Post mortem tissues were kindly provided by Neil Shneider (Columbia) and were collected from the following individuals: Sample 1 – age: 64, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 2 – age: 55, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 3 – age: 65, diagnosis: ALS, genotype: positive for C9ORF72 repeat expansion, Sample 4 – age: 65, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 5 – age: 50, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 6 – age: 50, diagnosis: control, genotype: negative for C9ORF72 repeat expansion, Sample 7 – age: 53, diagnosis: ALS, genotype: negative for C9ORF72 repeat expansion, Sample 8 - age: 64, diagnosis: ALS, genotype: negative for C9ORF72 repeat expansion. All donors except donor 7 (sample 7) were female. For immunofluorescence, 10 µm sections were sliced from flash frozen lumbar spinal cord tissues. Sections were then air dried and fixed with ice cold acetone for 10 minutes, and blocked with 10% normal goat serum/1% BSA/0.3% Triton-X/PBS at room temperature for 1 hour followed by incubation with NR1 antibody (1:200, BD Bioscience) in blocking buffer overnight at 4 ºC. Sections subsequently were blocked using avidin/biotin kit (Vector Lab), and washed with PBS. Then, sections were incubated with goat anti-rabbit IgG Biotin conjugate secondary antibody (1:750, Invitrogen) or with goat anti-mouse IgG Biotin conjugate secondary antibody (1:750, Invitrogen) for 1 hour at room temperature, washed and incubated with streptavidin-Alexa Fluor 488 conjugate (1:500, Invitrogen) in dark for 1 hour at room temperature. Sections were washed and blocked again in blocking buffer for 1 hour at room temperature. For neuronal marker staining, sections were incubated with Tu-20 antibody (1:1000, Abcam) or NeuN antibody (1:500, Abcam) at 37 ºC for 1 hour. Sections were washed with PBS and incubated with goat anti-mouse Alexa Fluor 546 (1:500, Invitrogen) or goat anti-rabbit Alexa Fluor 546 (1:500, Invitrogen) for 1 hour at room temperature. Lipofuscin autofluorescence was quenched by immersing sections in autofluorescence eliminator reagent (Millipore) for 4 minutes following manufacture’s instruction. Sections were then counterstained and mounted with Prolong Gold antifade mounting medium with DAPI (Invitrogen).
CRISPR/Cas9-mediated genome editing was performed in human iPSCs as previously described, using Cas9 nuclease62. To generate loss-of-function alleles of C9ORF72, control iPSCs were transfected with a sgRNA targeting exon 2 of the C9ORF72 gene. Colonies were picked on day 7 after transfection and genotyped by PCR amplification and sequencing of exon 2. Colonies containing a frameshift mutation were clonally purified on MEF feeders and the resulting clones were re-sequenced to verify the loss-of-function mutation in C9ORF72.

“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 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.
International Advisory Board: James Archibald (Translation Studies) - Hugo de Burgh (Chinese Media Studies) - Kristen Brustad (Arabic Linguistics) - Daniel Coste (French Language) - Luciano Curreri (Italian Literature) - Claudio Di Meola (German Linguistics) - Donatella Dolcini (Hindi Studies) - Johann Drumbl (German Linguistics) - Denis Ferraris (Italian Literature) - Lawrence Grossberg (Cultural Studies) - Stephen Gundle (Film and Television Studies) - Tsuchiya Junji (Sociology) - John McLeod (Post-colonial Studies) - Estrella Montolío Durán (Spanish Language) - Silvia Morgana (Italian Linguistics) - Samir Marzouki (Translation, Cultural Relations) - Mbare Ngom (Post-Colonial Literatures) - Christiane Nord (Translation Studies) - Roberto Perin (History) - Giovanni Rovere (Italian Linguistics) - Lara Ryazanova-Clarke (Russian Studies) - Shi-Xu (Discourse and Cultural Studies) - Srikant Sarangi (Discourse analysis) - Françoise Sabban, Centre d'études sur la Chine moderne et contemporaine (Chinese Studies) - Itala Vivan (Cultural Studies, Museum Studies)
(a) Production of Hb9::RFP+ iMNs and survival tracking by time-lapse microscopy. (b-d) Survival of control (CTRL) and C9ORF72 patient (C9-ALS) iMNs with neurotrophic factors (b) or in excess glutamate (shown with iMNs from all lines in aggregate (b, c) or for each individual line separately (d)). For (b-d), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. (e) iMNs at day 22 in excess glutamate. This experiment was repeated three times with similar results. (f-g) Survival of control and C9-ALS iMNs in excess glutamate with glutamate receptor antagonists (f) or without neurotrophic factors (g). For (f-g), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. (h) Survival of induced dopaminergic (iDA) neurons in excess glutamate. n=50 iMNs per line for 2 control and 2 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. Except in (d), each trace includes neurons from at least 2 donors with the specified genotype; see full detail in Methods. Scale bar: 100 μm (e). All iMN survival experiments were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. iMN survival experiments in (b-g) were performed in a Nikon Biostation and experiments in (h) were performed in a Molecular Devices ImageExpress.
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 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]
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
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.
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.

IPSC-MNs at differentiation D35 were harvested in cold Hypotonic buffer (20 mM HEPES pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1mM EGTA, 1 mM DTT and protease inhibitor cocktail (Roche)) and lysed by passing through G25 needles 25 times and then spun down at 700 x g for 10min at 4℃. The Supernatant was loaded onto pre-made 30% Percoll solution and re-centrifuged at 33,000 RPM using Beckman rotor SWI55 for 50min at 4℃. 300 ul aliquots were taken from top to bottom as fractions and all the collected samples were boiled with SDS-PAGE sample buffer and analyzed by western blot.
Importantly, our work establishes a new approach for suppressing DPR protein toxicity and blocking C9ORF72 pathogenesis: restoring or replacing C9ORF72 activity. Although high levels of C9ORF72 isoform A may have slightly detrimental effects on control motor neuron survival, we have only observed this in neurons without C9ORF72 repeat expansion. Thus, we would not anticipate a harmful effect of forced C9ORF72 expression in C9ORF72 patients. In addition, a better understanding of the effects of forced C9ORF72 expression could inform safe development of this therapeutic strategy. For example, determining if C9ORF72 accelerates turnover of DPR aggregates by stimulating autophagy could lead help to identify new therapeutic targets.
Because C9ORF72 activity is required to maintain normal lysosomal function, we measured the effect of C9ORF72 activity on PR50 clearance by monitoring the clearance of PR50-Dendra2 fusion proteins in C9ORF72−/− iPSC-derived fibroblasts with or without exogenous C9ORF72. Dendra2 is a green fluorescent protein that irreversibly converts to red fluorescence when exposed to blue light, enabling quantification of its degradation 49. PR50-Dendra2 formed discrete punctae within cells, indicating that Dendra2 did not prevent intracellular aggregation of PR50 (Supplementary Fig. 14c). Expression of C9ORF72-T2A-GFP in C9ORF72−/− iPSC-derived fibroblasts significantly enhanced the decay of PR50-Dendra2 fluorescence over GFP alone (Supplementary Fig. 14d). To determine if C9ORF72 activity modulates DPR aggregate clearance in human motor neurons, we compared the decay of PR50-Dendra2 in C9ORF72+/+ and C9ORF72+/− iMNs (Fig. 5e and Suppementary Fig. 14e). Consistent with the hypothesis that C9ORF72 activity promotes DPR aggregate clearance, PR50-Dendra2 decayed significantly slower in C9ORF72+/− iMNs (Fig. 5e).
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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).
Amongst four reproducible hit compounds, we identified a PIKFYVE kinase inhibitor (YM201636) that significantly increased C9ORF72 patient iMN survival (n=2 patients) (Fig. 6b, c and Supplementary Fig. 15a). PIKFYVE is a lipid kinase that converts phosphatidylinositol 3-phosphate (PI3P) into phosphtidylinositol (3,5)-bisphosphate (PI(3,5)P2)51(Fig. 6f). PI3P is primarily generated by PI3-kinases recruited to early endosomes by RAB5, and PI3P anchors EEA1 to early endosomes to drive endosomal maturation 52(Fig 6f). Following endosomal maturation into lysosomes, PI3P drives fusion of lysosomes with autophagosomes 53. PIKFYVE regulates PI3P levels by converting PI3P into PI(3,5)P2 52, which disfavors lysosomal fusion with endosomes and autophagosomes 53,54. Therefore, inhibition of PIKFYVEincreases autophagosome-lysosome fusion 53 and may compensate for reduced C9ORF72 activity and other disease processes by increasing PI3P levels to facilitate removal of glutamate receptors or DPRs (Fig. 6f). Interestingly, FIG4 is a phosphatase that opposes PIKFYVE kinase by converting PI(3,5)P2 to PI3P and loss-of-function mutations in FIG4 cause ALS 55. Thus, genetic evidence suggests that PIKFYVE inhibition may be capable of modulating ALS disease processes in humans.
To examine C9ORF72 function, we determined its localization in iMNs. We first used an HA-tagged C9ORF72 construct to verify that the C9ORF72 antibody specifically recognizes C9ORF72 in cells (Supplementary Fig. 7a). In iMNs, C9ORF72 co-localized to cytoplasmic puncta and ASO-mediated knockdown of C9ORF72 expression reduced the number of antibody-detected cytoplasmic puncta in iMNs, indicating that the antibody specifically recognizes C9ORF72 in these puncta (Supplementary Fig. 7b, c). Super-resolution microscopy and z-stack imaging showed that about 80% of the C9ORF72+ vesicles also expressed the early endosomal proteins RAB5 and EEA1 (Fig. 3a and Supplementary 7d-h). Only rarely did C9ORF72 co-localize with the lysosomal marker LAMP1 (20%)(Supplementary Fig. 7e), and control and patient iMNs showed similar C9ORF72 localization (Supplementary Fig. 7h). We performed density gradient centrifugation on lysates from iPSC-derived motor neurons to separate light (endosomal) and heavy (lysosomal) membrane fractions. C9ORF72 co-segregated with EEA1 and not LAMP1, supporting the notion that C9ORF72 localizes predominantly in early endosomes (Fig. 3b, Supplementary Fig. 5d). In addition, we found that C9ORF72 isoform B bound strongly to an immobilized N-terminal fragment of EEA1 (Supplementary Fig. 7i). C9ORF72 isoform A did not interact as strongly with EEA1 (Supplementary Fig. 7i). The fact that not all EEA1+ vesicles contained high levels of C9ORF72 is consistent with this hypothesis and suggests that C9ORF72 may not localize to all types of EEA1+ vesicles (Fig. 3a).
Eliminating C9ORF72 protein expression from one or both alleles reduced iMN survival to levels comparable to patient iMNs (Fig. 2f). Antisense oligonucleotide (ASO)-mediated suppression of C9ORF72 expression levels also reduced control iMN survival (Fig. 2g and Supplementary Fig. 4j), suggesting that reduced iMN survival was not due to an off-target effect of the CRISPR/Cas9 genome editing. Exogenously restoring C9ORF72 expression in C9ORF72+/− and C9ORF72−/− iMNs rescued survival (Supplementary Fig. 4k, l), verifying that depletion of C9ORF72 caused the observed neurodegeneration.

Wuqiao County (simplified Chinese: 吴桥县; traditional Chinese: 吳橋縣; pinyin: Wúqiáo Xiàn, literally "Wu Bridge") is a county of southeastern Hebei province, China, bordering Shandong province to the southeast. It is the southernmost county-level division of the prefecture-level city of Cangzhou. Wuqiao covers an area of 583 km2 (225 sq mi) with a population of 280,000 and 444 natural villages under its jurisdiction. Over a period of more than 1500 years, Wuqiao is an old county with a vivid and rich history and culture. Wuqiao is situated in the center of the Huabei Plains and has a pleasant climate most of the year round and it is possible to pleasurably visit here at almost any time of the year.[citation needed]
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.
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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