dataesr/scientific-paragraphs-categorization huggingface dataset:
8, 9  addition to pre-clinical studies, microdystrophin gene therapy is currently being investigated in clinical trials for the treatment of DMD (ClinicalTrials. gov: NCT03368742, NCT03769116, and NCT03362502). With the potential clinical success of a microdystrophin gene therapy program on the horizon, additional questions related to real-world scenarios should be explored. For example, what are the effects of increased physical activity, such as voluntary exercise, on overall treatment efficacy and durability? 1] [12] [13]  Pre-clinical studies evaluating exercise combined with microdystrophin may help inform guidelines for physical activity for treated DMD boys. The effects of exercise have been studied in the mdx mouse model of DMD across various modalities including voluntary wheel running,  [13] [14] [15]  forced treadmill running,  16, 17  and swimming  18, 19  to better understand how dystrophic muscles respond.
These data demonstrate that voluntary running exercise complemented the AAV9-CK8-microdystrophin by improving mitochondrial respiration in red and white muscles. Remarkably, only modest changes in mitochondrial metabolic enzyme activities were observed, with no differences evident between the mdxRGT and mdxGT muscles nor between the WTR and WT muscles. When the red gastrocnemius (and soleus) and white quadriceps were assayed, all enzymes had activities greater in red than white muscles, independent of group. Gene therapy alone did not change metabolic enzyme activities in red or white muscle, but when combined with running, red muscles of mdxRGT mice had greater citrate synthase activity versus WT and greater cytochrome c oxidase activity versus WT and mdx mice. These data also indicate that running plus gene therapy drives positive adaptations in mitochondria of red muscle.
Biodistribution In addition to whole blood, heart, diaphragm, left quadriceps, left gastrocnemius, and left extensor digitorum longus were harvested at sacrifice in a manner that prevented cross-contamination, snap frozen in liquid nitrogen, and stored at À80 C until gDNA was extracted. gDNA was isolated using a DNeasy blood and tissue kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions, and vector gDNA concentrations were determined using the NanoDrop One system (Thermo Fisher, Carlsbad, CA, USA). AAV vg copies present in gDNA were quantified by real-time PCR using the QuantStudio 3 Real-Time PCR system (Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer's instructions. Results were analyzed using the QuantStudio Design & Analysis v1. 4. 1 software with the threshold, start baseline, and stop baseline set automatically by the software.
Voluntary wheel running At time 0 for each cohort, mice in the WTR and mdxRGT groups were single housed in cages with running wheels (15-cm diameter). The wheels were instrumented to count revolutions every 2 min, and the values were recorded by a laptop computer into an excel spreadsheet using our lab's custom software. The software determined the distance per revolution (m) using the equation circumference = 2pr. Running data were collected continuously for 24 h each day for 7 days. At the end of the 7-day period, the resulting spreadsheet was downloaded for additional data manipulation (e. g. , distance, km/week) and analysis, and the program was restarted to collect data for the next week. Mice in the mdx, mdxGT, and WT groups were single housed in cages with locked running wheels. Relative running-wheel data were calculated as each respective mouse value at each time point/that mouse's week 1 running distance Â 100%.
Equipment and software In vivo contractile properties were determined with Aurora Scientific (ASI, Aurora, ON, Canada) equipment and software including a 5N dual-mode servomotor (305C-FP-M) with a foot pedal displacement maximum of $40 (±20 from neutral position) and a 701C high-power follow stimulator set to constant current. Current was optimized for each mouse for each in vivo experiment (range, 45-65 mA). Dynamic Muscle Control (DMC) software controlled the timing and frequency of the stimulations and collection of force, torque, and foot pedal displacement. As noted below, the DMC units were set to either torque (mN Â m) or force (mN) depending on the assay. The mouse platform was maintained at 37 C by an HTP-1500 heat-therapy pump (Adroit Medical Systems, Loudon, TN, USA). Dynamic Muscle Analysis (DMA) software was used to analyze the force, torque, and displacement data.
Isometric torque frequency Torque is determined by the force (mN) applied to, and multiplied by the length of, the foot pedal (m). DMC units were therefore set to mN Â m. Isometric torque was determined with the foot at 90 to the tibia (neutral position) over a series of stimulations at increasing frequencies: 1, 10, 30, 50, 65, 80, 100, 120, 150, 180, and 200 Hz. Data were plotted as torque normalized to mouse body mass (mN Â m/g) versus frequency. Relative torque data were calculated as each respective mouse value at each time point/that mouse's baseline torque Â 100%. Torque velocity and power DMC units were set to mN. 2 min following the end of the torque-frequency assay, maximum isometric tetanic forces were recorded at 150 Hz with the foot pedal in dorsiflexion (+20 ), in the neutral position (0 ), and plantarflexion (À20 ).
90 of the maximal dorsiflexion tetanic force value to subsequently calculate maximum torque, which typically occurs at 0. 40 fractional load. 24 Because the time to full displacement became shorter as the fractional load decreased, we modified the 150 Hz stimulation duration at the various fractional loads: 0. 2 s (0. 90); 0. 1 s (0. 75, 0. 50, 0. 40); and 0. 05 s (0. 30). At fractional loads less than 0. 30, there was insufficient time to capture the peak velocity before the pedal exceeded the displacement limit. Displacement in degrees was converted to mm, data were plotted as fractional load versus velocity in mm/s, and the Hill equation was used to fit a curve and generate an equation (GraphPad Prism, GraphPad Software, La Jolla, CA, USA). Power = force Â velocity. Power was determined as the product of a given load in mN and the associated velocity (mm/s) and then expressed in mW.
5-mM increments (data not shown). Each assay was completed before O 2 concentration was depleted below 150 mM to avoid O 2 diffusion limiting the reactions. 71 on completion of the assay, each fiber bundle was removed from the chamber, rinsed in dH 2 O for 5 min, blotted dry, frozen at À80 C, and lyophilized overnight. Mass of the dried fiber bundles was measured with a Thermo Cahn C-35 microbalance (Thermo Sci-entific, Waltham, MA, USA) to the nearest 0. 1 mg, and JO 2 measurements normalized to dry bundle mass.
Western blot Total protein was isolated from quadriceps and hearts of mdxGT and mdxRGT animals using RIPA buffer (Millipore, #20-188) containing protease inhibitors (Roche, #11836153001) (Roche, #05892970001). Proteins were transferred to nitrocellulose membranes and probed for microdystrophin using the NCL-DYSB primary antibody (Leica, 6052319, 1:50) and a horseradish peroxidase (HRP)-linked antimouse insulin growth factor (IgG) secondary antibody (Jackson Labs, #715-035-150, 1:4,000). Signal detection was performed using the Clarity Western ECL substrate (GE Life Sciences, #RPN2236) and measured by digital imaging using the ChemiDoc MP system (Bio-Rad). Quantification of band signal intensities normalized to b-tubulin (Abcam, Ab21058, 1:5,000) was performed with Image Lab Software (Bio-Rad).
Immunofluorescence Isopentane-frozen quadriceps muscle specimens were sectioned at 8mm thickness and immunostained per standard techniques for dystrophin (Leica, NCL-DYS2), microdystrophin (Leica, NCL-DYSB), laminin (Sigma, L9393), myosin heavy-chain type 1 (Developmental Studies Hybridoma Bank [DSHB], BA-D5), or myosin heavy-chain type 2A (DSHB, SC-71). Evaluation of percent dystrophin-or microdystrophin-positive fibers was performed by a neuropathologist using a standard fluorescent microscope and estimated to the nearest 5%. For fiber type analysis, slides were scanned with an Olympus VS120 whole slide scanner, and scanned images were evaluated with VisioPharm software using 20Â magnification. Immunostaining on the far-red channel highlighted laminin expression, which identified edges of myofibers.
Type 1 myosin heavy-chain expression was visualized on the red channel, type 2A myosin heavy-chain expression was visualized on the green channel, and together these fibers were assessed as the oxidative fiber population. 72 Fibers negative for type 1 and type 2A myosin heavy chain were assumed to be glycolytic and categorized separately. The Visiopharm software identified muscle fibers using laminin expression to determine cell edges and fiber size limits of between 5 and 110 mm, which was assessed by manual measurement of the smallest and largest fibers in samples from this dataset. Minferet diameter data was obtained for all muscle fibers and with respect to fibers that were positive for type 1, type 2A, or negative for both type 1 and 2A myosin heavy chain. Frequency histograms were constructed using Prism software (GraphPad Prism 8).
Fiber size data and fiber proportion data were analyzed using a two-way ANOVA with Prism software (GraphPad Prism 8). Determination of dystrophic grade H&E-stained sections of frozen quadriceps-muscle tissue were assessed by a board-certified neuropathologist with respect to findings associated with active dystrophic disease, including myofiber degeneration, active myofiber regeneration (basophilic fibers), and inflammation. The goal of this approach was to capture histological features that were indicative of myofiber degeneration and regeneration in the post-treatment period, while excluding indications of longer-standing muscle damage that may have occurred prior to treatment. The area of each sample displaying these findings was visually estimated, and the following grading scheme was applied: grade 0 = normal, grade 1 = chronic regenerative changes only, grade 1.
5 = very mild (<5% of muscle area with active dystrophic pathology), grade 2 = mild (6%-20% of muscle area with active dystrophic pathology), grade 2. 5 = mild to moderate (21%-30% of muscle area with active dystrophic pathology), grade 3 = moderate (30%-50% of muscle area with active dystrophic pathology), grade 3. 5 = moderate to severe (51%-60% of muscle area with active dystrophic pathology) and grade 4 = severe (>60% of muscle area with active dystrophic pathology). Areas indicative of chronic regeneration (internally nucleated fibers with appropriate eosinophilia, endomysial fibrosis, or fatty infiltration) were not integrated into the grading of dystrophic severity, as it is possible that the damage associated with these changes occurred before treatment. Dystrophic severity of each treatment group was analyzed with a repeated-measure one-way ANOVA using Prism software (GraphPad Prism 8).
Experimental Design In a first experiment, 30 mice were used to determine an acute effective dose of Safr'Inside TM in the forced swim test (FST). Based on a preliminary study testing three doses (6. 25, 150 and 600 mg/Kg; unpublished data), two of them were chosen for the present experiment: 6. 25 and 600 mg/Kg. The low one was calculated based on the ratio given by the United States Food and Drug Administration  [34]  to reflect, for a mouse, the equivalent of the effective dose classically administrated to humans, namely 30 mg/day  [15]. The high dose was selected on the basis of previously published data  [18]. In a second experiment, 20 naïve mice were used to investigate the effects of acute and chronic administration of Safr'Inside TM on depressive-like and anxiety-like behavior, and brain monoaminergic systems.
Briefly, mice were placed individually in a cylinder (diameter: 16 cm; height: 31 cm) half-filled with 25 °C (+/-1 °C) water for a 6-min test. Duration of swimming, climbing and immobility was determined during the last 4 min of the test  [38]. A mouse was judged to be immobile when it moved only slowly to remain floating, keeping its head above water. Increased duration of immobility has been proposed to reflect a state of helplessness that is reduced by ADs. Behavior was videotaped to be scored later by a trained observer blind to treatment conditions, using ''The Observer XT 11'' software (Noldus, Wageningen,The Netherlands). An immobility threshold that represents the average percentage of time spent immobile by control animals was calculated for each experiment. We then determined an immobility index reflecting the percentage of mice spending more or less immobility time than the immobility threshold.
Behavior was videotaped to be scored later by a trained observer blind to treatment conditions, using "The Observer XT 11" software (Noldus, Wageningen, The Netherlands). An immobility threshold that represents the average percentage of time spent immobile by control animals was calculated for each experiment. We then determined an immobility index reflecting the percentage of mice spending more or less immobility time than the immobility threshold. Anxiety-like behavior in the light/dark test (LDT). The LDT is a standardized test to assess anxiety in rodents. As previously described  [39] , it is based on their innate aversion to brightly lit areas, and their spontaneous exploratory behavior when exposed to a novel environment. In this procedure, an increase in time spent in the light compartment is considered to reflect an anxiolytic activity.
The flow rate of the pump allowing the circulation of the mobile phase (0. 1 M sodium acetate, 0. 1 M citric acid, 1 mM diethylamine, 1 mM sodium octyl sulfate and 0. 1 mM EDTA) was set at 0. 3 mL/min. The monoamines of interest, DA, 5-HT, and their metabolites, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), were identified according to their retention time, as compared to their respective standards injected daily using the Chromeleon integration 6. 8 software (Dionex, Sunnyvale, CA, US). The different standards were mixed in a standard solution, which therefore contained a known concentration of each monoamine (DAhydrochloride (H8502) and 5-HT-hydrochloride (H9523), Sigma, St. Quentin Fallavier, France) and each metabolite (DOPAC (850217), HVA (H1252) and 5-HIAA (H8876), Sigma, St.
We focused on genes coding for 5-HT receptors (5-Htr1a: Mm00434106_s1 and 5-Htr1b: Mm00439377_s1) and the 5-HT transporter (SERT: Mm00439391_m1) for 5-HT pathway, DA receptors (DRD1: Mm01353211_m1 and DRD2: Mm00438545_m1), the enzyme Catechol-O-Methyltransferase (COMT: Mm00514377_m1) and the DA transporter (DAT: Mm00438388_m1) for DA pathway, and the monoamine oxidases (Maoa: Mm00558004_m1 and Maob: Mm00555412_m1) that catabolize both 5-HT and DA. Fluorescence was determined on a Light cycler 480 II system (Roche Diagnostics, Meylan, France). The PCR program consisted of 1 cycle of 95 • C for 10 min, 50 cycles of 95 • C for 10 s and 60 • C for 30 s, and 1 cycle of 40 • C for 30 s. Data were analyzed using the comparative threshold cycle method as previously described  [42]  and results were expressed as relative fold change with GAPDH as a house-keeping gene (Mm99999915_g1).
Statistical Analysis All data are presented as means ± SEM. Data were analyzed using Statistica 6 software (StatSoft, Tulsa, OK, USA), and possible outliers were assessed with Graphpad Outlier Calculator  [43]  and removed from the analysis. Normality was assessed using the Shapiro-Wilk test. When distribution was normal data were analyzed with parametric statistics; unpaired t-test and one-way ANOVA, followed by Fisher LSD post-hoc test when appropriate, for comparison of 3 groups. For non-normal distribution, data were analyzed with non-parametric statistics; statistical validity was assessed with the Mann-Whitney U test for comparison of 2 groups and with the Kruskal-Wallis H test for comparison of 3 groups. The immobility indexes were analyzed using Fisher's exact test on contingency tables.
Supramaximal responses were gradually generated, and maximal responses were obtained with stimulus currents <2 mA. Data were acquired with a sensitivity between 10 and 20 μV/division with a sweep speed of 1 ms/division to ensure accurate measurement of lowamplitude responses. The latency was measured as the time from the given electrical stimulus to the appearance of a sensory nerve response corresponding to the initial deflection from the baseline. Baseline to peak amplitudes (μV) from the sensory nerve-evoked responses were measured and averaged, resulting in one averaged amplitude per animal, which was used for statistical analysis. Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously  (Oosthuyse et al. , 2001). Briefly, CMAPs were elicited with square pulses of 0.
2 ms duration with a repetition rate set at 1 Hz, delivered with a monopolar needle electrode to the sciatic nerve at the sciatic notch level. CMAPs were measured by two monopolar needle electrodes inserted in the gastrocnemius muscle, and the system was grounded by subcutaneously inserted monopolar needle electrode in the back of the animal. Data were acquired with a sensitivity between 1 and 5 mV/division with a sweep speed of 1 ms/ division to ensure accurate measurement of low-amplitude responses. Supramaximal responses were gradually generated, and maximal responses were obtained with stimulus currents <5 mA. The latency was measured as the time from the given electrical stimulus to the appearance of a muscle response -the initial CMAP negative deflection from the baseline. Amplitudes (mV) from the baseline to the maximal negative peak of the CMAPs were measured and averaged, resulting in one average CMAP amplitude per animal, which was used for statistical analysis.
RNA was prepared from tissue homogenenates following Trizol manufacturer's instructions. One microgram of total RNA was used to synthesize cDNA using iScript reverse transcriptase (iScriptTM Reverse Transcription Supermix for RT-qPCR, Bio-Rad, Hercules, CA) as specified by the manufacturer. Quantitative PCR was performed on a CFX96 real-time System (Bio-Rad) using iQ SYBR Green supermix (Bio-Rad). Three standard genes (H2AC: F-CAACGACGAGGAGCTCAACAAG, R-GAAGTTTCCGCAGATTCTGTT GC/ H2AX: F-TCCTGCCCAACATCCAGG, R-TCAGTACTCCTGAGAGGCC TGC/ H1H2BC: F-AACAAGCGCTCGACCATCA, R-GAATTCGCTACG-GAGGCTTACT) were used to compute a normalization factor using Genorm software v3. 5. The following primer sequences were used to assess muscle denervation: AchRalpha (F-CCACAGACTCAGGGGA-GAAG, R-AACGGTGGTGTGTGTTGATG), AchRbeta (F-GGCAAGTTCC TGCTTTTCGG, R-CGTCCGGAAGTGGATGTTCA), AchRdelta (F-CGCTGCTTCTGCTTCTAGGG, R-ATCAGTTGGCCTTCGGCTT) and Ach-Repsilon (F-CAATGCCAATCCAGACACTG, R-CCCTGCTTCTCC TGACACTC).
Immunocytochemistry was performed as described  (Sen et al. , 2019b) , blocking with 5% BSA for 30 min at RT. Primary antibodies were incubated O/N at 4 • C. Images from the different stainings were done with a Nikon Eclipse TE200-E confocal microscope. In all cases, Zstacks were processed with ImageJ software  (Schindelin et al. , 2012). Photoshop CS5. 1 was used to generate figures. Primary antibodies used were: ATXN2 (BD Transduction 611378, 1:50), IBA1 (Wako 019-19741, 1:1000), PABPC1 (Abcam ab21060, 1:100); TDP43 (Abcam ab41881, 1:100), TIA1 (Santa Cruz sc-1751, 1:100). Secondary antibodies used were: Alexa Fluor 488 goat antimouse IgG A11029, Alexa Fluor 488 goat anti-rabbit IgG A11034, Alexa Fluor 565 rabbit anti-goat IgG A11079, Alexa Fluor 568 donkey anti-sheep IgG A21099, Alexa Fluor 568 goat anti-rabbit IgG A11036 (all Invitrogen, 1:1000).
Quantitative reverse-transcriptase PCR in spinal cord Total RNA isolation from mouse cervicothoracic spinal cord tissue (4 WT vs. 4 KO; 6 WT vs. 5 KIN at 3 months; 7 WT vs. 8 KIN at 14 months) and RT-qPCR in technical duplicates were done as described previously  (Sen et al. , 2019b). RNA was quantified with the NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Tbp was used as the housekeeping gene. The data were analyzed using the 2 -ΔΔCt method  (Schmittgen and Livak, 2008). TaqMan assays from Thermo Scientific were used for Aif1 Mm00479862_g1, Ano3 Mm01270409_m1, Atxn2 Mm01199902_m1, C1qa Mm00432142_m1, C1qb Mm01179619 _m1, C1qc Mm00776126_m1, C3 Mm01232779_m1, Calb2 Mm00801461_m1, Casp3 Mm01195085_m1, Cdr1 Mm04178856_s1, Cirbp Mm00483336_g1, Clgn Mm00515526_m1, Cybb Mm01287743_ m1, Cyp46a1 Mm00487306_m1, Cyp51a1 Mm00490968_m1, Dcp1a Mm00460131_m1, Dcp1b Mm01183995-m1, Dcp2 Mm01264061_m1, Dcps Mm00510029_m1, Ddx1 Mm00506205_m1, Ddx6 Mm006193 26_m1, Dhcr24 Mm00519071_m1, Dhx15 Mm00492113_m1, Eif5a2 Mm00812570_g1, Elovl5 Mm00506717_m1, Fmr1 Mm01339582_m1, Gfap Mm01253033_m1, Gnal Mm01258217_m1, Gpnmb Mm013 28587_m1, Grn Mm00433848-m1, Hecw1 Mm00462346_m1, Hmgcs1 Mm01304569_m1, Hnrnpa2b1 Mm01332941_m1, Hnrnpd Mm01201314_m1, Idi1 Mm01337454_m1, Irak4 Mm00459443_m1, Kcna1 Mm00439977_s1, Kcna2 Mm00434584_s1, Kif5a Mm005152 65_m1, Kif5b Mm01225510_g1, Kif5c Mm00500464_m1, Lsm1 Mm01600253_m1, Msmo1 Mm00499390_m1, Parva Mm00480444_m1, Pcbp1 Mm00478712_s1, Pcbp2 Mm01296174_g1, Pcbp3 Mm011497 50_m1, Pcbp4 Mm00451991_g1, Prpf19 Mm01208295_m1, Ptbp1 Mm01731480_gH, Pura Mm01158049_s1, Puf60 Mm00505017_m1, Rbfox3 Mm01248771_m1, Ripk1 Mm00436354_m1, Rnaset2a/b Mm02601904_m1, Scn4b Mm01175562_m1, Srrm2 Mm00613771_m1, Tacr1 Mm00436892_m1, Tardbp Mm00523866_m1, Tbp Mm00446 973_m1, Tlr3 Mm01207404_m1, Tlr7 Mm00446590_m1, Tlr9 Mm00446193_m1, Trem2 Mm00451744_m1, Ttbk2 Mm00453709_m1, Tyrobp Mm00449152_m1, Uhmk1 Mm00495171_m1, Unc80 Mm0061 5703_m1, Ybx1 Mm00850878_g1.
Global transcriptomics by Clariom D microarrays Cervicothoracic spinal cord RNA integrity was controlled by the 2100 Bioanalyzer with the Nano Assay (Agilent Technologies, Santa Clara, CA). As recommended, 1 μg of RNA was pre-treated with DNase amplification grade (Invitrogen). The GeneChip TM WT PLUS Reagent Kit (Applied Biosystems, Foster City, CA) was used to generate singlestranded cDNA (ss-cDNA) following the manufacturer's instructions. The ss-cDNA was fragmented and labeled right before the hybridization to a Clariom D Array (Thermo Fisher Scientific). The arrays were scanned with the Affymetrix GeneChip Scanner and the data were processed with the Transcriptome Analysis Console (TAC) 4. 0. 1 (Applied Biosystems) using default algorithm parameters.
Gas chromatographic separation and detection of cholesterol and 5alpha-cholestane (ISTD) was performed on a DB-XLB 30 m x 0. 25 mm i. d. x 0. 25 μm film thickness (J&W Scientific Alltech, Folsom, CA, U. S. A. ) in an Agilent Technologies 7890B GC System (Agilent Technologies, Palo Alto, CA, U. S. A), equipped with an FID. The GC-FID instrument is controlled by a computer with the Agilent OpenLAB CDS Software version C. 01. 07. Authentic sterols and deuterium labeled oxysterols were separated on another DB-XLB column (30 m x 0. 25 mm i. d. x 0. 25 μm film thickness, J&W Scientific Alltech, Folsom, CA, U. S. A. ) in a Agilent Technologies 7890B GC System (Agilent Technologies, Waldbronn, Germany) connected with a direct capillary inlet system to a quadrupole mass selective detector 5977B inert MSD (Agilent Technologies, Waldbronn, Germany).
Both GC systems were equipped with Agilent Technologies 7693 series autosamplers and injector plus trays. The GC-MSD instrument is controlled by a computer with the Agilent MS MassHunter Software version B. 07. 05. After saponification with 2 mL 1 M 95% ethanolic sodium hydroxide solution (Merck KGaA, Darmstadt, Germany) at 60 • C for one hour, the free sterols and oxysterols were extracted three times with 3 mL cyclohexane each. The organic solvent was evaporated by a gentle stream of nitrogen at 60 • C on a heating block. The residue was dissolved in 80 μL n-decane (Merck KGaA, Darmstadt, Germany). An aliquot of 40 μL was incubated (1 h at 70 • C on a heating block) by addition of 20 μL of the trimethylsilylating (TMSi) reagent (chlortrimethylsilane [Merck KGaA, Darmstadt, Germany]/1. 1.