Analysis was performed with the statistical software package Prism Origin (GraphPad Software, La Jolla, USA). Statistical analysis of iMN survival experiments was performed using a two-sided log-rank test to account for events that did not occur (i.e. iMNs that did not degenerate before the end of the experiment). For each line, the survival data from 50 iMNs were selected randomly using Microsoft Excel, and these data were used to generate the survival curve. If all iMNs degenerated in a given experiment, statistical significance was calculated using a two-tailed Student’s t-test. For all other experiments, differences between two groups were analyzed using a two-tailed Student’s t-test, unless the data was non-normally distributed for which two-sided Mann-Whitney testing was used. Differences between more than two groups were analyzed by one way-ANOVA with Tukey correction for multiple testing. Significance was assumed at p < 0.05. Error bars represent the standard deviation unless otherwise stated.
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.
iMNs were fixed in 4% paraformaldehyde (PFA) for 1h at 4 ºC, permeabilized with 0.5% PBS-T overnight at 4 ºC, blocked with 10% FBS in 0.1% PBS-T at room temperature for 2 h, and incubated with primary antibodies at 4 ºC overnight. Cells were then washed with 0.1% PBS-T and incubated with Alexa Fluor® secondary antibodies (Life Technologies) in blocking buffer for 2 h at room temperature. To visualize nuclei, cells were stained with DAPI (Life Technologies) then mounted on slides with Vectashield® (Vector Labs). Images were acquired on an LSM 780 confocal microcope (Zeiss). The following primary antibodies were used: mouse anti-HB9 (Developmental Studies Hybridoma Bank); mouse anti-TUJ1 (EMD Millipore); rabbit anti-VACHT (Sigma); rabbit anti-C9ORF72 (Sigma-Aldrich); mouse anti-EEA1 (BD Biosciences); mouse anti-RAB5 (BD Biosciences); mouse anti-RAB7 (GeneTex); mouse anti-LAMP1 (Abcam); mouse anti-LAMP3 (DSHB, cat. no. H5C6); rabbit anti-LAMP3 (Proteintech, cat. no. 12632); mouse anti-LAMP2 (DSHB, cat. no. H4B4); mouse anti-M6PR (Abcam, cat. no. Ab2733); rabbit anti-GluR1 (EMD Millipore, cat. no. pc246); mouse anti-GluR1 (Santa Cruz); rabbit anti-NR1 (EMD Millipore); mouse anti-NR1 (EMD Millipore, cat. no. MAB363); chicken anti-GFP (GeneTex).
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.
The Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
The following antibodies were used in this manuscript: mouse anti-HB9 (Developmental Studies Hybridoma Bank); 81.5C10. chicken anti-TUJ1 (EMD Millipore); AB9354. rabbit anti-VACHT (Sigma); SAB4200559. rabbit anti-C9ORF72 (Sigma-Aldrich); HPA023873. rabbit anti-C9ORF72 (Proteintech); 25757–1-AP. mouse anti-EEA1 (BD Biosciences); 610457. mouse antiRAB5 (BD Biosciences); 610281. mouse anti-RAB7 (GeneTex); GTX16196. mouse anti-LAMP1 (Abcam); ab25630. mouse anti-M6PR (Abcam); ab2733. rabbit anti-GluR1 (EMD Millipore); pc246. mouse anti-NR1 (EMD Millipore); MAB363. chicken anti-GFP (GeneTex); GTX13970. rabbit anti-Glur6/7 (EMD Millipore); 04–921. mouse anti-FLAG (Sigma); F1804. mouse anti-GAPDH (Santa Cruz); sc-32233. chicken anti-MAP2 (Abcam); ab11267, rabbit anti-GLUR1 (Millipore, cat. no. 1504), mouse anti-NR1 (Novus, cat. no. NB300118), mouse anti-Transferrin receptor (Thermo, cat. no. 136800), mouse anti-LAMP3 (DSHB, cat. no. H5C6), rabbit anti-LAMP3 (Proteintech, cat. no. 12632), mouse anti-LAMP2 (DSHB, cat. no. H4B4), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), mouse anti-TUJ1 (Biolegend, cat. no. MMS-435P), rabbit anti-APP (Abcam, cat. no. ab32136), mouse anti-Tau5 (Thermo, cat. no. AHB0042), mouse anti-PSD-95 (Thermo, cat. no. MA1–045), mouse anti-p53 (Cell Signaling, cat. no. 2524S), anti-mouse HRP (Cell Signaling, cat. no. 7076S), anti-rabbit HRP (Cell Signaling, cat. no. 7074S).
The following antibodies were used in this manuscript: mouse anti-HB9 (Developmental Studies Hybridoma Bank); 81.5C10. chicken anti-TUJ1 (EMD Millipore); AB9354. rabbit anti-VACHT (Sigma); SAB4200559. rabbit anti-C9ORF72 (Sigma-Aldrich); HPA023873. rabbit anti-C9ORF72 (Proteintech); 25757–1-AP. mouse anti-EEA1 (BD Biosciences); 610457. mouse antiRAB5 (BD Biosciences); 610281. mouse anti-RAB7 (GeneTex); GTX16196. mouse anti-LAMP1 (Abcam); ab25630. mouse anti-M6PR (Abcam); ab2733. rabbit anti-GluR1 (EMD Millipore); pc246. mouse anti-NR1 (EMD Millipore); MAB363. chicken anti-GFP (GeneTex); GTX13970. rabbit anti-Glur6/7 (EMD Millipore); 04–921. mouse anti-FLAG (Sigma); F1804. mouse anti-GAPDH (Santa Cruz); sc-32233. chicken anti-MAP2 (Abcam); ab11267, rabbit anti-GLUR1 (Millipore, cat. no. 1504), mouse anti-NR1 (Novus, cat. no. NB300118), mouse anti-Transferrin receptor (Thermo, cat. no. 136800), mouse anti-LAMP3 (DSHB, cat. no. H5C6), rabbit anti-LAMP3 (Proteintech, cat. no. 12632), mouse anti-LAMP2 (DSHB, cat. no. H4B4), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), mouse anti-TUJ1 (Biolegend, cat. no. MMS-435P), rabbit anti-APP (Abcam, cat. no. ab32136), mouse anti-Tau5 (Thermo, cat. no. AHB0042), mouse anti-PSD-95 (Thermo, cat. no. MA1–045), mouse anti-p53 (Cell Signaling, cat. no. 2524S), anti-mouse HRP (Cell Signaling, cat. no. 7076S), anti-rabbit HRP (Cell Signaling, cat. no. 7074S).
The kung fu component of Li force is limited by one's physical condition. When a person passes his/her prime age, one's kung fu ability will pass the optimum level, too. The degree of kung fu will decline when muscles and bones are not as strong as they used to be. On the other hand, the kung fu aspect of Neijing is said to continually grow as long as one lives.[7]
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.
To provide a quantitative measure of (GGGGCC)n hexanuceotide expansion in C9ORF72, 100 ng of genomic DNA was amplified by touchdown PCR using primers shown in Supplementary Data Table 4, in a 28-µl PCR reaction consisting of 0.2 mM each of 7-deaza-2-deoxyguanine triphosphate (deaza-dGTP) (NEB), dATP, dCTP and dTTP, 7% DMSO, 1X Q-Solution, 1X Taq PCR buffer (Roche), 0.9 mM MgCl2, 0.7 µM reverse primer (four GGGGCC repeats with an anchor tail), 1.4 µM 6FAM-fluorescently labeled forward primer, and 1.4 µM anchor primer corresponding to the anchor tail of reverse primer (Supplementary Data Table 4). During the PCR, the annealing temperature was gradually decreased from 70 ºC and 56 ºC in 2 ºC increments with a 3 min extension time for each cycle. The PCR products were purified using the QiaQuick PCR purification kit (Qiagen) and analyzed using an ABI3730 DNA Analyzer and Peak Scanner™ Software v1.0 (Life Technologies).
Live imaging of iMNs expressing a M6PR-GFP fusion protein that localizes to M6PR+ vesicles 44 confirmed that C9ORF72 patient and C9ORF72-deficient iMNs possess increased numbers of M6PR+ vesicle clusters, and that overexpression of C9ORF72 isoform A or B rescues this phenotype (Supplementary Fig. 9c-g and Supplementary Videos 5-9). Clusters did not disperse over the time course of the assay, suggesting that they are relatively stable and not in rapid flux (Supplementary Videos 5-9). In addition, M6PR+ puncta moved with a slower average speed in C9ORF72 patient and C9ORF72+/− iMNs than controls (Supplementary Fig. 9h, i). Thus, reduced C9ORF72 levels lead to fewer lysosomes in motor neurons in vitro and in vivo, and this may be due in part to altered trafficking of M6PR+ vesicles.
The tomb murals of the Eastern Wei Dynasty (534–550) in the Southern and Northern Dynasties Period (386–581) unearthed from Xiaomachang Village of Wuqiao County in 1958 depict the performances of handstands, plate spinning, deft horsemanship and so on. However, it was after the Yuan Dynasty (1271–1368) that acrobatics of Wuqiao gained much reputation. Before that, acrobatics in Henan Province was much more influential. After the Yuan Dynasty was established, the capital was moved from Kaifeng of Henan to Beijing, and the acrobatics in Wuqiao of Hebei, which neighbors Beijing, began to prosper and was increasingly influential.
Although C9orf72 knockout mice do not show overt neurodegeneration, gain-of-function disease processes may trigger neurodegeneration through mechanisms induced by reduced C9ORF72 levels. For example, DPRs cause mis-splicing of the EAAT2 glutamate transporter in astrocytes, which couldincrease excitotoxicity in neurons with elevated glutamate receptor levels 12. To determine if DPRs alter glutamate uptake by astrocytes, we compared glutamate uptake in human primary astrocytes expressing either GFP or GR50 –GFP. Indeed, GR50 –GFP significantly impaired glutamate uptake by astrocytes (Supplementary Fig. 13h).
Our results highlight the importance of C9ORF72 protein function, RAB5 activity, PI3P levels, and lysosomeal function as key therapeutic targets for C9ORF72 ALS/FTD. By generating PI3P, RAB5 drives early endosomal maturation and the initial stages of lysosomal biogenesis (Fig. 6f)59. PI3P also plays important roles in autophagosome formation and autophagsome-lysosome fusion. Indeed, a previous study suggests that PIKFYVE inhibition may increase autophagic flux 53, and this should be investigated in the context of motor neurons. Loss-of-function mutations in two other genes whose proteins function to increase PI3P levels, ALS2 and FIG4, also cause ALS 1. ALS2 encodes the RAB5 guanine exchange factor ALSIN 60, while FIG4 converts PI(3,5)P2 into PI3P 55(Fig. 6f). In addition, proteins encoded by several other ALS genes play key roles in lysosomal biogenesis, including CHMP2B, OPTN, and SQSTM1 1. The fact that FIG4 and ALS2 loss-of-function mutations can cause ALS suggests that PIKFYVE inhibition or RAB5 activation may be capable of modulating ALS disease processes in humans.
Postsynaptic density extraction was done following a protocol published previously 63. Briefly, mouse spinal cord tissue or human cortical tissue was homogenized in cold Sucrose Buffer (320 mM Sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA, 30 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, and protease inhibitor cocktail (Roche)) using a tissue grinder and then spun down at 500 g for 6 min at 4℃. The supernatant was re-centrifuged at 10,000 g for 10 min at 4℃. The supernatant was collected as the “Total” fraction, and the pellet was resuspended in cold Triton buffer (50 mM HEPES pH 7.4, 2 mM EDTA, 50 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, 1% Triton X-100 and protease inhibitor cocktail (Roche)) and then spun down at 30,000 RPM using a Beckman rotor MLA-130 for 40 min at 4℃. The supernantant was collected as the “Triton” fraction and the pellet was resuspended in DOC buffer (50 mM HEPES pH 9.0, 50 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, 20 uM ZnCl2, 1% Sodium Deoxycholate and protease inhibitor cocktail (Roche)) and collected as the “DOC”, PSD-enriched fraction. Collected samples were boiled with SDS-PAGE sample buffer and analyzed by western blot. Purity of the PSD preps was analyzed by immunoblotting for PSD-95 (PSD), p53 (non-PSD), and synaptophysin (non-PSD).

Total RNA was extracted from sorted iMNs at day 21 post-transduction with Trizol RNA Extraction Kit (Life Technologies) and reverse transcribed with an Oligo dT primer using ProtoScript® II First Strand Synthesis Kit (NEB). RNA integrity was checked using the Experion system (Bio-Rad). Real-time PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) using primers shown in Supplementary Data Table 4.
Yingxiao Shi,#1,2,3 Shaoyu Lin,#1,2,3 Kim A. Staats,1,2,3 Yichen Li,1,2,3 Wen-Hsuan Chang,1,2,3 Shu-Ting Hung,1,2,3 Eric Hendricks,1,2,3 Gabriel R. Linares,1,2,3 Yaoming Wang,3,4 Esther Y. Son,5 Xinmei Wen,6 Kassandra Kisler,3,4 Brent Wilkinson,3 Louise Menendez,1,2,3 Tohru Sugawara,1,2,3 Phillip Woolwine,1,2,3 Mickey Huang,1,2,3 Michael J. Cowan,1,2,3 Brandon Ge,1,2,3 Nicole Koutsodendris,1,2,3 Kaitlin P. Sandor,1,2,3 Jacob Komberg,1,2,3 Vamshidhar R. Vangoor,7 Ketharini Senthilkumar,7 Valerie Hennes,1,2,3 Carina Seah,1,2,3 Amy R. Nelson,3,4 Tze-Yuan Cheng,8 Shih-Jong J. Lee,8 Paul R. August,9 Jason A. Chen,10 Nicholas Wisniewski,10 Hanson-Smith Victor,10 T. Grant Belgard,10 Alice Zhang,10 Marcelo Coba,3,11 Chris Grunseich,12 Michael E. Ward,12 Leonard H. van den Berg,13 R. Jeroen Pasterkamp,7 Davide Trotti,6 Berislav V. Zlokovic,3,4 and Justin K. Ichida1,2,3,†

Since glutamate receptor activation and neuronal firing both induce calcium influx, we determined their relative contributions to the increased Gcamp6 activation by. using the ion channel inhibitors TTX and TEA to block neuronal firing. C9ORF72+/− iMNs still displayed more frequent Gcamp6 activation than C9ORF72+/+ iMNs (Supplementary Fig. 13a), indicating that part of the hyperexcitability is due to increased glutamate receptor activation. To determine which receptors were responsible for the increased glutamate response, we tested small molecule agonists of specific glutamate receptor subtypes. Activation of NMDA, AMPA, and kainate receptors was higher in C9ORF72+/− iMNs than controls (Supplementary Fig. 13a).
With the four components of a chemical heat pump (two solid-gas reactors, an evaporator and a condenser), a cycle of the double-effect type can be applied to continuous refrigeration. The performance of this process is analysed, allowing the infinite sink temperature and the couples of reactive salts to be used, which depend on the production temperature envisaged, to be selected. The results are ... [Show full abstract]Read more
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.
On the other hand, the level of the Neijing force depends on the extent one can exercise over one's will power to release an inner qi energy. Within the framework of Chinese martial arts, every person is believed to possess the inborn energy of qi. Martial artists can harness the force of qi so that it is strong enough to be applied in combat. When qi is being directed by one's will, it is called Neijing.[4]
Human EEA1 (1–209) with an N-terminal GST tag in pGEX-6P-1 vector or GST only were expressed in E. Coli BL21 (DE3) cells (Thermo Fisher Scientific) for 12 hr at 18℃. Harvested cells were lysed by sonication in cold GST Purification Buffer (50 mM Tris pH 8.0, 200 mM NaCl, 2 mM DTT, 0.5 mg/ml Lysozyme, 0.2% Triton X-100 and protease inhibitor cocktail (Roche)). After centrifugation at 15,000 g for 30 min at 4℃, clarified lysate was incubated with Glutathione Sepharose 4B beads (GE Healthcare Life Science) for 3 hours to purify GST-EEA1 or GST. HEK cells were transfected with C-terminal 3XFLAG tagged C9ORF72 isoform A or B, or eGFP constructs and harvested 36–48 hr post-transfection in cold Lysis Buffer (25 mM HEPES pH 7.4, 100 mM NACl, 5 mM MgCl2, 1 mM DTT, 10% Glycerol, 0.1% Triton X-100 and protease inhibitor cocktail (Roche)).After centrifugation at 8,000 g for 10 min at 4℃, the clarified supernatant was incubated with washed GST-EEA1 or GST beads for 2 hr at 4℃ with end-to-end rotation. Beads were then boiled in 2X SDS-PAGE sample buffer and pulled-down protein was analyzed by western blot.
Total RNA was extracted from sorted iMNs at day 21 post-transduction with Trizol RNA Extraction Kit (Life Technologies) and reverse transcribed with an Oligo dT primer using ProtoScript® II First Strand Synthesis Kit (NEB). RNA integrity was checked using the Experion system (Bio-Rad). Real-time PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) using primers shown in Supplementary Data Table 4.

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Consistent with PIKFYVE being the relevant target in the iMN survival assay, Apilimod increased C9ORF72 patient, but not control, iMN survival in either neurotrophic withdrawal conditions (Fig. 6d) or excess glutamate (n=4 patients, Supplementary Fig. 15f (n=3 controls, Supplementary Fig. 15g). Automated neuron tracking software independently verified Apilimod efficacy on C9ORF72 patient iMNs (Supplementary Fig. 15h). As further confirmation that PIKFYVE was the active target, ASO-mediated suppression of PIKFYVE also rescued C9ORF72 patient iMN survival (Fig. 6d and Supplementary Fig. 15i). In addition, we synthesized a structural analog of Apilimod with a reduced ability to inhibit PIKFYVE kinase activity in a biochemical assay using purified PIKFYVE protein (Fig. 6b and Supplementary Fig. 15j, 16). The reduced activity analog was significantly less effective at rescuing C9ORF72 patient iMN survival (Fig. 6e). Thus, small molecule inhibition of PIKFYVE can rescue patient motor neuron survival.
Hb9::RFP+ C9ORF72 ALS/FTD iMNs were generated in 96-well plates. On Day 15 post transduction, neurotrophic factors and RepSox were withdrawn and the small molecule library was added (EMD Millipore kinase collection and Stemselect library, 3.3 µM final concentration) and added fresh every other day until the screen was terminated on Day 25 post-transduction. Identification of neuroprotective compounds was identified using SVcell 3.0 (DRVision Technologies) and further verification by manual iMN tracking.
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).
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