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WILD 20

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Most cited protocols related to «WILD 20»

1
Genomic Analysis of Strawberry Diversity
Cited 15 times
A Japanese variety, ‘Reikou’, and its progeny (S1) were subjected to genome sequencing as representatives of F. x ananassa. ‘Reikou’ was bred in the Chiba Prefectural Agriculture and Forestry Research Center. Like other strawberry varieties, ‘Reikou’ maintains heterozygosity in the genome. The S1 progenies were sequenced for SNP discovery within ‘Reikou’ for further analysis (data were not shown in this study). A total of 20 wild Fragaria species with a diverse range of polyploidy were used in phylogenetic analyses with SSR markers (Supplementary Table S1). Along with the 20 wild species, the following four Japanese F. x ananassa varieties were subjected to the phylogenic analysis: ‘Reikou’, ‘Hokowase’, ‘Sachinoka’, and ‘Akihime’. Whole-genome sequencing was subsequently performed for four wild species, F. iinumae, F. nipponica, F. nubicola, and F. orientalis. The genomic DNA was extracted from young leaves using a DNeasy Plant Mini Kit (Qiagen, Inc., CA, USA). DNA quantification and quality checks were performed using a Nanodrop ND1000 spectrophotometer (Nanodrop Technologies, DE, USA) and 0.8% agarose gel electrophoresis, respectively.
Hirakawa H., Shirasawa K., Kosugi S., Tashiro K., Nakayama S., Yamada M., Kohara M., Watanabe A., Kishida Y., Fujishiro T., Tsuruoka H., Minami C., Sasamoto S., Kato M., Nanri K., Komaki A., Yanagi T., Guoxin Q., Maeda F., Ishikawa M., Kuhara S., Sato S., Tabata S, & Isobe S.N. (2013). Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 21(2), 169-181.
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Publication 2013
Electrophoresis, Agar Gel Fragaria Genome Heterozygote Japanese Plants Polyploidy Strawberries WILD 20
2
Multi-tissue Transcriptional Profiling of Huntington's Disease Mouse Model
Cited 14 times
Striatum, cortex, and liver were selected for full profiling. Specifically, at each of 3 time points (2, 6, 10 months), four female and four male heterozygous KI mice from each of the 6 Htt CAG repeat lengths were profiled, resulting in 48 samples from each tissue and each time point. Additional samples from wild type littermates from the Q20 line were profiled as well (striatum at all 3 ages, cortex and liver at 2 and 10m only). In addition to the fully profiled tissues, 11 other tissues were selected for a smaller study (referred to as the Tissue Survey) involving 8 (4 male and 4 female) wild type mice from the 6-month Q=20 line, and 8 (4 male and 4 female) heterozygous mice from the 6-month Q175 line, for a total of 16 samples per tissue. The 11 tissues include 5 brain regions (brainstem, cerebellum, hippocampus, hypothalamus/thalamus, and corpus callosum) and 6 peripheral tissues (white gonadal adipose, white intestinal adipose, brown adipose, skin, heart, and gastrocnemius muscle). Messenger RNA was extracted and prepared using the Illumina TruSeq RNA sample prep kit and sequenced on an Illumina HiSeq2000 sequencer using strand-specific, paired end, 50-mer sequencing protocols to a minimum read depth of 40 million reads per sample. The sequencing was performed in 2 separate batches (6-month samples in batch 1, 2- and 10-month samples in batch 2). Clipped reads were aligned to mouse genome mm9 using the STAR aligner53 (link) using default settings. Read counts for individual genes were obtained using HTSeq54 (link).
Langfelder P., Cantle J.P., Chatzopoulou D., Wang N., Gao F., Al-Ramahi I., Lu X.H., Ramos E.M., El-Zein K., Zhao Y., Deverasetty S., Tebbe A., Schaab C., Lavery D.J., Howland D., Kwak S., Botas J., Aaronson J.S., Rosinski J., Coppola G., Horvath S, & Yang X.W. (2016). Integrated genomics and proteomics to define huntingtin CAG length-dependent networks in HD Mice. Nature neuroscience, 19(4), 623-633.
Publication 2016
Brain Brain Stem Cerebellum Corpus Callosum Cortex, Cerebral Females Genes Genome Gonads Heart Heterozygote Hypothalamus Intestines Liver Males Mice, House Muscle, Gastrocnemius Obesity RNA, Messenger Seahorses Skin Striatum, Corpus Thalamus Tissues WILD 20
3
Multi-tissue Transcriptional Profiling of Huntington's Disease Mouse Model
Cited 14 times
Striatum, cortex, and liver were selected for full profiling. Specifically, at each of 3 time points (2, 6, 10 months), four female and four male heterozygous KI mice from each of the 6 Htt CAG repeat lengths were profiled, resulting in 48 samples from each tissue and each time point. Additional samples from wild type littermates from the Q20 line were profiled as well (striatum at all 3 ages, cortex and liver at 2 and 10m only). In addition to the fully profiled tissues, 11 other tissues were selected for a smaller study (referred to as the Tissue Survey) involving 8 (4 male and 4 female) wild type mice from the 6-month Q=20 line, and 8 (4 male and 4 female) heterozygous mice from the 6-month Q175 line, for a total of 16 samples per tissue. The 11 tissues include 5 brain regions (brainstem, cerebellum, hippocampus, hypothalamus/thalamus, and corpus callosum) and 6 peripheral tissues (white gonadal adipose, white intestinal adipose, brown adipose, skin, heart, and gastrocnemius muscle). Messenger RNA was extracted and prepared using the Illumina TruSeq RNA sample prep kit and sequenced on an Illumina HiSeq2000 sequencer using strand-specific, paired end, 50-mer sequencing protocols to a minimum read depth of 40 million reads per sample. The sequencing was performed in 2 separate batches (6-month samples in batch 1, 2- and 10-month samples in batch 2). Clipped reads were aligned to mouse genome mm9 using the STAR aligner53 (link) using default settings. Read counts for individual genes were obtained using HTSeq54 (link).
Langfelder P., Cantle J.P., Chatzopoulou D., Wang N., Gao F., Al-Ramahi I., Lu X.H., Ramos E.M., El-Zein K., Zhao Y., Deverasetty S., Tebbe A., Schaab C., Lavery D.J., Howland D., Kwak S., Botas J., Aaronson J.S., Rosinski J., Coppola G., Horvath S, & Yang X.W. (2016). Integrated genomics and proteomics to define huntingtin CAG length-dependent networks in HD Mice. Nature neuroscience, 19(4), 623-633.
Publication 2016
Brain Brain Stem Cerebellum Corpus Callosum Cortex, Cerebral Females Genes Genome Gonads Heart Heterozygote Hypothalamus Intestines Liver Males Mice, House Muscle, Gastrocnemius Obesity RNA, Messenger Seahorses Skin Striatum, Corpus Thalamus Tissues WILD 20
4
Optimized Structural Characterization of EAAT1
Cited 9 times
We used fluorescence-detection size-exclusion chromatography (FSEC)50 (link) to screen solubilization conditions and
EAAT1 variants fused to enhanced green fluorescent protein (eGFP). EAAT1
N-terminal fusions solubilized in dodecanoyl sucrose (DDS, Anatrace) were found
to have good solubility and mono-dispersity by FSEC in clear lysates. However,
EAAT1 looses its transport activity and chromatographic monodispersity upon
purification. To increase its stability, we used consensus mutagenesis51 (link), and screened EAAT1 variants with
different consensus mutations in the predicted transmembrane helices by FSEC.
The apparent melting temperature (Tm) of the most stable EAAT1 construct was
>20 °C over that of the wild-type EAAT1, but the
mutated transporter was still refractory to crystallization. We reasoned that
the divergent extracellular region between TM3-4c could interfere with
crystallization, and changed it for the equivalent region in ASCT2, the shortest
one among human SLC1 members (Extended Data
Fig.1). In addition, we mutated the two predicted N-glycosylation
sites of the transporter (N155T and N204T mutations) to further improve
crystallizability.
Canul-Tec J.C., Assal R., Cirri E., Legrand P., Brier S., Chamot-Rooke J, & Reyes N. (2017). Structure and allosteric inhibition of excitatory amino acid transporter 1. Nature, 544(7651), 446-451.
Publication 2017
Chromatography Crystallization enhanced green fluorescent protein Fluorescence Helix (Snails) Homo sapiens Membrane Transport Proteins Molecular Sieve Chromatography Mutation Sucrose WILD 20
5
Fluorescent Labeling of HA-Displaying Virus Particles
Cited 6 times

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Publication 2018
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol 2000) Biotin Buffers Cells Centrifugation Codon, Terminator CY5.5 cyanine dye HIV-1 Lipids Pellets, Drug Plasmids Proteins Protomers Sodium Chloride Tromethamine Ultracentrifugation Virion Virus Western Blotting WILD 20

Most recents protocols related to «WILD 20»

1
Influenza Virus Infection in Mice
After 21 days following immunization, the mice were inoculated intranasally with 20 µL of wild-type D/OK (1.0 × 105 PFU). After three days following the challenge (day 24), mice (four animals per group; n = 12) were euthanized, and organs containing nasal turbinates, the tracheas, and the lungs were collected for virus titration via the plaque assay.
Ishida H., Murakami S., Kamiki H., Matsugo H., Katayama M., Sekine W., Ohira K., Takenaka-Uema A, & Horimoto T. (2023). Generation of a recombinant temperature-sensitive influenza D virus. Scientific Reports, 13, 3806.
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Publication 2023
Animals Biological Assay Dental Plaque Lung Mus Titrimetry Trachea Turbinates Vaccination Virus WILD 20
2
Purification of aCcr1 and Mutants from E. coli
To purify the aCcr1 (SiRe_0197) and its mutant proteins from E. coli, cells harbouring plasmids pET22b-aCcr1-C-His (for the wild type cellular protein), pET22b-aCcr1-R2A-C-His, pET22b-aCcr1-K7A-C-His, pET22b-aCcr1-R27A-C-His (for the DNA binding site deficient cellular protein mutants) and pET22b-ATV_gp29-C-His and pET22b-SMV3_gp63-C-His (for virus-derived aCcr1 homologs) were grown in 2 L of LB medium at 37°C with shaking until the optical density OD600 reached 0.4∼0.6, when 1.0 mM IPTG was added into the cultures and the cells were then grown at 37°C for 4 h with shaking. The cells were harvested by centrifugation at 7000 g for 10 min and then resuspended in the lysis buffer A (50 mM Tris-HCl [pH 7.4], 200 mM NaCl and 5% glycerol). Then, the cells were crushed with an ultrasonic crusher at 40% power, working at 5 s intervals for 5 s until the cell lysate became clear and cell debris was removed by centrifugation at 12 000 g for 15 min. The supernatant was incubated at 70°C for 20 min, centrifuged at 12 000 g for 15 min again, and then filtered through a membrane filter (0.45 μm). The samples were loaded on to a Ni-NTA agarose column (Invitrogen) pre-equilibrated with buffer A. Finally, the target protein was eluted with buffer A containing 300 mM imidazole. The eluted sample was analysed using a 18% SDS-PAGE gel. The protein samples were concentrated by ultrafiltration using an Amicon Ultra-3KDa concentrator (Millipore). For further purification, size exclusion chromatography was performed using a Superdex 200 increase 10/300 column (GE Healthcare). The protein concentration was determined by the Bradford method using bovine serum albumin as the standard. To assay the oligomeric status, 20 μl of wild-type aCcr1 protein (1 mg/ml) were incubated with increasing concentrations of glutaraldehyde (0.01–0.16%) on ice at 4°C for 15 min. The reaction was then stopped by the addition of SDS-PAGE loading buffer, after which the samples were electrophoresed by 20% SDS–PAGE, and the gel was stained with Coomassie blue R-250.
Yang Y., Liu J., Fu X., Zhou F., Zhang S., Zhang X., Huang Q., Krupovic M., She Q., Ni J, & Shen Y. (2023). A novel RHH family transcription factor aCcr1 and its viral homologs dictate cell cycle progression in archaea. Nucleic Acids Research, 51(4), 1707-1723.
Publication 2023
ATP8A2 protein, human Biological Assay Buffers Cells Centrifugation Coomassie brilliant blue R DNA-Binding Proteins Escherichia coli Proteins Glutaral Glycerin imidazole Isopropyl Thiogalactoside Molecular Sieve Chromatography Plasmids Proteins SDS-PAGE Sepharose Serum Albumin, Bovine Sodium Chloride Tissue, Membrane Tromethamine Ultrafiltration Ultrasonics Virus Vision WILD 20
3
Tissue Collection from C57BL/6 Mice
A total of 20 8-week-old male wild-type C57BL/6 mice weighing 20-25 g were purchased from Jackson Laboratory. Mice were housed in standard plastic cages in controlled conditions at 23±2˚C with a humidity of 55±10% under a 12/12-h light/dark cycle, with ad libitum access to food and water. For tissue collection, mice were anesthetized with 3% isoflurane (for induction) and euthanized by cervical dislocation. Death was pronounced by ascertaining cardiac and respiratory arrest. All animal studies were performed using protocols approved by the Institutional Animal Care and Use Committee of Chonnam National University (Gwangju, Korea; approval no. CNU IACUC-YB-2018-02).
Jang A.R., Lee H.N., Hong J.J., Kim Y.M, & Park J.H. (2023). Ethanol extract of Chrysanthemum zawadskii inhibits the NLRP3 inflammasome by suppressing ASC oligomerization in macrophages. Experimental and Therapeutic Medicine, 25(3), 128.
Publication 2023
Animals Food Heart Humidity Institutional Animal Care and Use Committees Isoflurane Joint Dislocations Males Mice, House Mice, Inbred C57BL Neck Respiratory Rate WILD 20
4
Molecular Dynamics Simulation of Protein Mutants
Molecular dynamics simulation of wild type, non-cleavable form and mutants were performed with Gromacs 5.0.2 software under Amber ff99SB-LIDN forcefield. Each initial structure was centered in a cubic box with 1.0 nm distance from the box edge. Periodic boundary conditions were applied to avoid artificial effects due to finite size of simulation box. The box was then filled with appropriate amount of TIP3P water molecules. System was made electrically neutral by replacing water molecules with required number of counter-ions using the genion tool of Gromacs package ( 31 (link)
). Energy minimization was done using steepest descent algorithm. The energy-minimized system was equilibrated by the position restrained simulation under canonical ensemble (NVT) followed by isothermal isobaric ensemble (NPT) in 500 and 1000 ps respectively. The initial velocities were derived from Maxwell-Boltzman distribution at 300 k. Productive unrestrained MD simulations were carried out in the NPT ensemble for 20 ns and bond lengths constrained with LINCS algorithm. The leapfrog algorithm with 2 fs time step was applied to integrate the equation of motion. Conditions of constant pressure 1 bar and temperature 300 K were accomplished by application of the Parinello-Rahman barostat and Nosé-Hoover thermostat. The particle mesh Ewald (PME) method was used for calculating the long-range electrostatics interactions. The utilities of Gromacs 5.0.2 such as gmx rms, gmx rmsf, gmx gyrate, gmx sasa, gmx density and gmx hbond were used to analyze MD simulation trajectories ( 32 (link)
, 33 (link)
). The program DSSP was used to study the secondary structure fluctuations ( 34 (link)
). Principal components analysis (PCA) reduces the dimensionality of the data obtained from MD simulations. PCA is described in detailed by Giuliani ( 35 (link)
). Here, it was performed to examine the protein global motions. The Gromacs utility “gmx covar” was used to calculate and diagonalize the covariance matrix of Cα atomic positions from the 20 ns trajectories of the wild type r-PA and its mutants. Then, the eigenvalues and corresponding eigenvectors of the matrix were produced. The gmx anaeig was used for analysis and plotting the eigenvectors ( 35 (link)
, 36 (link)
). All graphs were plotted using Xmgrace ( 37
). The gmx sham was used to calculate the free energy (G) by the first two eigenvectors extracted after PCA. 2D and 3D representation of the free energy landscape (FEL)
were plotted using GNUplot 5.2 (www.gnuplot.info). The dynamic cross-correlation matrix (DCCM) that shows the fluctuations of the fluctuations between any two pair of Cα atoms
were calculated by the Bio3D package.
Haji-Allahverdipoor K., Jalali Javaran M., Rashidi Monfared S., Khadem-Erfan M.B., Nikkhoo B., Bahrami Rad Z., Eslami H, & Nasseri S. (2023). Insights Into The Effects of Amino Acid Substitutions on The Stability of Reteplase Structure: A Molecular Dynamics Simulation Study. Iranian Journal of Biotechnology, 21(1), e3175.
Publication 2023
Amber Cuboid Bone Electricity Electrostatics Eye Movements Familial Mediterranean Fever Ions Pressure Proteins Sasa STEEP1 protein, human WILD 20
5
Transfection Efficiency Optimization for Cell Lines
Human embryonic kidney cell line 293T and astrocytic cell lines U373 and SVGA were purchased from American Tissue Culture Collection (Manassas, VA) and maintained at 37°C with 5% CO2 in Dulbecco's Modified Eagle's Medium (Corning Inc., Corning, NY) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich, Burlington, MA) and 10% fetal bovine serum (R&D Systems, Minneapolis, MN). Mouse primary astrocytes were collected from the brain tissues of prenatal day 18-20 wild-type mouse fetus, cultured in F12K media (Corning) for two passages, and used. Human fetal primary astrocytes (HPA) were isolated from aborted human fetal brain tissues provided by the Laboratory of Developmental Biology, supported by the NIH Award Number 5R24HD000836 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. 293T and SVGA were transfected by the standard calcium phosphate precipitation transfection method and estimated to have a transfection efficiency of >95% and ~75%, respectively. The calcium phosphate transfection method for U373 and HPA cells resulted in low efficiency of gene expression (~20% and ~5%). To circumvent this, U373 and HPA were transfected by Lipofectamine 3000 transfection reagents (Cat. # L3000015, ThermoFisher Scientific, Waltham, MA) and media was replaced 16 hr post transfection the cells were cultured for 24-48 hr with an estimated >95% and ~80% transfection efficiency. For both calcium phosphate and lipofectamine 3000 methods, a total of 2.5ug DNA was used and transfection efficiency was estimated by co-transfection of pC3.GFP plasmid and counting the GFP+ cells under a fluorescence microscope.
Wilson K.M, & He J.J. (2023). HIV Nef Expression Down-modulated GFAP Expression and Altered Glutamate Uptake and Release and Proliferation in Astrocytes. Aging and Disease, 14(1), 152-169.
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Publication 2023
Astrocytes Brain Calcium Phosphates Cell Lines Culture Media DNA, A-Form Embryo Fetal Bovine Serum Fetus Fetuses, Aborted Gene Expression Histocompatibility Testing Homo sapiens Kidney Lipofectamine Microscopy, Fluorescence Mus Penicillins Plasmids Streptomycin Tissues Transfection WILD 20

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More about "WILD 20"

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