐based sorting signal (ExxxLL) present in the C‐terminal cytoplasmic domain of ATP8a1 that is necessary for its proper targeting to LB of AT2. To dissect the relative roles of AP‐1 and AP‐3 in the transport of ATP8a1 to LB, we have developed MLE‐15 cell lines in which a subunit of AP‐1 (Ap1γ1) and/or AP‐3 (Ap3β1) has been inactivated using CRISPR/Cas9 gene editing and tested the targeting of ATP8a1 in these mutated cell lines. The lack of AP‐3 in MLE‐15 reduced efficient LB delivery of ATP8a1, while deletion of the AP‐1 subunit had no effect on ATP8a1 delivery to LB. P4‐;ATPases typically flip aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of organelle membranes. PS is highly enriched in REs and is essential for endosomal membrane traffic. Here, we show that the cytoplasmic leaflet of LB in MLE‐15 and human AT2 are highly enriched in PS using a GFP‐LactC2 probe; in contrast, AP‐3‐deficient cells showed impaired distribution of PS, favoring RE locazliation. These data provide evidence that ATP8a1 is dependent on AP‐3 for trafficking from RE to LB in AT2, and suggest that loss of ATP8a1 targeting to LB in HPS2 impairs PS localization to LB in AT2 cells.Support or Funding InformationNational Institutes of Health (GM108807/SG, MM and LG). SG is the Julia Carell Stadler Professor of Pediatrics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tale of the two domains in twinfilin: Deciphering phagocytosis through actin dynamics
Authors: RATH PRAGYAN PARIMITA , KUMAR NITESH , BABUTA MRIGYA , BHATTACHARYA ALOK , GOURINATH SAMUDRALA 
Published: June 2021

Phagocytosis is an indispensable cellular function in phagocytic cells or protists. It is choreographed by actin and its surfeit of regulatory proteins. This process is critical in Entamoeba histolytica’s lifecycle as it relies primarily on phagocytosis for its nutritional uptake as well as its pathogenesis. In our studies we have tried to puzzle out the role of Twinfilin‐ an actin binding protein in E. histolytica's pathogenesis. Twinfilin is an Actin Depolymerizing Factor (ADF)/cofilin family member, containing two ADF‐homology domains, one each as N‐terminal (NTD) and C‐terminal (CTD).We have solved a 1.5 Å resolution crystal structure of EhTwinfilin NTD and determined that it contains typical ADF homology fold i.e., central β sheets surrounded by α helices. With the help of fluorimetric and SPR studies we have shown the actin binding activities of both the domains and interestingly we found that NTD stabilized F‐actin whereas CTD depolymerized it. NTD has stronger G‐actin binding while CTD binds to F‐actin with much higher affinity; this is rather role reversal of the studies published on twinfilin from other organisms. Structural and actin binding studies of EhTwinfilin provides explanation for some of the functional differences observed with respect to the human and Plasmodium falciparum's ADF such as the ability to bind G‐actin. Cellular localization studies established its significant role during phagocytosis. GFP tagged EhTwinfilin was visualized using confocal imaging, it was found to colocalize with F‐actin at phagocytic cups. Down‐regulation of EhTwinfilin expression leads to an increased rate of phagocytosis. This is the second protein so far that was seen to be present throughout the phagocytosis, up till phagosome maturation and first protein to perform actin severing activity in E. histolytica.Support or Funding InformationSERB, Department of Science and Technology, IndiaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Seeking new molecular targets to control mitochondrial biogenesis
Authors: Silveira Leonardo R. , Pauletti Bianca A. , Paes Leme Adriana F. , Lima Tanes I. 
Published: June 2021

Mitochondrial number and shape are constantly changing in response to increased energy demands. The ability to synchronize mitochondrial signaling pathways with environmental stimuli is a central aspect against obesity in mammals. The binding and regulation of transcriptional complexes at the promoter of mitochondrial genes are critical components of the mitochondrial biogenesis process. Specifically, these promoters contain cis‐regulatory elements that lead to the recruitment of selected nuclear receptors (NR) and transcriptional factors (TF). Although different molecular regulators of this system have been described, only a small number of nuclear receptors and transcription factors have been associated with the mitochondrial biogenesis process. In this study, therefore, we proposed to seek new targets for the regulation of the mitochondrial biogenesis process in C2C12 cells during differentiation through the integration of proteomic and transcriptomic analysis. As expected. mitochondrial function and biogenesis were strongly induced during C2C12 cell differentiation (Figure 1). By evaluating the signaling pathways and molecular networks during myogenesis using RNAseq data extracted from GEO datasets (GSE20846), we revealed the enrichment of several signaling pathways associated with mitochondrial biogenesis and oxidative metabolism such as OXPHOS and TCA cycle (Figure 2). These results showed a strong enrichment in the PPAR signaling which was validated through PPRE transactivation experiments and qPCR (Figure 2). Accordingly, Venn diagram analysis showed an overlap of 170 genes associated with mitochondrial function from upregulated genes during myogenesis and PPAR targets suggesting the involvement of PPAR activity in mitochondrial biogenesis (Figure 3). In addition, proteome analysis during C2C12 differentiation exhibited 609 proteins positively regulated, from which 246 overlapped with PPAR targets and also presented enrichment of mitochondrial signaling pathways. To investigate the involvement of other uncharacterized transcription factors and nuclear receptors in mitochondrial biogenesis accompanying skeletal muscle cell differentiation, we performed a over‐represented conserved transcription binding sites analysis (oPOSSUM 3.0) in the set of upregulated mitochondrial associated genes and proteins previously analyzed using GO‐term of cellular components (DAVID). These analyses revealed the role of several unrelated NRs and TFs in mitochondrial biogenesis accompanying skeletal muscle cell differentiation (Table 1).Support or Funding InformationThis work was supported by grants from Fundação de Amparo à Pesquisa do estado de São Paulo – FAPESP (2013/22733‐0; 2016/23008‐5) and CNPq (444323/2014).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The genetic origin of a rare mitochondrial disorder
Authors: Bartl Lucas , DeVries Brooklyn , Reider Joshua , Vitiello Seasson P. 
Published: June 2021

A pediatric patient with hypotonia, cerebellar atrophy, psychomotor delay, and increased blood‐lactate underwent whole exome sequencing. The patient harbors two novel variants of the TARS2 gene, which encodes the mitochondrial threonyl‐tRNA synthetase (mtThrRS). The aminoacylation catalyzed by mtThrRS is necessary for the translation of proteins encoded by mitochondrial DNA. One variant in the patient contains a putative splice‐site mutation, while the other variant contains a missense mutation. Both mutations are predicted to cause loss of mtThrRS function, suggesting that these novel variants of the TARS2 gene cause mitochondrial dysfunction and are therefore causal for the disorder. We aimed to assess the effects of these variants on mitochondria and energy production, and to determine the molecular effects of the TARS2 splice‐site mutation. This work contributes to the understanding of the connections between tRNA synthetase function and health.Support or Funding InformationNIH/INBRE (NIGMS) South Dakota INBRE Program (P20GM103443)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation
Authors: Yang Loretta , Wu Shengcheng , Cooper J. Christopher , Paul Mallory K. , Cummings Amanda L. , Eletr Ziad M. , Ben‐Arye Shani Leviatan , Padler‐Karavani Vered , Samli Kausar N. , Woods Robert J. 
Published: June 2021

Glycans have distinct properties that make them appealing as disease biomarker targets, and novel highly specific reagents are essential to overcome current limitations in the discovery and exploitation of disease‐related glycans.Lectenz® Bio is engineering glycan‐processing enzymes into catalytically inactive, high affinity glycan binding reagents with tunable specificities. These novel lectin‐like, enzyme‐derived reagents called Lectenz® are being developed for a variety of glycan detection and enrichment applications including affinity chromatography, Western blot, and immunohistochemistry. The conversion of such enzymes into affinity reagents is facilitated by computationally‐guided directed evolution. Using site‐directed mutagenesis and yeast display selection of a site‐saturation mutagenesis library, multiple Lectenz® candidates can be identified.Here we present two novel sialic acid recognizing Lectenz® engineered from a sialidase enzyme: 1) the pan‐specific sialic acid reagent, Sia‐PS1 Lectenz®, which recognizes sialic acid in a linkage independent manner; and 2) the Sia‐3S1 Lectenz®, which is specific for α2,3‐linked sialoglycans. The data demonstrate that the Sia‐PS1 Lectenz® reagent recognizes glycans terminating in α2,3‐, α2,6‐, and α2,8‐linked sialic acid sequences, but not Gal‐terminating sequences. With the Sia‐3S1 Lectenz®, we demonstrate binding to Neu5Acα2,3Galβ1,4Glc, but not Neu5Acα2,6 Galβ1,4Glc or Galβ1,4Glc.Support or Funding InformationNational Institutes of Health (R41GM113351)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Boston Naming Test Predicts Deterioration Of Cerebrospinal Fluid Biomarkers In Pre‐Symptomatic Alzheimer's Disease
Authors: Wilder Charleen , Moncrieffe Kristina , Nolty Anne , Arakaki Xianghong , Fonteh Alfred , Harrington Michael 
Published: June 2021

Background and ObjectivesIdentifying predictors of Alzheimer's disease provides an opportunity for early treatment, which can minimize the negative impact of the disease. The current longitudinal study examines neuropsychometric changes in cognitively healthy pre‐symptomatic elderly participants using cerebrospinal fluid (CSF) amyloid and tau measurements, established biomarkers of Alzheimer's disease pathology. Participants were classified as having normal amyloid/tau (NAT) or pathological amyloid/tau (PAT). By examining the neuropsychological differences between cognitively healthy individuals with normal biochemistry (NATs) and those who convert over time to abnormal biochemistry (PATs), early neuropsychological predictors may be identified. Our hypothesis is that the differences in neuropsychological data will predict the change in biochemistry in participants who convert from NATs to PATs.MethodsA total of 63 elderly participants (70–95 years) were classified using the Uniform Data Set, Version 2, as cognitively healthy. They were tested at two time points: at an average initial age of 80, and again 40 months later. At each visit, participants underwent a series of clinical, biochemical, and neuropsychological assessments.Results & DiscussionOut of the 23 NATs that were reassessed clinically, all remained cognitively healthy as assessed by consensus conferencing and extensive neuropsychological evaluation, while 40% of the PATs had cognitive decline. From their CSF, 14 NATs remained NAT (NAT‐NAT) while 9 converted to PATs (NAT‐PAT) at the second time point. Mann Whitney U‐test analyses indicated that there was a significant difference between those who on reassessment were NAT‐NAT vs. NAT‐PAT on the Boston Naming Test (BNT) at the first time point. Our neuropsychological data suggest that cognitively healthy individuals who convert to abnormal CSF biochemistry performed worse on the initial BNT than cognitively healthy individuals who have persistently normal CSF biochemistry. Small population size is a limitation of this study. We propose that performance on the BNT may predict changes in amyloid and tau biochemistry in cognitively healthy individuals; 40% of these CSF converters (compared to none of the non‐converters) have declined clinically in 4 years.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.