To determine if glutamate receptor accumulation occurs on C9ORF72 patient motor neurons in vivo, we measured glutamate receptor expression in ventral horn neurons in lumbar spinal cord samples from 3 C9ORF72 ALS patients and 3 unaffected controls. We identified motor neurons by size and confirmed that most neurons selected in this manner were CHAT+ and SMI-32+ (Supplementary Fig. 12d). Spinal motor neurons from the C9ORF72 ALS patients displayed higher NR1 levels than control neurons (Supplementary Fig. 12e). In addition, post-synaptic densities isolated from the motor corticesof C9ORF72 patients had higher levels of NR1 and GLUR1 than controls (Fig. 4k, l and Supplementary Fig. 5k).
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,†
Primary chick myoblasts were dissected from D11 chick embryos and plated onto plastic dishes pre-coated with 0.1% gelatin. After 3 days of culture in muscle medium containing F10 (Life Technologies), 10% horse serum, 5% chicken serum (Life Technologies), 0.145 mg/ml CaCl2 (Sigma), and 2% Penicillin/Streptomycin, myoblasts were trypsinized and replated onto iMNs which were at days 15–18 post-transduction. The co-culture was maintained in neuronal medium containing DMEM/F12, 2% B27, 1% GlutaMax and 1% Penicillin/Streptomycin, supplemented with 10ng/ml BDNF, GDNF, and CNTF for 7 days in order to allow neuromuscular junctions to form. Videos were taken using Nikon Eclipse Tis microscope with NIS Element AR software. Light-stimulated contraction shown in Supplementary Figure 2j are representative of contraction observed in 2 biological replicates, with 5 contractile sites per replicate.
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.
Base text for this translation. ___. Wang Meng’ou’s , ed. Tangren xiaoshuo jiaoshi . Taipei: Zhongzheng Shuju, 1983, 2319-38. For other texts and editions see footnote 1. Translations Birch, Cyril. “The Curly-bearded Hero,” in Anthology of Chinese Literature, v. 1, New York, 1965, pp. 314-322. Chai, Ch’u, and Winberg Chai. “The Curly-Bearded Guest,” in A Treasury of Chinese Literature, New York, 1965, pp. 117-124. Hsu Sung-nien. “Biographie d’un preux barbu,” Anthologie de la littérature chinoise.Paris: Delagrave, 1933, pp. 241-6. Levenson, Christopher, tran., The Golden Casket. Harmondsworth, Middlesex: Penguin Books, 1967, pp. 137-47. Lévy, André. “Barbe-bouclée, L’étranger à la barbe et aux favoris bouclés,” in Histoires extraordinaires et récits fantastiques de la Chine ancienne.Paris: Flammarion, 1993, pp. 177-195 (with notes). Lin Yutang. “Curly-Beard,” in Famous Chinese Short Stories. New York: John Day (Cardinal), 1953, pp. 3-22. Schafer, E.H. “Three Divine Women of South China,” CLEAR, 1 (1979), pp. 31-42. Wang, Elizabeth Te-chen, tran. “The Curly-Bearded Guest,” in Wang’s Ladies of the Tang: 22 Classical Chinese Stories. Taipei: Mei Ya Publications, 1973, pp. 133-50.
Our results highlight the importance of C9ORF72 protein function, RAB5 activity, PI3P levels, and lysosomeal function as key therapeutic targets for C9ORF72 ALS/FTD. By generating PI3P, RAB5 drives early endosomal maturation and the initial stages of lysosomal biogenesis (Fig. 6f)59. PI3P also plays important roles in autophagosome formation and autophagsome-lysosome fusion. Indeed, a previous study suggests that PIKFYVE inhibition may increase autophagic flux 53, and this should be investigated in the context of motor neurons. Loss-of-function mutations in two other genes whose proteins function to increase PI3P levels, ALS2 and FIG4, also cause ALS 1. ALS2 encodes the RAB5 guanine exchange factor ALSIN 60, while FIG4 converts PI(3,5)P2 into PI3P 55(Fig. 6f). In addition, proteins encoded by several other ALS genes play key roles in lysosomal biogenesis, including CHMP2B, OPTN, and SQSTM1 1. The fact that FIG4 and ALS2 loss-of-function mutations can cause ALS suggests that PIKFYVE inhibition or RAB5 activation may be capable of modulating ALS disease processes in humans.
To generate Dox-NIL iMNs, the Dox-NIL construct was integrated into the AAVS1 safe harbor locus of the control, C9+/−, and C9-ALS patient iPSC lines using CRISPR/Cas9 editing (gRNA sequence shown in Supplementary Table 4). Dox-NIL iMNs were generated by plating at ~25% confluency on matrigel coated plates and adding 1 μg/ml of doxycylin in N3 media +7.5 μM RepSox 1 day after plating. Mouse primary mixed glia were added to the cultures at day 6, and doxycyline was maintained throughout conversion. iMN cultures were harvested at day 17.
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.
To determine if patient iMN degeneration resulted from bona fide ALS disease processes specific for motor neurons, we measured the survival of induced dopaminergic neurons (iDAs) generated by expression of FoxA2, Lmx1a, Brn2, Ascl1, and Myt1l 29. These neurons expressed high levels of tyrosine hydroxylase, indicating they had established a key aspect of the dopamine synthesis pathway and were distinct from iMNs, which do not express this enzyme 24 (Supplementary Fig. 3m, n). Unlike iMN cultures, iDA cultures from C9ORF72 patients (n=2 patients) did not show reduced survival compared to controls (n=2 controls) in either glutamate treatment and neurotrophic factor withdrawal conditions (Fig. 1h and Supplementary Fig. 3o), indicating that the in vitro neurodegenerative phenotype elicited by the C9ORF72 mutation is selective for motor neurons.
Biotinylation of plasma membrane localized glutamate receptors was performed using the Piece™ Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, Dox-NIL iMNs were incubated with 0.25mg/ml Sulfo-NHS-SS-Biotin in cold room for 1~2 hrs with end-to-end shaking. After quenching, cells were harvested by scraping and lysed with lysis buffer from the Piece™ Cell Surface Protein Isolation Kit or the M-PER™ mammilian protein extraction buffer (Thermo Fisher Scientific). Cell lysate was incubated with High Capacity NeutrAvidin™ agorase beads (Thermo Fisher Scientific), and the bound protein was eluted in 2X SDS-PAGE sample buffer supplemented with 50mM DTT for 1 hr at room temperature with end-to-end rotation, and further analyzed by western blot.
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 also found that Reduced C9ORF72 activity also induces iMN hypersensitivity to DPRs by impairing their clearance. This uncovers a more direct form of cooperative pathogenesis between gain- and loss-of-function mechanisms in C9ORF72 ALS/FTD. Through a potentially similar mechanism, reduced C9orf72 levels can also facilitate cytoplasmic TDP-43 accumulation in mouse neurons 20.
iPSC motor neurons were generated as described previously28, with slight modifications. On day 0, iPSCs were dissociated with Accutase (Life Technologies) and 300,000 iPSCs were seeded into one Matrigel (Corning)-coated well of a 6-well plate in mTeSR medium (Stem Cell Technologies) with 10 μM Rock Inhibitor (Selleck). On day 1, the medium was changed to Neural Differentiation Medium (NDM) consisting of a 1:1 ratio of DMEM/F12 (Genesee Scientific) and Neurobasal medium (Life Technologies), 0.5x N2 (Life Technologies), 0.5x B27 (Life Technologies), 0.1 mM ascorbic acid (Sigma), 1x Glutamax (Life Technologies). 3 μM CHIR99021 (Cayman), 2 μM DMH1 (Selleck), and 2 μM SB431542 (Cayman) were also added. On day 7, cells were dissociated with Accutase and 4.5 million cells were seeded into Matrigel coated 10cm dishes in NDM plus 1 μM CHIR99021, 2 μM DMH1, 2 μM SB431542, 0.1 μM RA (Sigma), 0.5 μM Purmorphamine (Cayman), and 10 μM Rock Inhibitor. Rock inhibitor was removed on day 9. On day 13, cells were dissociated with Accutase and seeded at a density of 40 million cells per well in a non-adhesive 6 well plate (Corning) in NDM plus 0.5 μM RA, 0.1 μM Purmorphamine, and 10 μM Rock Inhibitor. On day 19, the media was changed to NDM plus 1 μM RA, 1 μM Purmorphamine, 0.1 μM Compound E (Cayman), and 5 ng/ml each of BDNF, GDNF and CNTF (R&D Systems). Cells were used for experiments between days 25–35 of differentiation.
(a) Super-resolution microscopy images of immunofluorescence shows NR1+ puncta on neurites of iMNs overexpressing eGFP or C9ORF72 isoform B-eGFP. Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (b-d) Number of NR1+ puncta per unit area in control (b-d), patient (b), C9ORF72+/− (c), and C9ORF72+/− (d) iMNs. Mean ± s.d. Each grey open circle represents the number of NR1+ puncta per area unit on a single neurite (one neurite quantified per iMN). For (b), n=75 (CTRL + GFP), 84 (C9-ALS + GFP), 95 (C9-ALS + isoA), and 111 (C9-ALS + isoB) iMNs quantified from two biologically independent iMN conversions of 3 CTRL or 4 C9-ALS lines. For (c), n=37 (CTRL + GFP), 37 (C9ORF72+/− + GFP), 25 (C9ORF72+/− + isoA), and 27 (C9ORF72+/− + isoB) iMNs quantified from two biologically independent iMN conversions per condition. For (d), n=37 (CTRL + GFP), 37 (C9ORF72−/− + GFP), 38 (C9ORF72−/− + isoA), and 23 (C9ORF72−/− + isoB) iMNs quantified from two biologically independent iMN conversions per condition. One-way ANOVA with Tukey correction for all comparisons. F-value (DFn, DFd): (3, 360) = 56.63 (b), (3, 122) = 13.42 (c), (3, 131) = 17.11 (d). (e-h) Immunoblotting analysis of surface NR1 after surface protein biotinylation in control (e-h), C9ORF72+/− (e-f), and C9-ALS patient (g-h) iMNs generated with 3 factors (NGN2, ISL1, and LHX3). In (f), n=4 biologically independent iMN conversions from CTRL2 and 2 biologically independent iMN conversions from the C9ORF72+/− line. Mean +/− s.d. In (h), two-tailed Mann-Whitney test. n=11 biologically independent motor neuron cultures from 11 independent control lines and 4 biologically independent motor neuron cultures from 4 independent C9-ALS patient lines. Experiments in (e-h) were repeated twice with similar results. Mean +/− s.e.m. (i-j) Immunoblotting analysis of surface Nr1 and Glur1 in post-synaptic densities (PSDs) from C9orf72 control and knockout mice, two-tailed t-test. t-value: 4.424 (Nr1), 4.632 (Glur1), degrees of freedom: 4 (Nr1), 4 (Glur1). n= 3 control PSD preparations isolated from 3 control mice and 3 C9orf72−/− PSD preparations isolated from 3 C9orf72−/− mice. This experiment was repeated twice with similar results. Mean +/− s.e.m. (k-l) Immunoblotting analysis of surface NR1 and GLUR1 in post-synaptic densities (PSDs) from post mortem control and C9-ALS patient motor cortices, n=3 control and 2 C9-ALS patient PSD preparations isolated from 3 control and 2 C9-ALS patients. This experiment was repeated twice with similar results. Mean +/− s.d. (m) Average Ca2+ flux in the presence of glutamate per minute. n=28 (CTRL1), 15 (CTRL2), 15 (CTRL3), 26 C9-ALS1), 20 (C9-ALS2), 24 (C9-ALS3), and 15 (C9ORF72+/−) iMNs analyzed from two biologically independent iMN conversions for each line. Mean ± s.e.m. One-way ANOVA with Tukey correction between all controls and all patients and C9ORF72+/−. F-value (DFn, DFd): (6, 136) = 11.21.

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 advanced traditional Chinese kung fu (martial arts), Neijing (Traditional Chinese: 內勁; pinyin: nèijìng) refers to the conscious control of the practitioner's qi, or "life energy", to gain advantages in combat.[1] Nèijìng is developed by using "Neigong" (Traditional Chinese: 內功; pinyin: nèigōng) (內功), or "internal exercises," as opposed to "wàigōng" (外功), "external exercises."
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).
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).

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]


RNA sequencing output was aligned to the GRCh38 Reference Genome and quantified using the STAR aligner.65 Genes were annotated against the GENCODE version 23 Comprehensive Gene Annotation. Quality control was performed using Picard Tools AlignmentSummaryMetrics. Samples passing quality control and having RNA Integrity Number (RIN) > 5 were used in downstream analysis. To identify differentially expressed genes, the R package DESeq2 was used as previously described.66 The function DESeq was used to estimate size factors, estimate dispersion, fit the data to a negative binomial generalized linear model, and generate differential expression statistics using the Wald test. KEGG enrichment analysis was performed for internal analysis using the R package clusterProfiler.67
Minerals 2017, 7, 57; doi:10.3390/min7040057 www.mdpi.com/journal/minerals Article Migration Behavior of Lithium during Brine Evaporation and KCl Production Plants in Qarhan Salt Lake Weijun Song 1,2, Hongze Gang 1, Yuanqing Ma 4, Shizhong Yang 1 and Bozhong Mu 1,3,* 1 Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, Shanghai 200237, China; [email protected] (W.S.); [email protected] (H.G.); [email protected] (S.Y.) 2 School of Chemical Engineering, Qinghai University, Xining 810016, China 3 Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, China 4 Qinghai Salt Lake Industry Group Co. Ltd., Golmud 816000, China; [email protected] * Correspondence: [email protected] Academic Editor: Javier Sánchez-España Received: 8 January 2017; Accepted: 2 April 2017; Published: 11 April 2017 Abstract: Lithium-brine is an important potential source of lithium. Much research and investigation has been carried out aimed at lithium recovery from brine. Although the distribution and occurrence status of lithium in brine have important implications for lithium recovery, few reports had correlated to this issue. In this article, a study was carried out to explore the lithium migration behavior during brine evaporation and KCl production process at Qarhan Salt Lake. The occurrence status of lithium both in fresh mined brine and residual brine after evaporation were also speculated by means of lithium concentration evaluation and theoretical calculation based on the Pitzer electrolyte solution theory. Results showed that, for Qarhan brine mined from the Bieletan region, most lithium was enriched in the residual brine during the brine evaporation process. The concentration of lithium in the residual brine could be more than 400 mg/L. More than 99.93% lithium ions in residual brine exist in free ions state and lithium does not precipitate from brine with a density of 1.3649 g/mL. The results also revealed that lithium concentration in wastewater discharged from KCl plants can reach a level of 243.8 mg/L. The investigation results provide a theoretical basis for comprehensive development and utilization of lithium resources in Qarhan Salt Lake. Keywords: lithium migration; occurrence status; Qarhan Salt Lake 1. Introduction As an energy metal of the twenty-first century, lithium had attracted more and more attention in the past few decades. Lithium has been widely applied in high energy batteries, controlled thermonuclear reactions, the manufacturing of ceramic and glass, and other fields [1–7]. Lithium consumption for batteries had increased most significantly due to the development of the electric vehicle industry and the popularity of portable electronic products. Stimulated by the political affairs, economic requirements, and environmental conservation, lithium resources have become the focus of the international mining market and lithium’s position as a strategic resource is becoming more prominent. Salt lake brine, thermal spring, and oilfield water are important geological sources of lithium. The commercial exploitation of the lithium resource of brine began at the Searles Lake in the US in 1936. Since then, more focus has been placed on recovering lithium from salt lake brine because of its low economical cost and low environmental impact [8–10]. As a country possessing huge amount of
HEK 293T cells were used to produce retrovirus, lentivirus, and C9ORF72 protein. HEK cells were used for these purposes based on previous published studies using HEK cells in order to produce viral particles and mammalian proteins. HEK cells were obtained from American Type Culture Collection, catalog number CRL-11268. HEK and iPS cells were tested for mycoplasma before, during, and after the study and were negative.
To determine if glutamate receptor accumulation occurs on C9ORF72 patient motor neurons in vivo, we measured glutamate receptor expression in ventral horn neurons in lumbar spinal cord samples from 3 C9ORF72 ALS patients and 3 unaffected controls. We identified motor neurons by size and confirmed that most neurons selected in this manner were CHAT+ and SMI-32+ (Supplementary Fig. 12d). Spinal motor neurons from the C9ORF72 ALS patients displayed higher NR1 levels than control neurons (Supplementary Fig. 12e). In addition, post-synaptic densities isolated from the motor corticesof C9ORF72 patients had higher levels of NR1 and GLUR1 than controls (Fig. 4k, l and Supplementary Fig. 5k).
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.
Biotinylation of plasma membrane localized glutamate receptors was performed using the Piece™ Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, Dox-NIL iMNs were incubated with 0.25mg/ml Sulfo-NHS-SS-Biotin in cold room for 1~2 hrs with end-to-end shaking. After quenching, cells were harvested by scraping and lysed with lysis buffer from the Piece™ Cell Surface Protein Isolation Kit or the M-PER™ mammilian protein extraction buffer (Thermo Fisher Scientific). Cell lysate was incubated with High Capacity NeutrAvidin™ agorase beads (Thermo Fisher Scientific), and the bound protein was eluted in 2X SDS-PAGE sample buffer supplemented with 50mM DTT for 1 hr at room temperature with end-to-end rotation, and further analyzed by western blot.
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.

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.
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.
Consistent with PIKFYVE being the relevant target in the iMN survival assay, Apilimod increased C9ORF72 patient, but not control, iMN survival in either neurotrophic withdrawal conditions (Fig. 6d) or excess glutamate (n=4 patients, Supplementary Fig. 15f (n=3 controls, Supplementary Fig. 15g). Automated neuron tracking software independently verified Apilimod efficacy on C9ORF72 patient iMNs (Supplementary Fig. 15h). As further confirmation that PIKFYVE was the active target, ASO-mediated suppression of PIKFYVE also rescued C9ORF72 patient iMN survival (Fig. 6d and Supplementary Fig. 15i). In addition, we synthesized a structural analog of Apilimod with a reduced ability to inhibit PIKFYVE kinase activity in a biochemical assay using purified PIKFYVE protein (Fig. 6b and Supplementary Fig. 15j, 16). The reduced activity analog was significantly less effective at rescuing C9ORF72 patient iMN survival (Fig. 6e). Thus, small molecule inhibition of PIKFYVE can rescue patient motor neuron survival.
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.[citation needed]
Lithium-brine is an important potential source of lithium. Much research and investigation has been carried out aimed at lithium recovery from brine. Although the distribution and occurrence status of lithium in brine have important implications for lithium recovery, few reports had correlated to this issue. In this article, a study was carried out to explore the lithium migration behavior during brine evaporation and KCl production process at Qarhan Salt Lake. The occurrence status of lithium both in fresh mined brine and residual brine after evaporation were also speculated by means of lithium concentration evaluation and theoretical calculation based on the Pitzer electrolyte solution theory. Results showed that, for Qarhan brine mined from the Bieletan region, most lithium was enriched in the residual brine during the brine evaporation process. The concentration of lithium in the residual brine could be more than 400 mg/L. More than 99.93% lithium ions in residual brine exist in free ions state and lithium does not precipitate from brine with a density of 1.3649 g/mL. The results also revealed that lithium concentration in wastewater discharged from KCl plants can reach a level of 243.8 mg/L. The investigation results provide a theoretical basis for comprehensive development and utilization of lithium resources in Qarhan Salt Lake.
(a) Super-resolution microscopy images of control iMNs showing colocalization (arrows) of C9ORF72 (green) with EEA1 (red). Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (b) Immunoblot against C9ORF72, EEA1, and LAMP1 on lysates from iPSC-derived motor neurons separated into light (endosomal) and heavy (lysosomal) membrane fractions using percoll gradient centrifugation. This experiment was repeated twice with similar results. (c) Super-resolution microscopy images of LAMP1 immunostaining in iMNs of specified genotypes expressing eGFP or C9ORF72 (isoform A or B)-eGFP. Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (d-f) Number of LAMP1+ vesicles in control (d-f), patient (d), C9ORF72+/− (e), and C9ORF72−/− (f) iMNs overexpressing eGFP or C9ORF72 (isoform A or B)-eGFP. Each grey open circle represents a single iMN, Mean ± s.d. For (d), n=80 (CTRL + GFP), 80 (C9-ALS + GFP), 64 (C9-ALS + isoA), and 61 (C9-ALS + isoB) iMNs quantified from two biologically independent iMN conversions of 3 CTRL or 4 C9-ALS lines. For (e), n=20 (CTRL + GFP), 15 (C9ORF72+/− + GFP), 12 (C9ORF72+/− + isoA), and 13 (C9ORF72+/− + isoB) iMNs f quantified from two biologically independent iMN conversions per condition. For (f), n=20 iMNs quantified from two biologically independent iMN conversions per condition. One-way ANOVA with Tukey correction between CTRL2 and C9ORF72+/− and C9ORF72−/− (e, f), one-way ANOVA with Tukey correction between controls and patient conditions (d). F-value (DFn, DFd): (3, 273)=12.12 (d), (3, 57)=5.64 (e), (3, 77)=6.091 (f). Dotted lines outline iMNs. (g) Representative electron micrographs of control, C9ORF72−/−, and patient iMNs showing lysosomes as electron-dense spherical perinuclear structures (arrows). This experiment was repeated twice with similar results. Scale bar: 1 μm. (h-i) Number of electron-dense spheres per square micron of perinuclear cytosol in control (h-i), C9ORF72−/− (h), and patient iMNs (i) Median ± interquartile range, each data point represents a single cell, Two-sided Mann-Whitney test). For (h), n=20 (CTRL2) and 19 (C9ORF72+/−), and for (i) n=20 (CTRL2) and 26 (C9ORF72 patient) cells quantified from two biologically independent iMN conversions of one line per genotype. (j) Super-resolution microscopy images of Lamp1 immunoreactivity in control and C9-KO mouse spinal neurons. This experiment was repeated twice with similar results. Scale bar: 5 μm. (k) Number of Lamp1+ punctae in Chat+ mouse spinal neurons. Median ± interquartile range, two-tailed t-test. t-value: 3.681. Degrees of freedom: 113. n=59 (CTRL2) and 56 (C9ORF72−/−) cells quantified from sections of two mice per genotype.
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).

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.
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.
In advanced traditional Chinese kung fu (martial arts), Neijing (Traditional Chinese: 內勁; pinyin: nèijìng) refers to the conscious control of the practitioner's qi, or "life energy", to gain advantages in combat.[1] Nèijìng is developed by using "Neigong" (Traditional Chinese: 內功; pinyin: nèigōng) (內功), or "internal exercises," as opposed to "wàigōng" (外功), "external exercises."
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