Samples were first fixed in 4% PFA (1x PBS) overnight at 4°C and were subsequently washed three times with 1x PBS. Next, cells were permeabilized with 0.3% Triton X-100 (1x PBS) for 10 min at room temperature, followed by three washes with 1x PBS for 10 min each. After permeabilization, the samples were equilibrated in 1x SSC buffer for 10 min at room temperature and then transferred into 50% formamide (2x SSC) for 10 min at 60°C. The repeat expansion-targeting probe and the negative control probe were ordered from Exiqon 58. During this step, the probe mixture (1µl salmon sperm (10 µg/µl), 0.5 µl E. coli tRNA (20 µg/µl), 0.4 µl probe (25 µM), 25 µl 80% formamide/per sample) was made and placed at 95°C for at least 10 min. The samples were submerged in 200 µl of hybridization buffer (4ml 100% formamide, 0.5 ml 20x SSC, 1 ml BSA fraction V, 0.5ml RVC (20 mM), 1ml NaPO4 (0.1 M), 3 ml nuclease-free water) and 27 µl of the probe mixture was added to each sample and incubated for 1 hour at 60°C. After probe hybridization, the samples were washed twice with 50% formamide (2x SSC) for 20 min each at 65°C and once more with 40% formamide (1x SSC) for 10 min at 60°C. The remaining formamide was removed by briefly washing with 1x SSC three times. A final crosslinking step was performed by first incubating the samples with 1x Tris-Glycine for 5 minutes followed by a 5 min incubation in 4% PFA. Samples were washed three times with 1x PBS, stained with DAPI, and imaged using a Zeiss LSM 800 confocal microscope.
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. The contact point only represents the gateway to conduct Neijing energy at the point of attack.
Shi Y1,2,3, Lin S1,2,3, Staats KA1,2,3, Li Y1,2,3, Chang WH1,2,3, Hung ST1,2,3, Hendricks E1,2,3, Linares GR1,2,3, Wang Y3,4, Son EY5, Wen X6, Kisler K3,4, Wilkinson B3, Menendez L1,2,3, Sugawara T1,2,3, Woolwine P1,2,3, Huang M1,2,3, Cowan MJ1,2,3, Ge B1,2,3, Koutsodendris N1,2,3, Sandor KP1,2,3, Komberg J1,2,3, Vangoor VR7, Senthilkumar K7, Hennes V1,2,3, Seah C1,2,3, Nelson AR3,4, Cheng TY8, Lee SJ8, August PR9, Chen JA10, Wisniewski N10, Hanson-Smith V10, Belgard TG10, Zhang A10, Coba M3,11, Grunseich C12, Ward ME12, van den Berg LH13, Pasterkamp RJ7, Trotti D6, Zlokovic BV3,4, Ichida JK1,2,3.
Although studies in mice have indicated that C9orf72 activity is important for myeloid cell function 14,18, a report in mouse neurons and zebrafish suggests that C9orf72 isoform A may modulate poly-Q Ataxin-2 toxicity 20, and studies have implicated C9ORF72 in regulating autophagy 20,31,32,38, our study provides the first direct evidence showing that gain- and loss-of-function C9ORF72 mechanisms cooperate to cause the degeneration of human motor neurons. Recent studies have shown that C9ORF72 isoform A, but not isoform B, can form a functional complex with SMCR8 and WDR41 20. However, since both C9ORF72 isoforms rescue C9-ALS iMN degeneration in our assay, other mechanisms or protein interactions may underlie the rescue of patient iMNs.
Mice were anesthetized with i.p. ketamine (100 mg ⁄ kg) and xylazine (10 mg ⁄ kg), and body temperature kept at 36.9 ± 0.1°C with a thermostatic heating pad. Mice were placed in a stereotactic apparatus (ASI Instruments, USA) and the head is fixed accordingly. A burr hole was drilled, and an injection needle (33 gauge) was lowered into the hippocampus between CA1 and the dentate gyrus (AP −2.0, ML +1.5, DV −1.8). NMDA (20 nmol in 0.3 μl of phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL, USA). Simultaneously, or independently, Apilimod (0.3 μl of 20 μM in phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL, USA). The needle was left in place for an additional 8 min after the injection. Animals were euthanized 48 h later. Brains were quickly removed, frozen on dry ice, and stored at −80°C until processing. Thirty-micrometer-thick coronal sections were prepared using a cryostat. Every fifth section 1 mm anterior and posterior to the site of injection was stained with cresyl violet. The lesion area was identiﬁed by the loss of staining, measured by NIH ImageJ software and integrated to obtain the volume of injury.
Human lymphocytes from healthy subjects and ALS patients were obtained from the NINDS Biorepository at the Coriell Institute for Medical Research and reprogrammed into iPSCs as previously described using episomal vectors61. Briefly, mammalian expression vectors containing Oct4, Sox2, Klf4, L-Myc, Lin28, and a p53 shRNA were introduced into the lymphocytes using the Adult Dermal Fibroblast Nucleofector™ Kit and Nucleofector™ 2b Device (Lonza) according to the manufacturer’s protocol. The cells were then cultured on mouse feeders until iPSC colonies appeared. The colonies were then expanded and maintained on Matrigel (BD) in mTeSR1 medium (Stem Cell Technologies).