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]
Consistent with previous studies 3,4,6–8, patient iMNs (n=5 patients) had reduced C9ORF72 expression compared to controls (n=3; Fig. 2a and Supplementary Fig. 4a, 5b). While previous studies have linked low C9ORF72 levels to changes in vesicle trafficking or autophagy 18,20,30–33, it remains unknown if loss of C9ORF72 protein directly contributes to degeneration. Thus, we re-expressed C9ORF72 (isoform A or B) in iMNs using a retroviral cassette (Supplementary Fig. 4b) and found that both isoforms rescued C9ORF72 patient iMN survival in response to glutamate treatment (n=3 patients Fig. 2b and Supplementary Fig. 4c). This effect was specific for C9ORF72 iMNs, as forced expression of C9ORF72 did not rescue SOD1A4V iMN survival (Fig. 2c), nor did it improve the survival of control iMNs (n=2 controls Fig. 2d and Supplementary Fig. 4d).
We compared the differential expression results from our data to other transcriptomic datasets in ALS, obtained from the Gene Expression Omnibus (GEO). Raw Affymetrix array data (.CEL files) were downloaded for dataset GSE56504, and preprocessed using a standard exon array pipeline implemented using the R Bioconductor package oligo. For GSE56504, only the laser-capture microdissection samples were included/ Differential expression was calculated using the R Bioconductor package limma. RNA-seq counts data was obtained for dataset GSE67196. For GSE67196, only the frontal cortex samples were included. Normalization and differential expression analysis were performed using DESeq2.
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.

To determine if a deletion of C9ORF72 or the C9ORF72 repeat expansion caused changes in endosomal trafficking in motor neurons, we examined the number of early endosomes (RAB5+, EEA1+), late endosomes (RAB7+), and lysosomes (LAMP1+, LAMP2+, LAMP3+) in control, C9ORF72 patient, C9ORF72+/−, and C9ORF72−/− iMNs. We observed the most significant difference in the lysosomal population, with C9ORF72 patient iMNs (n=4 patients) having fewer LAMP1+, LAMP2+, and LAMP3+ vesicles than control iMNs (n=4 controls)(Fig. 3c, d and Supplementary Fig. 8a-d). C9ORF72+/− and C9ORF72−/− also harbored fewer LAMP1+, LAMP2+, and LAMP3+ vesicles than isogenic control iMNs, indicating that reduced C9ORF72 levels alone leads to a loss of lysosomes (Fig. 3c, e, f and Supplementary Fig. 8a-d). ASO-mediated knockdown of C9ORF72 expression also decreased lysosome numbers in iMNs (Supplementary Fig. 8e). Although membrane fractionation showed that control and patient iMNs have similar amounts of LAMP2 in the lysosomal membrane fraction (Supplementary Fig. 8f), analysis of the immunofluorescence intensity of LAMP proteins suggests that this is likely due to the fact that C9ORF72 patient and C9ORF72+/− iMNs have a higher concentration of LAMP proteins in their lysosomal membranes, possibly as a result of fewer lysosomes being present (Supplementary Fig. 8g). Using electron microscopy to identify lysosomes by their high election density 40, we verified that the vesicles reduced in C9ORF72-deficient cells were lysosomes (Fig. 3g-i). Forced expression of either C9ORF72 isoform restored the number of LAMP1+, LAMP2+, and LAMP3+ lysosomes in patient (n=4 patients) and C9ORF72-deficient iMNs (Fig. 3c-f and Supplementary Fig. 8a-h). To determine if loss of C9ORF72 activity reduces lysosome numbers in motor neurons in vivo, we measured the number of lysosomes in spinal motor neurons in Nestin-Cre-Stop-Flox-C9orf72 mice 22. C9orf72−/− motor neurons contained significantly fewer Lamp1+ lysosomes than control motor neurons (Fig. 3j, k).
Brand serves as commitment, as trust and as standard. It is the voting of the customers for a company and the natural sifter for customers to tell the good from the bad. Xuerong is convinced that quality is what comprises the brand. During the last 15 years we have cautiously and conscientiously created a brand channel partners and customers can trust. Today Xuerong can proudly claim that it is the leading role in edible mushroom and the market can also admit and prove our effort by higher value.
All experiments involving live vertebrates (cortical glial isolation) performed at USC were done in compliance with ethical regulations approved by the USC IACUC committee. All animal use and care at the University Medical Center Utrecht were in accordance with local institution guidelines of the University Medical Center Utrecht (Utrecht, the Netherlands) and approved by the Dierexperimenten Ethische Commissie Utrecht with the protocol number DEC 2013.I.09.069.
To determine if Pikfyve inhibition rescues gain-of-function processes in vivo, we measured DPR levels in C9-BAC transgenic mice 58 with or without Apilimod treatment. Although it was not previously reported 58, we observed significantly higher levels of GR+ punctae in hippocampal neurons in C9-BAC mice than controls (Fig. 6j) using a previously-validated poly(GR) antibody 11. These data are consistent with findings in another published C9-BAC mouse model 14, suggesting that poly(GR) may be a common feature of C9-BAC mice. We also detected a low level of poly(GR) in neurons from control mice (Fig. 6j), which may be derived from other repeat regions or proteins with short poly(GR) sequences. Nevertheless, GR+ punctae levels were significantly higher in C9-BAC mouse neurons than in controls (Fig. 6j). Importantly, Apilimod treatment significantly reduced the number of GR+ punctae in hippocampal neurons in C9-BAC mice after 48 hrs (Fig. 6i, j). Therefore, small molecule inhibition of Pikfyve rescues both gain- and loss-of-function disease processes induced by C9ORF72 repeat expansion in vivo.
Consistent with previous studies 3,4,6–8, patient iMNs (n=5 patients) had reduced C9ORF72 expression compared to controls (n=3; Fig. 2a and Supplementary Fig. 4a, 5b). While previous studies have linked low C9ORF72 levels to changes in vesicle trafficking or autophagy 18,20,30–33, it remains unknown if loss of C9ORF72 protein directly contributes to degeneration. Thus, we re-expressed C9ORF72 (isoform A or B) in iMNs using a retroviral cassette (Supplementary Fig. 4b) and found that both isoforms rescued C9ORF72 patient iMN survival in response to glutamate treatment (n=3 patients Fig. 2b and Supplementary Fig. 4c). This effect was specific for C9ORF72 iMNs, as forced expression of C9ORF72 did not rescue SOD1A4V iMN survival (Fig. 2c), nor did it improve the survival of control iMNs (n=2 controls Fig. 2d and Supplementary Fig. 4d).
“The Tale of the Curly-Bearded Guest” 231Studies Bian, Xiaoxuan . “Lun ‘Qiu ran ke zhuan’ de zuozhe, zuonian ji zhengzhi beijing” , in Dongnan daxue xuebao. Vol. 3, 2005, pp. 93-98. Cai, Miaozhen . “Chongtu yu jueze — ‘Qiu ran ke zhuan’ de renweu xingge suzao ji qi yihan” in Xingda renwen xuebao . Vol. 34, 2004, pp. 153-180. Zhang, Hong . “Du Guangting ‘Qiu ran ke zhuan’ de liuchuan yu yingxiang” in Zhongguo daojiao, vol. 1, 1997, pp. 28-31. Liu, Zhiwei . “Gujin ‘Qiu ran ke zhuan’ de yanjiu fansi” in Xibei daxue xuebao. Vol. 1, 2000. Sun, Yiping . Du Guangting pingzhuan. Nanjing: Nanjing daxue chubanshe, 2005. ___. “‘Qiu xu ke’ yu ‘Qiu ran ke’” in Zhongguo daojiao. vol. 6, 2005, pp. 14-17. Luo, Zhengming . Du Guangting daojiao xiaoshuo yanjiu . Chengdu: Bashu shushe, 2005. Wang, Meng’ou . “Qiuran ke yu Tang zhi chuangye chuangshuo” in Tangren xiaoshuo yanjiu siji. Taipei: Yiwen chubanshe, 1978, p. 254. Xu, Jiankun . “‘Qiu ran ke zhuan’ jili jiegou xintan” in Donghai zhongwen xuebao . Vol. 11, 1994, pp. 61-72. Ye, Qingbing . “‘Qiu ran ke zhuan’ de xiezuo jiqiao” in Zhongguo gudian wenxue yanjiu congkan — Xiaoshuo zhi bu . Taipei: Juliu, 1977, pp. 167-79.
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]
During lysosomal biogenesis, lysosomal proteins are transported in Mannose-6-Phosphate Receptor (M6PR)+ vesicles from the trans-Golgi Network to early and late endosomes for eventual incorporation into lysosomes 41. Disruption of M6PR+ vesicle trafficking can lead to a reduction in lysosome numbers 42 and altered localization of M6PR+ vesicles 43. In control iMNs (n=3 controls), M6PR+ vesicles were distributed loosely around the perinuclear region and to a lesser extent in the non-perinuclear cytosol (Supplementary Fig. 9a, b). In contrast, C9ORF72 patient (n=4 patients), C9ORF72+/−, and C9ORF72−/− iMNs frequently harbored densely-packed clusters of M6PR+ vesicles (Supplementary Fig. 9a, b). This was not due to a reduced number of M6PR+ vesicles in patient and C9ORF72-deficient iMNs (Supplementary Fig. 9c). Forced expression of C9ORF72 isoform B restored normal M6PR+ vesicle localization in patient (n=4 patients) and C9ORF72-deficient iMNs, confirming that a lack of C9ORF72 activity induced this phenotype (Supplementary Fig. 9a, b).
The GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for about 10% of each disease worldwide 1–4. In the central nervous system (CNS), neurons and microglia express the highest levels of C9ORF72 5, suggesting that C9ORF72 acts in part cell autonomously and effects in neurons are a key source of disease etiology. Studies showing that the repeat expansion generates neurotoxic species including nuclear RNA foci 6–8, RNA/DNA G-quadruplexes 9, and dipeptide repeat proteins (DPRs) 10–12 have oriented the field towards a therapeutic focus on blocking the toxicity of these products 6–8,13,14. However, these strategies have not fully rescued the degeneration of patient-derived neurons 7,13. Moreover, tandem GGGGCC repeats are transcribed from over 80 other genomic locations within human spinal motor neurons (Supplementary Tables 1 and 2), yet genetic studies have not linked repeat expansions in these regions to ALS/FTD. In addition, hexanucleotide repeat-mediated toxicity in mice requires supraphysiological expression levels or a specific genetic background 14–16. These observations suggest that there are additional pathogenic triggers caused by repeat expansion within C9ORF72.

Local field potentials (LFPs) were recorded from iPSC-derived motor neurons on days 17–21 in culture in 6-well multielectrode chips (9 electrodes and 1 ground per well) using a MultiChannel Systems MEA-2100 multielectrode array (MEA) amplifier (ALA Scientific) with built-in heating elements set to 37°C. Cells were allowed to acclimate for 5 minutes after chips were placed into the MEA amplifier, and after glutamate addition (10 μM final concentration). For 1 μM Apilimod treatments, chips were incubated for 35 min in a humidified incubator in the presence of the particular drug, then returned to the MEA amplifier and acclimated for 5 min before beginning recordings. For each condition, recordings (5 min baseline, 10 min glutamate and/or drug, 40 kHz sampling rate) were filtered between 1–500 Hz, and average LFP frequency per well was determined using the accompanying MC Rack software.
GCaMP6 was cloned into the pMXs-Dest-WRE retroviral vector and transduced into reprogramming cultures concurrently with the motor neuron factors. To assess GCaMP6 activity, 1.5 μm glutamate was added to iMN cultures and cells were imaged continuously for 2 minutes at 24 frames per second. GFP flashes were scored manually using the video recording. At least 3 different fields of view from three independent cultures, totalling 50–100 iMNs, were scored per condition.
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.
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.
×