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.
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.
Near the cities Beijing, Tianjin, Shijiazhuang, and Jinan, Wuqiao County has many transportation connections. There are many rail and bus services operating in the town. Wuqiao was the first Chinese city to open up its doors to the world under the "Open Door" policy and over many years development, Wuqiao has become a flourishing city with a favorable investment environment.
All animal use and care 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. Wild-type C57BL6/J (strain: 000664), C9ORF72 KO (C57BL/6J-3110043021Rikem5Lutzy/J, strain: 027068), and C9-BAC (C57BL/6J-Tg(C9orf72_i3)112Lutzy/J, strain: 023099) were purchased from Jackson Laboratories (The Jackson Laboratory, Bar Harbor, USA). Mice were housed in standard conditions with food and water ad libitum in the conventional vivarium at the University of Southern California. All animal use and care were in accordance with local institution guidelines of the University of Southern California and the IACUC board of the University of Southern California (Los Angeles, USA) with the protocol numbers 20546 and 11938.
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.
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,†
(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.
The repeat expansion suppresses the production of C9ORF72 protein by inhibiting transcription 3,4,6,7,9,17, raising the possibility that haploinsufficiency for C9ORF72 activity triggers disease pathogenesis. Consistent with this hypothesis, elimination of C9orf72 activity alters myeloid cell behavior in mice 14,18,19 and in vitro studies suggest that C9ORF72 activity may enhance autophagy 20,21.
(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.
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.
(a) Phenotypic screening for small molecules that enhance the survival of C9-ALS iMNs. (b) Chemical structure of the PIKFYVE inhibitors YM201636 and Apilimod, and a reduced-activity analog of Apilimod. (c) Live cell images of iMNs at day 7 of treatment with DMSO or YM201636 (scale bar: 1 mm). This experiment was performed 3 times with similar results. (d) Survival effect of scrambled or PIFKVYE ASOs on C9-ALS iMNs in excess glutamate. n=50 iMNs per condition, iMNs quantified from 3 biologically independent iMN conversions per condition. (e) Survival effect of Apilimod and the reduced-activity analog on C9-ALS patient iMNs with neurotrophic factor withdrawal. n=50 iMNs per condition, iMNs quantified from 3 biologically independent iMN conversions per condition. All iMN survival experiments in (d, e) were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. (f) Activities of therapeutic targets in C9ORF72 ALS. (g, h) The effect of 3 μM Apilimod on NMDA-induced hippocampal injury in control, C9orf72+/−, or C9orf72−/− mice. (Mean +/− s.e.m. of n=3 mice per condition, one-way ANOVA with Tukey correction across all comparisons, F-value (DFn, DFd): (3, 8)=43.55, AP = Apilimod, red dashed lines outline the injury sites). (i, j) The effect of 3 μM Apilimod on the level of GR+ puncta in the dentate gyrus of control or C9-BAC mice. Mean +/− s.d. of the number of GR+ puncta per cell, each data point represents a single cell. n=20 (wild-type + DMSO), 20 (wild-type + Apilimod), 87 (C9-BAC + DMSO), and 87 (C9-BAC + Apilimod) cells quantified from 3 mice per condition, one-way ANOVA with Tukey correction for all comparisons, F-value (DFn, DFd): (3, 180) = 16.29. Scale bars = 2 μm, dotted lines outline nuclei, and white arrows denote GR+ punctae (i). (k) Model for the mechanisms that cooperate to cause neurodegeneration in C9ORF72 ALS/FTD. Proteins in red are known to be mutated in ALS or FTD. iMN survival experiments in (d, e) were performed in a Molecular Devices ImageExpress.
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To verify that PIKFYVE is the functional target of the inhibitor, we first confirmed PIKFYVE expression by qPCR in control and patient (n=3 patients) iMNs (Supplementary Fig. 15b). Next, we verified that YM201636 rescued C9ORF72 patient iMN survival in a dose-dependent manner (Supplementary Fig. 15c). We then asked if Apilimod, a structurally distinct PIKFYVE inhibitor, could rescue patient iMN survival 51(Fig. 6b). To verify target engagement by Apilimod in iPSC-derived motor neurons, we administered Apilimod for three hours and measured EEA1+ early endosome size. PIKFYVE inhibition increases PI3P levels, leading to increased recruitment of EEA1 to early endosomes, more homotypic early endosomal fusion, and larger EEA1+ early endosomes 54. As expected, Apilimod treatment increased EEA1+ endosome size in a dose-dependent manner, verifying target engagement in motor neurons (Supplementary Fig. 15d, e).
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.
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).
To determine if the survival difference between C9ORF72 patient iMNs and controls was specific to our transcription factor-based reprogramming approach, we also measured the survival of Hb9::RFP+ control and C9ORF72 patient motor neurons derived from iPSCs by small molecule activation of the Sonic Hedgehog and retinoic acid signaling pathways 28 (Supplementary Fig. 3g, h). Similarly to iMNs, morphogen-generated motor neurons showed a significant survival difference between C9ORF72 patients and controls (Supplementary Fig. 3i-l).
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
Journalistic genres in China have acquired distinctive characteristics and have shaped original sub-genres that are unique to the local journalistic tradition. While many studies analyzing their characteristics have been written in Chinese, works on the subject in other languages are still scarce. This contribution aims to fill this void by presenting the two main genres in which written journalistic production can be understood, i.e., “news” and “views”, as well as their sub-genres, and showing how they are interpreted in Chinese media studies. The analysis is based on a corpus of recent academic publications that represent the current Chinese scholarly interpretations of local genres of journalism. In doing so, the paper also offers insights on recent theoretical reflections about the functions of journalistic writing in the People’s Republic of China.
In September 2012, Xuerong biotechnology join hand in hand with China Space, becoming the only edible fungi enterprise in the industry that won the honor, the only food partner of China Space in Shanghai area. Xuerong will establish Xuerong space ecological mushroom laboratory with the Chinese Astronaut Center Space Nutrition and Food Laboratory, promote the concept of Xuerong's "Ecological Beauty Restored by Science and Technology" to acme, bring safe healthy, nutritious, fresh, delicious top space level mushrooms to consumers.
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).
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).