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1.
Development ; 148(6)2021 03 23.
Article in English | MEDLINE | ID: mdl-33658223

ABSTRACT

The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus, it is a crucial parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine/disease modelling applications. However, the in vitro generation of posterior MNs corresponding to the thoracic/lumbosacral spinal cord has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well defined. Here, we determine the signals guiding the transition of human NMP-like cells toward thoracic ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal pre-/early neural state, whereas suppression of TGFß-BMP signalling pathways promotes a ventral identity and neural commitment. Based on these results, we define an optimised protocol for the generation of thoracic MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the comparison of hPSC-derived spinal cord cells of distinct axial identities.


Subject(s)
Cell Differentiation/genetics , Mesoderm/growth & development , Neural Stem Cells/metabolism , Spinal Cord/growth & development , Animals , Body Patterning/genetics , Bone Morphogenetic Proteins/genetics , Cell Lineage/genetics , Chick Embryo , Fibroblast Growth Factors/genetics , Gene Expression Regulation, Developmental/genetics , Humans , Mesoderm/metabolism , Motor Neurons/metabolism , Neural Stem Cells/cytology , Pluripotent Stem Cells/cytology , Signal Transduction/genetics , Spinal Cord/metabolism , Transforming Growth Factor beta/genetics , Wnt Proteins/genetics
2.
SLAS Discov ; 25(6): 605-617, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32441189

ABSTRACT

Modified messenger RNAs (mRNAs) hold great potential as therapeutics by using the body's own processes for protein production. However, a key challenge is efficient delivery of therapeutic mRNA to the cell cytosol and productive protein translation. Lipid nanoparticles (LNPs) are the most clinically advanced system for nucleic acid delivery; however, a relatively narrow therapeutic index makes them unsuitable for many therapeutic applications. A key obstacle to the development of more potent LNPs is a limited mechanistic understanding of the interaction of LNPs with cells. To address this gap, we performed an arrayed CRISPR screen to identify novel pathways important for the functional delivery of MC3 lipid-based LNP encapsulated mRNA (LNP-mRNA). Here, we have developed and validated a robust, high-throughput screening-friendly phenotypic assay to identify novel targets that modulate productive LNP-mRNA delivery. We screened the druggable genome (7795 genes) and validated 44 genes that either increased (37 genes) or inhibited (14 genes) the productive delivery of LNP-mRNA. Many of these genes clustered into families involved with host cell transcription, protein ubiquitination, and intracellular trafficking. We show that both UDP-glucose ceramide glucosyltransferase and V-type proton ATPase can significantly modulate the productive delivery of LNP-mRNA, increasing and decreasing, respectively, with both genetic perturbation and by small-molecule inhibition. Taken together, these findings shed new light into the molecular machinery regulating the delivery of LNPs into cells and improve our mechanistic understanding of the cellular processes modulating the interaction of LNPs with cells.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genetic Therapy/trends , Nanoparticles/chemistry , RNA, Messenger/genetics , Gene Transfer Techniques/trends , Genome, Human/genetics , High-Throughput Screening Assays/methods , Humans , Lipids/chemistry , Lipids/genetics , Lipids/therapeutic use , Nanoparticles/therapeutic use , RNA, Messenger/therapeutic use
3.
J Biol Chem ; 291(13): 6796-812, 2016 Mar 25.
Article in English | MEDLINE | ID: mdl-26841862

ABSTRACT

The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases.


Subject(s)
Adenoviridae/immunology , Adenovirus E3 Proteins/chemistry , Cell Membrane/immunology , Epithelial Cells/immunology , Fibroblasts/immunology , Adenoviridae/chemistry , Adenoviridae/pathogenicity , Adenovirus E3 Proteins/genetics , Adenovirus E3 Proteins/immunology , Amino Acid Motifs , Brefeldin A/pharmacology , Cell Line, Tumor , Cell Membrane/virology , Endosomes/immunology , Endosomes/virology , Epithelial Cells/drug effects , Epithelial Cells/virology , Fibroblasts/drug effects , Fibroblasts/virology , Gene Expression , Gene Expression Regulation , Host-Pathogen Interactions/immunology , Humans , Immunomodulation , Jurkat Cells , Lysosomes/immunology , Lysosomes/virology , Molecular Sequence Data , Primary Cell Culture , Protein Structure, Tertiary , Proteolysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Signal Transduction , Transfection , trans-Golgi Network/immunology , trans-Golgi Network/virology
4.
Toxicol Sci ; 145(2): 396-406, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25820236

ABSTRACT

We sought to investigate whether drug-induced changes in contractility were affected by pacing rates that represent the range of heart rates encountered in vivo. Using the cell geometry measurement system (IonOptix), we paced dog cardiomyocytes at different cycle lengths (CLs) of 2000, 1000, 500, and 333.3 ms, before and after exposure to 13 inotropic drugs. Time course data using vehicle control (0.1% dimethyl sulfoxide (DMSO)) demonstrated stability of the system at all CLs tested. Seven positive inotropes (eg isoproterenol) exerted rate-dependent increases in sarcomere shortening (Sarc. short.; maximal effect at a CL of 333.3 ms [0.1 µM isoproterenol increased Sarc. short. by 41.1% and 145.9% at 2000 and 333.3 ms, respectively]). Omecamtiv mecarbil showed an atypical profile (increased Sarc. short. at 2000 ms [106.9%] and decreased at 333.3 ms [IC(50) = 0.64 µM]). Four negative inotropes (eg flecainide) showed rate-independent inhibition of Sarc. short. (IC(50)s: 3.3 µM [2000 ms] versus 2.3 µM [333.3 ms]). The remaining negative inotropes, verapamil, and BTS (N-benzyl-p-toluene sulphonamide) produced an increase (IC(50)s: 3.9 µM [2000 ms] versus 0.043 µM [333.3ms]) and decrease (IC(50)s: 18.3 µM [2000 ms] versus 34.0 µM [333.3 ms]) in potency, respectively. Negative inotropes (eg flecainide, BTS, and verapamil) decreased the area of the Ca(2+) transient versus Sarc. short. hysteresis loop, although rate dependency was seen with verapamil only. Positive inotropes (eg isoproterenol and levosimendan) induced a rate-dependent increase in the area, however Omecamtiv mecarbil increased and decreased the area at CLs of 2000 and 333.3 ms, respectively. Thus, the use of different pacing rates may improve the detection of inotropes in early drug discovery and illustrate the potential for finger-printing different mechanisms of action.


Subject(s)
Cardiotonic Agents/pharmacology , Excitation Contraction Coupling/drug effects , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Animals , Calcium/metabolism , Cardiac Pacing, Artificial , Dogs , Dose-Response Relationship, Drug , Female , Heart Rate , Myocytes, Cardiac/metabolism , Sarcomeres/drug effects , Sarcomeres/metabolism , Time Factors
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