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.

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.


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.
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.

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.
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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)
To confirm that reduced C9ORF72 protein levels are sufficient to cause neurodegeneration, we used CRISPR/Cas9-mediated genome editing to introduce a frameshift mutation into one or both alleles of C9ORF72 in control iPSCs (Fig. 2e and Supplementary Fig. 4e). qPCR showed that targeting one allele reduced C9ORF72 transcript levels due to nonsense-mediated decay and transcript levels were more severely reduced in homozygous mutant cells (Supplementary Fig. 4f). Frameshift mutations also decreased C9ORF72 protein expression (Supplementary Fig. 4g, 5c). RNA sequencing of flow-purified Hb9::RFP+ iMNs showed that targeting C9ORF72 did not significantly alter the expression of the top 10 genes with predicted off-target sites for the CRISPR guide RNA (Supplementary Fig. 4h and Supplementary Table 7). In addition, expression levels of the 20 genes nearest C9ORF72 on chromosome 9 were largely unperturbed in either the C9ORF72+/− and C9ORF72−/− iMNs, indicating that this approach specifically inactivated C9ORF72 (Supplementary Fig. 4i).
For experiments other than the comparison of Apilimod and the reduced-activity analog, Apilimod was purchased from Axon Medchem (cat. no. 1369). For the reduced-activity analog assays, Apilimod and the reduced activity analog were synthesized at Icagen, Inc. according to the schemes shown in Supplementary Fig. 16. PIKFYVE kinase inhibition was measured using the ADP-Glo kinase assay from SignalChem according to the manufacturer’s instructions, using purified PIKFYVE kinase (SignalChem cat. no. P17–11BG-05).
An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its pathogenic mechanism remains unclear. Here we use human induced motor neurons (iMNs) to show that repeat-expanded C9ORF72 is haploinsufficient in ALS. We show that C9ORF72 interacts with endosomes and is required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduces C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms leads to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescues patient neuron survival and ameliorates neurodegenerative processes in both gain- and loss-of function C9ORF72 mouse models. Thus, modulating vesicle trafficking can rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN, and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS/FTD.
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
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.
Removal of TTX and TEA during glutamate receptor agonist treatment revealed additional increases in Gcamp6 activation in C9ORF72+/− iMNs compared to controls, suggesting that C9ORF72+/− iMNs also fire action potentials more frequently than controls (Supplementary Fig. 13a), although we did not detect large changes in sodium or potassium current amplitudes in C9ORF72+/− iMNs (Supplementary Fig. 13b, c). To determine if increased neuronal activity due in part to elevated glutamate receptor levels contributes to neurodegeneration in C9ORF72 patient and C9ORF72+/− iMNs, we measured iMN survival in the presence or absence of retigabine. Retigabine is approved by the U.S. Food and Drug Administration for the treatment of epilepsy and reduces neuronal excitability by activating Kv7 potassium channels 48. In the glutamate treatment assay, retigabine increased the survival of C9ORF72 patient (n=2 patients) and C9ORF72-deficient iMNs, but not controls (n=2 controls)(Supplementary Fig. 13d-g).
iMNs from healthy controls and ALS patients were collected on day 21 post-transduction in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Roche). Protein quantity was measured by the BCA assay (Pierce) and samples were run on a 10% SDS gel at 4 °C. The gel was transferred onto an Immobilon membrane (Millipore). The membrane was blocked with 5% milk in 0.1% PBS-Tween 20 (PBS-T)(Sigma-Aldrich), incubated with primary antibodies overnight at 4 °C, washed three times with 0.1% PBS-T, then incubated with horseradish peroxidase (HRP)-conjugated (Santa Cruz). After three washes with 0.1% PBS-T, blots were visualized using an Amersham ECL Western Blotting Detection Kit (GE) or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) and developed on X-ray film (Genesee). The following primary antibodies were used: rabbit anti-C9ORF72 (Proteintech, cat. no. 22637–1-AP), mouse anti-GAPDH (Santa Cruz, cat. no. sc-32233), chicken anti-MAP2 (Abcam, cat. no. ab11267), mouse anti-FLAG (Sigma, cat. no. F1804), rabbit anti-GLUR1 (Millipore, cat. no. 1504), mouse anti-NR1 (Novus, cat. no. NB300118), mouse anti-Transferrin receptor (Thermo, cat. no. 136800), mouse anti-LAMP3 (DSHB, cat. no. H5C6), rabbit anti-LAMP3 (Proteintech, cat. no. 12632), mouse anti-LAMP2 (DSHB, cat. no. H4B4), mouse anti-LAMP1 (Abcam, cat. no. Ab25630), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), mouse anti-EEA1 (BD Biosciences, cat. no. BD610457), mouse anti-TUJ1 (Biolegend, cat. no. MMS-435P), rabbit anti-APP (Abcam, cat. no. ab32136), mouse anti-Tau5 (Thermo, cat. no. AHB0042), mouse anti-PSD-95 (Thermo, cat. no. MA1–045) , mouse anti-p53 (Cell Signaling, cat. no. 2524S), anti-mouse HRP (Cell Signaling, cat. no. 7076S), anti-rabbit HRP (Cell Signaling, cat. no. 7074S). For C9ORF72 western blots, to generate enough motor neurons for C9ORF72 protein detection, we used a directed differentiation method described previously 28.
The Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
GCaMP6 was cloned into the pMXs-Dest-WRE retroviral vector and transduced into reprogramming cultures concurrently with the motor neuron factors. To assess GCaMP6 activity, 1.5 μm glutamate was added to iMN cultures and cells were imaged continuously for 2 minutes at 24 frames per second. GFP flashes were scored manually using the video recording. At least 3 different fields of view from three independent cultures, totalling 50–100 iMNs, were scored per condition.
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