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.
Reprogramming was performed in 96-well plates (8 × 103 cells/well) or 13mm plastic coverslips (3.2 × 104 cells/coverslip) that were sequentially coated with gelatin (0.1%, 1 hour) and laminin (2–4 hours) at room temperature. To enable efficient expression of the transgenic reprogramming factors, iPSCs were cultured in fibroblast medium (DMEM + 10% FBS) for at least 48 hours and either used directly for retroviral transduction or passaged before transduction for each experiment. 7 iMN factors or 5 iDA factors were added in 100–200 µl fibroblast medium per 96-well well with 5 μg/ml polybrene. For iMNs, cultures were transduced with lentivirus encoding the Hb9::RFP reporter 48 hours after transduction with transcription factor-encoding retroviruses. On day 5, primary mouse cortical glial cells from P1 ICR pups (male and female) were added to the transduced cultures in glia medium containing MEM (Life Technologies), 10% donor equine serum (HyClone), 20% glucose (Sigma-Aldrich), and 1% penicillin/streptomycin. On day 6, cultures were switched to N3 medium containing DMEM/F12 (Life Technologies), 2% FBS, 1% penicillin/streptomycin, N2 and B27 supplements (Life Technologies), 7.5 µM RepSox (Selleck), and 10 ng/ml each of GDNF, BDNF, and CNTF (R&D). The iMN and iDA neuron cultures were maintained in N3 medium, changed every other day, unless otherwise noted.
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.
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The results of an experimental investigation of the effects of container geometry on the recovery of product water from indirectly frozen salt water are presented. Salt water was frozen in containers having circular or rectangular cross-section, then allowed to melt and drain until the residual ice was potable. Thin rectangular cross-sections were found to be more effective than circular ... [Show full abstract]Read more
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.
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.
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.
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.
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
The following antibodies were used in this manuscript: mouse anti-HB9 (Developmental Studies Hybridoma Bank); 81.5C10. chicken anti-TUJ1 (EMD Millipore); AB9354. rabbit anti-VACHT (Sigma); SAB4200559. rabbit anti-C9ORF72 (Sigma-Aldrich); HPA023873. rabbit anti-C9ORF72 (Proteintech); 25757–1-AP. mouse anti-EEA1 (BD Biosciences); 610457. mouse antiRAB5 (BD Biosciences); 610281. mouse anti-RAB7 (GeneTex); GTX16196. mouse anti-LAMP1 (Abcam); ab25630. mouse anti-M6PR (Abcam); ab2733. rabbit anti-GluR1 (EMD Millipore); pc246. mouse anti-NR1 (EMD Millipore); MAB363. chicken anti-GFP (GeneTex); GTX13970. rabbit anti-Glur6/7 (EMD Millipore); 04–921. mouse anti-FLAG (Sigma); F1804. mouse anti-GAPDH (Santa Cruz); sc-32233. chicken anti-MAP2 (Abcam); ab11267, 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), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), 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).