“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.
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.
(a-b) Survival of control and CRISPR-mutant iMNs without excess glutamate with overexpression of eGFP or PR(50)-eGFP (a) or GR(50)-eGFP (b). (c-d) Survival of control and C9-ALS iMNs without excess glutamate with overexpression of eGFP or PR(50)-eGFP (c) or GR(50)-eGFP (d). For (a), n=50 (CTRL1 + GFP AND CTRL1 + PR(50)), 49 (C9ORF72+/− + GFP), and 47 (C9ORF72+/− + PR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line. For (b), n=50 (CTRL1 + GFP AND CTRL1 + GR(50)), 49 (C9ORF72+/− + GFP), and 40 (C9ORF72+/− + GR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line. For (c), n=50 (CTRL1 + GFP AND CTRL1 + PR(50)), 50 (from each of two C9-ALS lines + GFP), and 41 (from each of two C9-ALS lines + PR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line per condition. For (d), n=50 (CTRL1 + GFP AND CTRL1 + GR(50)), 50 (from each of two C9-ALS lines + GFP), and 46 and 47 (from two C9-ALS lines + GR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line per condition. All iMN survival experiments in (a-d) were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. Survival curves for the “+GFP” condition were included as a reference, but were not used in statistical analyses. (e) Relative decay in Dendra2 fluorescence over 12 hours in iMNs of specified genotypes. Mean +/− s.e.m. n = 18 (control) and 24 (C9ORF72+/−) iMNs quantified from two biologically independent iMN conversions each, two-tailed t-test with Welch’s correction between data points at each time point, t-value: 2.739, degrees of freedom: 25.62). (f-h) Immunostaining to determine endogenous PR+ puncta in control or C9-ALS iMNs with or without overexpression of C9ORF72 isoform A or B. Scale bar = 2 μm. This experiment was repeated twice with similar results. (g) Mean +/− s.d. n= 4 biologically independent iMN conversions generated from two different iPSC lines per genotype. Quantified values represent the average number of PR+ puncta in 40 iMNs from a single iMN conversion. Two-tailed t-test, t-value: 5.908, degrees of freedom: 6. (h) Mean +/− s.e.m. n= 3 biologically independent iMN conversions per condition. Quantified values represent the average number of PR+ puncta in 40 iMNs from a single iMN conversion. One-way ANOVA with Tukey correction, F-value (DFn, DFd): (2, 6)=10.5. iMN survival experiments in (a-d) were performed in a Molecular Devices ImageExpress.
For all experiments, sample size was chosen using a power analysis based on pilot experiments that provided an estimate of effect size (http://ww.stat.ubc.ca/~rollin/stats/ssize/n2.html). Mice used for immunohistochemical analysis were selected randomly from a set of genotyped animals (genotypes were known to investigators). Mouse and human tissue sections used for immunohistochemical analysis were selected randomly. For mouse tissues, sections were prepared using an approximately equal representation of all levels of the spinal cord, and of those, all were imaged and quantified. The sections were only not used if NeuN or Chat immunostaining failed. For iMN survival assays, assays were repeated at least twice, with each round containing 3 biologically independent iMN conversions. iMNs from the 3 biologically independent iMN conversions in one representative round was used to generate the Kaplan-Meier plot shown. iMN survival times were confirmed by manual longitudinal tracking by an individual who was blinded to the identity of the genotype and condition of each sample. To select 50 iMNs per condition for analysis, >50 neurons were selected for tracking randomly at day 1 of the assay. Subsequently, the survival values for 50 cells were selected at random using the RAND function in Microsoft Excel. For quantification of immunofluorescence, samples were quantified by an individual who was blinded to the identity of the genotype of each sample.
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)
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.

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).
To verify that PIKFYVE-dependent modulation of vesicle trafficking was responsible for rescuing C9ORF72 patient iMN survival, we tested the ability of a constitutively active RAB5 mutant to block C9ORF72 patient iMN degeneration. Active RAB5 recruits PI3-kinase to synthesize PI3P from PI and therefore, like PIKFYVE inhibition, increases PI3P levels 56. Constitutively active RAB5 did not improve control iMN survival (n=2 controls)(Supplementary Fig. 15k), but successfully rescued C9ORF72 patient iMN survival (n=3 patients)(Supplementary Fig. 15l). In constrast, dominant negative RAB5, wild-type RAB5, or constitutively active RAB7 did not rescue C9ORF72 patient iMN survival (n=1, 3, 3 patients, respectively)(Supplementary Fig. 14m-o).
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.
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).
However, C9orf72-deficient mice do not display overt neurodegenerative phenotypes 14,18,19,22. Moreover, no studies have shown that reduced C9ORF72 activity leads to the degeneration of C9ORF72 ALS patient-derived motor neurons, nor have any provided direct evidence identifying a cellular pathway through which C9ORF72 activity modulates neuronal survival. Additionally, a patient homozygous for the C9ORF72 repeat expansion had clinical and pathological phenotypes that were severe but nonetheless did not fall outside the range of heterozygous patients, leaving it uncertain if reductions in C9ORF72 protein levels directly correlate with disease severity 23. Thus, the role of the C9ORF72 protein in C9ORF72 ALS/FTD disease pathogenesis remains unclear.
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.
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).
Structured illumination microscopy (SIM) images were acquired using a Zeiss Elyra PS.1 system equipped with a 100X 1.46 NA or 63X 1.4NA objective. Acquisition was performed with PCO edge sCMOS camera and image reconstruction was done with built-in structured illumination model. Confocal microscopy images were acquired using Zeiss LSM800 microcopy with 63X 1.4NA objective or Zeiss LSM780 microcopy with 40X 1.1NA objective. Z stack images were done with a step size of 2.5 um. Further image process was done with Fiji.
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]
To verify that PIKFYVE-dependent modulation of vesicle trafficking was responsible for rescuing C9ORF72 patient iMN survival, we tested the ability of a constitutively active RAB5 mutant to block C9ORF72 patient iMN degeneration. Active RAB5 recruits PI3-kinase to synthesize PI3P from PI and therefore, like PIKFYVE inhibition, increases PI3P levels 56. Constitutively active RAB5 did not improve control iMN survival (n=2 controls)(Supplementary Fig. 15k), but successfully rescued C9ORF72 patient iMN survival (n=3 patients)(Supplementary Fig. 15l). In constrast, dominant negative RAB5, wild-type RAB5, or constitutively active RAB7 did not rescue C9ORF72 patient iMN survival (n=1, 3, 3 patients, respectively)(Supplementary Fig. 14m-o).
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