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  1. Article: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement.

    Martin, Emily-Rose / Gandawijaya, Josan / Oguro-Ando, Asami

    Frontiers in pharmacology

    2022  Volume 13, Page(s) 943627

    Abstract: The human SH-SY5Y neuroblastoma cell line is widely used in neuroscience research as a neuronal cell model. Following differentiation to a neuron-like state, SH-SY5Y cells become more morphologically similar to neurons and form functional synapses. ... ...

    Abstract The human SH-SY5Y neuroblastoma cell line is widely used in neuroscience research as a neuronal cell model. Following differentiation to a neuron-like state, SH-SY5Y cells become more morphologically similar to neurons and form functional synapses. Previous studies have managed to differentiate SH-SY5Y cells towards cholinergic, dopaminergic and adrenergic fates. However, their application in disease modeling remains limited as other neuronal subtypes (e.g., glutamatergic, GABAergic) are also implicated in neurological disorders, and no current protocols exist to generate these subtypes of differentiated SH-SY5Y cells. Our study aimed to evaluate the use of a xeno-free version of B-27, a supplement commonly used in neuronal culture, for SH-SY5Y maintenance and differentiation. To evaluate the proliferative capacity of SH-SY5Y cells cultured in B-27, we performed growth curve analyses, immunocytochemical staining for Ki-67 and qRT-PCR to track changes in cell cycle progression. SH-SY5Y cells cultured in FBS or under serum-starved conditions were used as controls. We observed that SH-SY5Y cells show reduced growth and proliferation rates accompanied by decreased
    Language English
    Publishing date 2022-10-20
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2587355-6
    ISSN 1663-9812
    ISSN 1663-9812
    DOI 10.3389/fphar.2022.943627
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM).

    D'Atri, Ilaria / Martin, Emily-Rose / Yang, Liming / Sears, Elizabeth / Baple, Emma / Crosby, Andrew H / Chilton, John K / Oguro-Ando, Asami

    Neuroscience letters

    2024  Volume 830, Page(s) 137778

    Abstract: The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of ... ...

    Abstract The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins. In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart. To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.
    MeSH term(s) Humans ; Endoplasmic Reticulum/metabolism ; Sigma-1 Receptor ; Receptors, sigma/metabolism ; Receptors, sigma/genetics ; Unfolded Protein Response ; HEK293 Cells ; Mitochondria/metabolism ; Mitochondria/genetics ; Mitochondrial Membranes/metabolism ; Retinitis Pigmentosa/metabolism ; Retinitis Pigmentosa/genetics ; Retinitis Pigmentosa/pathology
    Chemical Substances Sigma-1 Receptor ; Receptors, sigma
    Language English
    Publishing date 2024-04-15
    Publishing country Ireland
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ZDB-ID 194929-9
    ISSN 1872-7972 ; 0304-3940
    ISSN (online) 1872-7972
    ISSN 0304-3940
    DOI 10.1016/j.neulet.2024.137778
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Identification and single-base gene-editing functional validation of a cis-EPO variant as a genetic predictor for EPO-increasing therapies.

    Harlow, Charli E / Gandawijaya, Josan / Bamford, Rosemary A / Martin, Emily-Rose / Wood, Andrew R / van der Most, Peter J / Tanaka, Toshiko / Leonard, Hampton L / Etheridge, Amy S / Innocenti, Federico / Beaumont, Robin N / Tyrrell, Jessica / Nalls, Mike A / Simonsick, Eleanor M / Garimella, Pranav S / Shiroma, Eric J / Verweij, Niek / van der Meer, Peter / Gansevoort, Ron T /
    Snieder, Harold / Gallins, Paul J / Jima, Dereje D / Wright, Fred / Zhou, Yi-Hui / Ferrucci, Luigi / Bandinelli, Stefania / Hernandez, Dena G / van der Harst, Pim / Patel, Vickas V / Waterworth, Dawn M / Chu, Audrey Y / Oguro-Ando, Asami / Frayling, Timothy M

    American journal of human genetics

    2022  Volume 109, Issue 9, Page(s) 1638–1652

    Abstract: Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are currently under clinical development for treating anemia in chronic kidney disease (CKD), but it is important to monitor their cardiovascular safety. Genetic variants can be used as ... ...

    Abstract Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are currently under clinical development for treating anemia in chronic kidney disease (CKD), but it is important to monitor their cardiovascular safety. Genetic variants can be used as predictors to help inform the potential risk of adverse effects associated with drug treatments. We therefore aimed to use human genetics to help assess the risk of adverse cardiovascular events associated with therapeutically altered EPO levels to help inform clinical trials studying the safety of HIF-PHIs. By performing a genome-wide association meta-analysis of EPO (n = 6,127), we identified a cis-EPO variant (rs1617640) lying in the EPO promoter region. We validated this variant as most likely causal in controlling EPO levels by using genetic and functional approaches, including single-base gene editing. Using this variant as a partial predictor for therapeutic modulation of EPO and large genome-wide association data in Mendelian randomization tests, we found no evidence (at p < 0.05) that genetically predicted long-term rises in endogenous EPO, equivalent to a 2.2-unit increase, increased risk of coronary artery disease (CAD, OR [95% CI] = 1.01 [0.93, 1.07]), myocardial infarction (MI, OR [95% CI] = 0.99 [0.87, 1.15]), or stroke (OR [95% CI] = 0.97 [0.87, 1.07]). We could exclude increased odds of 1.15 for cardiovascular disease for a 2.2-unit EPO increase. A combination of genetic and functional studies provides a powerful approach to investigate the potential therapeutic profile of EPO-increasing therapies for treating anemia in CKD.
    MeSH term(s) Anemia/drug therapy ; Anemia/genetics ; Coronary Artery Disease/genetics ; Genome-Wide Association Study ; Humans ; Mendelian Randomization Analysis ; Myocardial Infarction/genetics ; Renal Insufficiency, Chronic/genetics
    Language English
    Publishing date 2022-09-02
    Publishing country United States
    Document type Journal Article ; Meta-Analysis ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ZDB-ID 219384-x
    ISSN 1537-6605 ; 0002-9297
    ISSN (online) 1537-6605
    ISSN 0002-9297
    DOI 10.1016/j.ajhg.2022.08.004
    Database MEDical Literature Analysis and Retrieval System OnLINE

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