J.K.I. and P.A. are co-founders of Acurastem, Inc. P.A. is an employee of Icagen Corporation. J.K.I. and P.A. declare that they are bound by confidentiality agreements that prevent them from disclosing details of their financial interests in this work. S-J.L. is a founder of DRVision Technologies and T-Y.C. is an employee of DRVision Technologies. A.Z. and J.A.C. are co-founders of Verge Genomics and V.H-S., N.W., and T.G.B. are employees of Verge Genomics.
Therapeutic strategies in development for C9ORF72 ALS/FTD target gain-of-function mechanisms. These include ASOs 6–8 and small molecules 13 that disrupt RNA foci formation. However, these approaches have not fully rescued neurodegeneration in human patient-derived neurons 6–8,13, indicating that replacing C9ORF72 function or new therapeutic targets may be required.
Practitioners of kung fu refer to two separate forms of personal force: Li (Traditional Chinese: 力) refers to the more elementary use of tangible physical (or "external") force, such as that produced by muscles. Neijing (Traditional Chinese:內勁) or Neigong (Traditional Chinese: 內功), in contrast, refer to "internal" forces produced via advanced mental control over psychic energy (the qi).
Dirigido a blogueros, personas influyentes, funcionarios de relaciones públicas, personalised de marketing, aspirantes a periodistas o cualquier persona que quiera aprender más sobre el oficio de la escritura, el curso enseña las habilidades básicas de la escritura profesional: la introducción, la pirámide invertida, las 5 W, las 3 C y, lo más importante de todo, la narración de cuentos.
Primary chick myoblasts were dissected from D11 chick embryos and plated onto plastic dishes pre-coated with 0.1% gelatin. After 3 days of culture in muscle medium containing F10 (Life Technologies), 10% horse serum, 5% chicken serum (Life Technologies), 0.145 mg/ml CaCl2 (Sigma), and 2% Penicillin/Streptomycin, myoblasts were trypsinized and replated onto iMNs which were at days 15–18 post-transduction. The co-culture was maintained in neuronal medium containing DMEM/F12, 2% B27, 1% GlutaMax and 1% Penicillin/Streptomycin, supplemented with 10ng/ml BDNF, GDNF, and CNTF for 7 days in order to allow neuromuscular junctions to form. Videos were taken using Nikon Eclipse Tis microscope with NIS Element AR software. Light-stimulated contraction shown in Supplementary Figure 2j are representative of contraction observed in 2 biological replicates, with 5 contractile sites per replicate.
We thank the NINDS Biorepository at Coriell Institute for providing the following cell lines for this study: ND12133, ND03231, ND01751, ND11976, ND03719, ND00184, ND5280, ND06769, ND10689, ND12099, ND14954, ND08957, ND12100, and ND014587. We thank Helena Chui and Carol Miller at the University of Southern California Alzheimer’s Disease Research Center and Neil Shneider at the Columbia University Medical Center for control and C9ORF72 patient tissue. We thank the Choi Family Therapeutic Screening Facility for chemical screening support and the Translational Imaging Center at USC for imaging support. We thank Max Koppers, Youri Adolfs, Christiaan van der Meer, and Mark Broekhoven for help with mouse breeding and kainate injection experiments. We thank Prof. Satoshi Waguri for providing the M6PR-GFP construct. We thank Christopher Buser for assistance with electron microscopy. We also thank Sam Alworth (DRVision Technologies, LLC), Katja Hebestreit, and Raj Bhatnagar (Verge Genomics), Bob Baloh, Jacqueline O’Rourke, Christopher Donnelly, Chang Tong, Andrew McMahon and Qing Liu-Michael for reagents, technical support, and discussions. E.Y.S. is a Walter V. and Idun Berry Postdoctoral Fellow. K.A.S. was supported in part by a Muscular Dystrophy Association Development Grant. L.M. was supported by NIH grant T32DC009975–04. This work was supported by NIH grants AG039452, AG023084, and NS034467 to B.V.Z. R.J.P. was supported by grants from ALS Foundation Netherlands (TOTALS), Epilepsiefonds (12–08, 15–05), and VICI grant Netherlands Organisation for Scientific Research (NWO). This work was also supported by NIH grants R00NS077435 and R01NS097850, U.S. Department of Defense grant W81XWH-15–1-0187, and grants from the Donald E. and Delia B. Baxter Foundation, the Tau Consortium, the Frick Foundation for ALS Research, the Muscular Dystrophy Association, the New York Stem Cell Foundation, the USC Keck School of Medicine Regenerative Medicine Initiative, the USC Broad Innovation Award, and the Southern California Clinical and Translational Science Institute to J.K.I. J.K.I. is a New York Stem Cell Foundation-Robertson Investigator.
HEK 293T cells were used to produce retrovirus, lentivirus, and C9ORF72 protein. HEK cells were used for these purposes based on previous published studies using HEK cells in order to produce viral particles and mammalian proteins. HEK cells were obtained from American Type Culture Collection, catalog number CRL-11268. HEK and iPS cells were tested for mycoplasma before, during, and after the study and were negative.
The degree of Li force one can employ in kung fu depends on several variables such as resilience of muscles, strength of bones, speed and timing of attack and so on. An effective way to enhance the Li force is to exercise one's muscles and bones by applying increasing pressure on them (weight training, gym exercises, etc.).[2] The stronger one's muscles and bones become, the more powerful and skillful the level of kung fu is.[3]

The degree of Li force one can employ in kung fu depends on several variables such as resilience of muscles, strength of bones, speed and timing of attack and so on. An effective way to enhance the Li force is to exercise one's muscles and bones by applying increasing pressure on them (weight training, gym exercises, etc.).[2] The stronger one's muscles and bones become, the more powerful and skillful the level of kung fu is.[3]
To measure the effect of dipeptide repeat protein expression on iMN survival, PR50 and GR50 were cloned into the pHAGE lentiviral vector as fusions with GFP to allow tracking of protein expression. iMN cultures were transduced with PR50 and GR50 lentiviruses at day 17 of reprogramming and longitudinal survival analysis was started the same day. 10 ng/ml of GDNF, BDNF, and CNTF was maintained throughout the experiment, and glutamate treatment was not performed. To measure PR50 turnover, PR50 was cloned into the pHAGE lentiviral vector as a fusion with Dendra2 (Addgene). iPSC-derived fibroblasts were generated according to Daley and colleagues64. Briefly, when C9ORF72−/− iPSC cultures reached 80% confluence, the medium was switched from mTeSR1 (Stem Cell Technologies) to human fibroblast medium containing DMEM (Life Technologies), 10% fetal bovine serum (FBS)(Thermo Fisher Scientific), and 1% penicillin/streptomycin (Life Technologies). Cells were passaged 2 to 3 times using Accutase (Life Technologies) before use in experiments. iPSC-derived fibroblasts were transduced with either pMXs-eGFP or pMXs-C9ORF72 isoform B-T2A-eGFP retrovirus and treated with 10 μg/ml mitomycin C for 3 hrs to inhibit cell proliferation. The cells were then transduced with the PR50–Dendra2 lentivirus and exposed to blue light for 1.5 sec using a lumencor LED light source to initiate photoconversion. The amount of decay (as a fraction of the starting level) of the red fluorescent punctae was monitored by longitudinal time lapse imaging in a Molecular Devices ImageExpress and analyzed using SVCell 2.0 (DRVision Technologies). Fluorescence was quantified at t = 0 and 12 hours after photoconversion. Distinct photoconverted punctae were treated as discrete objects for analysis (n = 20 each for +eGFP and +C9ORF72-T2A-eGFP). For each object, background fluorescence was subtracted and fluorescence was normalized according to object size. The fractional decay was statistically analyzed by two-tailed Student’s t-test. ** - p<.01.
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