183 Matching Annotations
  1. Aug 2021
    1. RRID:ZFIN_ZDB-ALT-040716-16

      DOI: 10.7554/eLife.44431

      Resource: (ZFIN Cat# ZDB-ALT-040716-16,RRID:ZFIN_ZDB-ALT-040716-16)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-040716-16


      What is this?

  2. Jul 2021
  3. Jun 2021
    1. We explore them in other chapters and in particular in Macroeconomic Policy Around the World.

      Reference to OS 19 (not currently mapped in our AL)--remove?

  4. Mar 2021
    1. Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 4.7

      AssayResultAssertion: Normal

      StandardErrorMean: 0.11

    2. A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

      HGVS: NM_024675.3:c.1600T>G p.(Ser534Ala)

    1. SUPPLEMENTARY DATA

      AssayResult: 102.6

      AssayResultAssertion: Not reported

      PValue: > 0.9999

      Comment: Exact values reported in Table S3.

    2. To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

      HGVS: NM_024675.3:c.2865T>A p.(Ser955Arg)

    1. Source Data

      AssayResult: 64.92

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 8.7

      Comment: Exact values reported in “Source Data” file.

    2. Source Data

      AssayResult: 77.45

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardDeviation: 6.2

      StandardErrorMean: 4.38

      Comment: Exact values reported in “Source Data” file.

    3. We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

      HGVS: NM_024675.3:c.1865T>C p.(L622P)

    1. Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

      AssayResult: 23.2

      AssayResultAssertion: Abnormal

      ReplicateCount: 14

      StandardErrorMean: 7.1

      Comment: This variant had partial loss of function of peak current (10-50% of wildtype) and a >10mV loss of function shift in Vhalf activation, therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

    2. we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

      HGVS: NM_198056.2:c.2254G>A p.(Gly752Arg)

  5. Feb 2021
    1. Supplemental material

      AssayResult: 61

      AssayResultAssertion: Abnormal

      Comment: See Table S3 for details

    2. Supplemental material

      AssayResult: 3.1

      AssayResultAssertion: Abnormal

      Comment: See Table S3 for details

    3. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

      HGVS: NM_000546.5:c.375G>A p.?

  6. Sep 2020
  7. Jun 2020
    1. vous

      Pourquoi est ce que pour cette question il a fallu calculer a nouveau le rayon de la terre alors qu'elle était déjà donnée dans un document et que nous l'avons calculer dans la question 13 ? Et que les 3 valeurs ( valeur donnée dans le doc et dans les calculs ) ne sont pas les mêmes

  8. Jan 2020
    1. RRID:ZFIN_ZDB-ALT-170522-16

      DOI: 10.7554/eLife.42881

      Resource: (ZFIN Cat# ZDB-ALT-170522-16,RRID:ZFIN_ZDB-ALT-170522-16)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-170522-16


      What is this?

  9. Jun 2019
    1. chains were generated in the same way as for the isolated a-chains. Thus the model consisted of eight polypeptide chains ( 4 a-chains and 4 13-chains) with the central two a and two 13 chains making up the axial contact interface. The simulations of the above fiber models were carried out without explicit solvent using the GROMOS96 vacuum force field as implemented within the GROMACS suite of programs. Models of HbS mutants were generated using the program SCWRL and the mutant fiber models were generated as for the native HbS model. SCWRL replaces only the side chains of desired residues with the best possible rotamer of the mutated amino acids. Thus the initial backbone conformation of the isolated a-chains and the fiber models remained identical in the native HbS and the mutants. The MD simulation protocol for the fiber models consisted of an initial steepest descent energy minimization for 1000 steps followed by full MD simulation for 1.2ns at 300 K. Essential parameters of the simulation like the radius of gyration, root mean squared deviation from the initial structure as well as the kinetic and potential energies are summarized in Chapter I, Table 4. It was observed that all the global indicators of the simulation stabilized to their average values within 0.2ns. Hence data from 0.2ns till the end of the simulations were used for all subsequent analysis
    2. MD simulations on the a-chain of HbS and the mutants were carried out using the GROMACS suite of programs (Lindahl eta!, 2001). The initial structure of the native protein was taken from the high-resolution x-ray crystal· structure (Harrington et a!, 1997) of HbS (PDB entry: 2HBS). The structures of the mutants were generated interactively using INSIGHT-II. The initial model structures were placed in a simulation box of size 42.8 x 31.5 x 42.7 A. The closest distance from any protein atom to the walls of the box was not less than 9 A. The system was then solvated by adding a bath of SPC (Berendson eta!, 1981) waters in such a way that the density of the system was as close to 1 as possible. The overall charge of the system was neutralized by placing suitable counter ions wherever necessary (Chapter 1, Table 4). The resulting system was then energy minimized for 1000 steps using the steepest descent algorithm. This was followed by 0.3ns of position restrained MD during which the solvent and counter ions were allowed to move freely but the protein atoms were harmonically restrained to their initial positions. Finally, normal MD was run for 3ns using the default GROMACS force field. Bond lengths were restrained to their equilibrium values using the LINCS (Hess et a!, 1997) algorithm and a cut-off radius of 0.9nm was used for non-bonded interaction calculation. The temperature of the system was maintained close to 300K by weak coupling to an external temperature bath with a coupling constant of 0.1 ps. The integration time step used throughout the simulation was 1 fs. MD simulations of single mutant a-chains (K 16Q, E23Q and H20Q) as well as the double mutants (K16Q/H20Q, K16Q/E23Q and H20Q/E23Q) were carried out in a similar fashion for 3ns. In order to directly analyze the effects of the mutations on the contact interface, we also carried out MD simulations on a miniature model of the sickle hemoglobin fiber consisting of a complex of two hemoglobin tetramers. In the low salt crystal structure of deoxyhemoglobin S (Harrington et a!, 1997) the asymmetric unit consists of two HbS tetramers that pack as two strands of HbS molecules running parallel to the crystallographic a axis. Axial contacts occur between two tetramers of the same strand and lateral contacts occur between tetramers of different strands. A canonical fiber model was generated by taking one of the two tetramers in the asymmetric unit together with its neighbor translated along the crystallographic a axis [Figure 7 (A, B)]. Fiber models incorporating mutant a
    3. Molecular dynamics (MD) simulatio
  10. May 2019
    1. temperature for 2h. The nitrocellulose membrane was washed extensively with PBST and developed using chemiluminescence substrate from Pierce (USA).
    2. The proteins separated by SDS-PAGE were transferred from the gel to· nitrocellulose membrane using a blotting apparatus (Bio-Rad, USA). In brief, after removal of the stacking gel, the resolving gel was placed over nitrocellulose membrane and sandwiched with Whatman 3 mm filter paper in a cassette. The cassette was submerged in transfer buffer and transfer was carried out at 150 rnA for 3h at 4°C. Following the transfer, the membrane was carefully removed from the blotting apparatus and blocked with 3% non-fat dry milk protein for Ih. The membrane was washed thrice with PBST and incubated overnight with the primary antibody at 4°C. Following incubation, the membrane· was washed thrice with PBST and incubated with appropriate HRP-labeled secondary antibody at room
    3. Western Blot
    1. bacteria. The cells were washed once with ice-cold PBS and the uptake of labeled bacteria was analyzed by flow-cytometry
    2. The phagocytic ability of macrophages was determined by monitoring the uptake of Bioparticles® Alexa fluor 488 labeled dead E. coli (Molecular Probes, Eugene, OR). 2X105 THP-1 macrophages were plated per well in a 24 well plate. Alexa fluor 488 labeled dead E. coli particles were opsonized with an opsonizing reagent obtained from Molecular Probes. These opsonizing reagents are derived from purified rabbit polyclonal IgG antibodies that are specific for E.coli. These opsonized bioparticles® were transferred to the macrophage culture at an multiplicity of infection (MOl) of 1:10, i.e., 10 bacteria per macrophage. The plates were briefly centrifuged at 250 x g to allow the bacteria to settle at the bottom of the plate and were then transferred to an incubator maintained at 37°C and 5% C02 in air for 1 h. The culture medium was aspirated to remove excess unbound bacteria and the cells were washed 3x with ice-cold PBS. To eliminate fluorescence from non-phagocytosed bacteria adhering to the macrophage membrane, 0.25 mg/mL Trypan blue was added and incubated for 10 min to quench the fluorescence of extracellula
    3. Measurement of phagocytic ability of macrophages
    1. Cell-free protein synthesis inhibitory activity of restrictocin was assayed as described by Harlow and Lane ( 1988). The frozen rabbit reticulocyte lysate was thawed on ice in the presence of 30 J..LI of haemin solution per 0.5 ml vial. The toxin was diluted in 0.2% RNase free BSA and several concentrations were incubated with 10 J..LI lysate, I mM ATP, 0.2 mM GTP, 75 mM KCI, 2 mM magnesium acetate, 3 mM glucose, 10 mM Tris-HCI, pH 7.6, 4 J..LM amino acid mixture without leucine, 0.16 J..LCi eH] leucine, 1.33 mg/ml creatine phosphokinase, and 2.66 mg/ml creatine phosphate in a reaction volume of 30 J.LI. The reaction was carried out at 30 °C for one hour and terminated by adding 0.25 ml of I N NaOH containing 0.2% H202• The reaction mixture was further incubated for I 0 min. at 37 °C, BSA added to a final concentration of 65 J..Lg/ml, and the proteins were precipitated with 15% trichloroacetic acid. The mixture was left on ice for 30 min. for complete precipitation and harvested onto 26 mm glass fibre filters. The filter discs were placed in a manifold harvester (millipore) and rinsed with chilled 5% TCA, before the addition of reaction contents. The filters were thoroughly washed with chilled acetone and dried at 37 °C, for one hour. The dried filters were immersed in organic scintillation fluid, and counted using a liquid scintillation counter (Packard).
    2. Assay for Inhibition of in Vitro Protein Synthesis
    1. Following transfection with calcium phosphate or lipofectin, the cells were selected for neomycin resistance by using the analogue G418 Geniticin in the culture medium. After 48 ·hours post -transfection, the cells were harvested and replated at a lower density ( 0.5 x 104 cells I 60 mm dish). Culture medium containing G418 was then added to the cells. G418 was used at two concentrations -400 ug I ml and 500 ug 1 ml. The cells were cultured in G418 containing medium for 2 - 3 weeks. During this period, the mock transfected cells and the cells transfected with plasmid lacking neo gene, died and the transformed cells formed colonies. Individual G418 resistant colonies were picked up and propagated as independent clones. The culture supernates from these clones were analysed by RIA for BhCG. The stability of the BhCG secreting clones was assessed by culturing with several passages over a few weeks in media with or without G418.
    2. Isolation of stable clones.
    1. RFFIT is used for detennination of rabies virus neutralizing antibody (RVNA) titers. It is an in vitro cell culture based technique in which foci of virus-infected cells are observed by fluorescent antibody staining. In brief, mouse neuroblastoma (MNA) cells were cultured in T-25 tissue culture flasks in DMEM supplemented with 10% FCS at 35°C in humidified atmosphere of0.5% C02. For subculturing, cells were trypsinized (0.5% trypsin + 0.2% EDT A in DMEM without FCS), centrifuged at 150 X g for 10 min, resuspended in DMEM supplemented with 10% FCS and aliquoted into T -25 flasks. Sera from immunized mice were heat inactivated at 56°C for 30 min and the RVNA titers were determined by RFFIT as described previously (Smith . et al., 1996). Briefly, 100 111 of various dilutions of the reference (Standard Rabies Immune Globulin, Biological Research and Reviews, FDA, Maryland, US) and the test sera were mixed with 100 111 Challenge Virus Strain-11 of rabies virus (containing 50 FFD50) in 8-well tissue culture chamber slides and incubated at 35°C in presence of 0.5% C02 for 90 min. After the incubation period, 0.2 ml of MNA cells ( 1 x 1 05) were added to each well and the slides incubated for 40 h following which these were fixed in chilled acetone and stained with FITC conjugated anti-rabies MAb (Centocor Inc, USA) for 45 min. The slides were washed three times with PBS, mounted in glycerol : PBS (9 : 1 ), and examined under fluorescence microscope (Optiphot, Nikon, Japan). Data was expressed as neutralizing antibody titer that is the reciprocal of the serum dilution resulting in a 50% reduction in the number of the virus infected cells in the presence of the test serum.
    2. RAPID FLUORESCENT FOCUS INHIBITION TEST (RFFIT)
    1. Immediatelyafterisolation,thetissueswereweighedandsubjectedtolipidextractionthatwascarriedoutinduplicateaccordingtoFolchetal.(1957)
    2. Totallipids
    3. Salineextracts(0.89%)oftissueswerepreparedinTeflonglasshomogenizer.ThecarbohydratecontentoftheextractwasdoneaccordingtothemethodofShibkoetal.(1967)
    4. Carbohydrates
    5. TotalproteincontentwasdeterminedbytheFolin-CiocalteaumethodofLowryetal.(1951)asmodifiedbyZakandCohen(1961).Bovinecrystallinealbuminwasusedasa referencestandard
    6. Totalproteins
    7. Aftereffluentexposure,thecontrolandexperimentalfisheswerekilledbyhammeringonheadanddissectedimmediately.Excisedbrain,liver,muscle,gill,kidneyandair-breathingorgans werepooled incoldcondition andusedforbiochemicalestimations.
    8. BiochemicalAnalysis
    1. Strains were streaked on LBON agar plates and after an overnight incubation at 42°C growth was monitored (compared to that on LBON at 30°C as control). Absence of single colony growth was taken to reflect temperature sensitivity. Whenever needed the phenotype was also quantitatively assessed by plating dilutions of cultures on LBON agar plates and the drop in plating efficiency was scored after overnight incubation at 30°C and 42°C
    2. LBON(Ts) phenotype
    3. This test was therefore used for two purposes: (i) to distinguish relA+ from relA− strains, and (ii) as a qualitative measure of transcriptional polarity relief at the ilv locus. Growth in the presence of amino acids Serine, Methionine, and Glycine (SMG) was scored on glucose-minimal A plates supplemented with each of the amino acids at 100 μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype
    4. The E. coli relA mutants exhibit SMG-sensitive (SMGS) phenotype i.e. growth-inhibition in the presence of Serine, Methionine and Glycine at 1 mM concentration each (Uzan and Danchin, 1978) and is proposed to be a consequence of transcriptional polarity exerted by a frameshift mutation in the ilvG gene on the expression of downstream genes of the ilvGMEDA operon (Lopes et al., 1989). It was observed in another study that the rho and nusG mutants that are defective for transcription termination conferred SMG-resistant (SMGR) phenotype in a relA1 strain (Harinarayanan and Gowrishankar, 2003)
    5. SMG resistance
    6. Lac+ colonies were distinguished from Lac− on MacConkey-lactose plates or on Xgal indicator plates. Xgal is a non-inducing colourless substrate of β-galactosidase enzyme which upon hydrolysis yields dark blue indolyl moieties and hence, the Lac+ colonies on Xgal indicator plates are seen as dark blue colonies. Xgal was prepared as a stock solution of 5 mg/ml in dimethyl formamide and used at a final concentration of 25 μg/ml. On MacConkey-lactose medium (pH around 7.1) on the other hand, Lac+ strains can utilize the lactose sugar present in the medium to lower the pH of the medium to 6.8, resulting in a pink coloured colony while Lac─ strains are unable to utilize lactose to give a white colour
    7. Lac phenotype
    8. Scoring for phenotypes
    1. injection, programmed at 20°C min-1 to 200 °C and held for 10 min, then at 10 °C min-1 to 230 °C, and finally at 5 °C min-1 to 320 °C and held for 5 min. Injection temperature was set at 260 °C. High purity helium was used as carrier gas of 1.0 mL min-1 flow-rate. The spectrophotometers were operated in electron-impact (EI) mode, full scan of 40-550 amu or selected ion monitoring (SIM) was used, the ionization energy was 70 e V. Calibration curve was generated using n-hexane stock solutions of standard ergosterol dilutions (5-300 ng/mL) in duplicates and plotting the peak area versus the concentration (Yang et al., 2009).
    2. Gas chromatography (GC) is a common type of chromatography used in analytic chemistry for separating and analysing compounds that can be vaporised without decomposition, while Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles and thus determining masses for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules. Combined together the technique of GC-MS can be used to identify individual components in a mixture and also quantitate them. The sterol extracts prepared in 3.2.C.14 were dried under nitrogen (N2) gas, resuspended in n-hexane and derivatized with BSTF A (N,O-bis (trimethylsilyl) trifluoroacetamide) containing 1% TMCS (trimethylchlorosilane) at 70 °C for 1 hr. The derivative mixture was dried under N2 (gas) to remove excess BSTFA and subsequently re-dissolved in n-hexane. The column temperature was set at 100°C and held for 5 min for
    3. GC-MS analysis of ergosterol
    4. Leishmania! expression vector pXG-GFP+2 was obtained as a kind gift from Dr. Stephen S. Beverley (Washington University). Full length CYP5122A1 (Ld27) had been amplified by PCR using primers (F27P3/F27P2, Table 3.4) and cloned into pGEM-T Easy vector. The ORF was then excised from pGEMT using Notl and cloned into pXG-GFP+2 vector. The transformants were selected for the insertion of the gene in the correct orientation using restriction digestion. Standard cloning techniques were used (Sambrook et al., 1989).
    5. Generation of pXG-GFP+2-Ld27 (CYP5122Al)
    1. fluorescence by excitation at 440 (pH-independent) and 490 nm (pH-dependent) with emission at 535 nm. Ratio offluorescence intensity at 490 to440 nm was used tocalculatethe vacuolar pH. Background fluorescence was removed by subtracting the fluorescence intensity values of cells without BCECF-AM from the fluorescence intensity values of the probe-loaded cells
    2. Vacuole pH inyeast cells was determined asdescribed previously (Padilla-López and Pearce, 2006). Briefly, log-phase,YPD medium-grown yeast cells were harvested and suspended in 200 μl YPD medium containing 50 μM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Invitrogen # B1150) to the final cell density of 4 x 107 cells. Cells were incubated at 30 ̊C for 30 min at room temperaturefollowed by three washeswith YPD medium. Washed cells were resuspended in 1 ml YPD medium and 200 μl cell suspension was used for recording
    3. Measurement of vacuole pH
    1. After restriction enzyme digestion, digested products wereresolved on agarose gels and desired DNA fragmentswereextracted from the gel. Concentration of gel-extracted DNA fragments was determined usingspectrophotometerand ligation reactions were set up using a molar ratio of vector to insert of 1:3 and 1:1 for sticky and blunt end ligations, respectively. Ligation mixwas incubatedeither at 22ºC for 4 hor at 16°Cfor 14-16 h. After incubation,T4DNA ligase was inactivatedat 65ºC for 20 min
    2. Ligation
    3. Genomic mapping of disrupted locusin Tn7insertion mutants was carried out as describedpreviously(Kaur et al., 2004).C. glabratamutants carrying Tn7insertionswere grown in YPD-liquid medium and genomic DNA was isolated fromovernight cultures. 10 μg genomic DNA was digested either with restriction enzyme MfeIor SpeI.Restriction enzyme-digestedDNA was precipitated with 1 ml ethanol and 1/10thvolume of sodiumacetate (3 M,pH 5.2). DNA pellet was washed twice with ice-cold 70% ethanol, air driedand was resuspended in sterilewater. DNA was recircularized with T4 DNA ligase.Resultant circular DNA carriedTn7cassette flanked on bothsidesby the disrupted locus oftheC. glabratagenome. CircularDNA wastransformed in E. coliBW23473 strainwhich contains protein Π (the product of the pirgene) required by R6Kγorifor replication.Twoverified transformants were grown overnight in LB-kanamycin medium and plasmids were extracted. Purified plasmids were sequenced withprimers reading outwards (OgRK 183 and OgRK 184) from both ends ofTn7cassette.Sequences obtained were compared,usingBLAST,against C. glabratagenome sequence database and regionsof Tn7insertions in C. glabratawere mapped
    4. Mutant rescue
    1. The mixture was then incubated at 37oC for 45 min to radiolabel the oligonucleotide. Simultaneously, the Sephadex G-50 column was prepared in 1 ml syringe. The reaction mixture was loaded on the column and the eluting fractions were collected in the microfuge tube by loading 200 μl milli-Q water on the top of the column. After collecting 5-6 fractions, the tubes were analysed using a GM counter for the amount of radioactivity. The fractions having specific activity between 3.5-4.5 X 106cpm/pmoles were pooled. To this, 100 pM of the complimentary strand of the oligonucleotide was added and heated at 95°C for 5 minutes. The mixture was allowed to annealat room temperature for 1 h and further used in Gel shiftassay
    2. The oligonucleotides of different transcription factors such as NF-B, AP-1, p53 and SP-1 were 5′-end labelled using radioactive γ32-ATP (obtained from BRIT, BARC, Mumbai, India) and Polynucleotide kinase, as per manufacturers’ protocol. The reaction mixture containing the different components, described in the table 2.2below was added in a microfuge tube.Table 2.2: Various components of oligonucleotide labeling reaction mixture
    3. Radioactive labelling of oligonucleotides
    1. After 14-16 h incubation, hybridization buffer was decanted to a radioactive liquid waste container. Membranes were washed twice with 2X SSC (saline-sodium citrate) containing 0.1% SDS for 15 min at 55°C followed by two washes with 1X SSC containing 0.1% SDS for 15 min at room temperature. Post washes, membranes were rinsed with 1X SSC buffer at room temperature exposed to a phosphorimager screen for 1 h and scanned using a phosphorimager (Fuji Film FLA-9000). The data were analysed by densitometry using Fuji Film Multigauge software V3.11 and graphs were plotted using GraphPad Prism5 software.Note:Depending on signal saturation or non-specificity, high stringency washes were performed starting from 0.5X SSC followed by 0.2X SSC or 0.1X SSC wash buffers containing 0.1% SDSat room temperature
    2. Post-hybridization washes
    1. The reactions were carried out at 30 °C for 15 min in 25μlof ubiquitylation reaction buffer (40mM Tris-HCl at pH 7.6, 2mM DTT, 5mM MgCl2, 0.1M NaCl, 2mM ATP) containing the following components: 100μM ubiquitin, 20nM E1 (UBE1), 100nM UbcH5b (all from Boston Biochem). The bacterially purified MBP-WWP2 and MBP-WWP1 E3 ligases were added to the reaction mixture. The bacterially purified and GST bound GST-protein, GST-p73, and GST-ΔNp73 were used as the substrate in the reaction. After the ubiquitylation reaction, the GST beads werewashed five times with 1X NETN buffer and boiled withan equal volume ofSDS-PAGE loading buffer. The ubiquitination of the substrates was determined by western blotting with the substrate-specific antibody
    2. In vitroubiquitylation assay
    1. grown culture was inoculated in fresh PS medium with or without 50 μM 2, 2’-dipyridyl and grown at 28°C. At regulartime intervals, 1 ml culture was removed to determine OD at 600 nm. Furthermore, for GUS assay, 1 ml culture was centrifuged to obtain the pellet, which was washed once in sterile miliQ water, and resuspended in 250 μl volume of 1 mM MUG (4-methylumbelliferyl β-D-glucuronide) extraction buffer (50 mM sodium dihydrogen phosphate [pH 7.0], 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium lauryl sarcosine, and 10 mM β-mercaptoethanol),and incubated at 37°C (Jefferson et al., 1987). After appropriate time intervals, 75 μl aliquotes were taken from each reaction mixture, and reaction was terminated by adding 675 μl Na2CO3 (0.2 M). Fluorescence was measured against 4-methyl-umbelliferone as the standard at excitation/emission wavelength of 365/455 nm, respectively. Likewise, GFP activity was measured in Varioscan flash (Thermoscientific) at exitation/emission wavelength of 472/512 nm, respectively by taking 200 μl of culture directly
    2. For reporter assay, GUS and GFP marked Xanthomonas oryzaepv. oryzicolastrains and control strains were grown overnight in PS medium. 0.2
    3. Reporter assays with β-Glucuronidase (GUS) and green fluorescent protein (GFP)
    1. and pelleted down at 1200 rpm for 5 min. The supernatant containing 10% FBS and trypsin were discarded and the cell pellet was resuspended in media containing 1% FBS. 5×104cells suspended in 200 μLDMEM (1% FBS) was added to the upper chamber and carefully transferred to the well of the companion plate containing 700 μLof complete DMEM (10% FBS) to serve as chemotactic agent. Inserts were lifted with sterile forceps and placed in the companion plate by avoiding any bubbles at the bottom surface of the insert. Cells were incubated at 37ºC with 5% CO2for 18 h (MEFs) and 24 h (HeLa and HCT116 cells) to allow migration. At the indicated time, cells on the upper surface of the filter were removed carefully by scrubbing with wet cotton swabs and inserts were dunked twice in excess PBS. Cells that migrated to the lower surface of the filter were rinsed twice with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature. Post fixation inserts were rinsed in PBS twice and air dried at room temperature. Filters were cut carefully through the edges using a scalpel blade and placed inverted on a clean slide so that the migrated cells faced the upward direction. Filters were mounted with vectashield DAPI and covered with a clean coverslip. Migrated cells stained with DAPI were counted by imaging multiple fields using anepifluorescence inverted microscope (Olympus lX51,Image-Pro AMS 6.0 acquisition software, 20x 0.45 N.A. objective).The number of cells migrated to the lower surface was quantified by counting the total number of DAPI positive nuclei in at least 10 random fieldsper sample
    2. Transwell migration assays were conducted as described previously (Raoet al., 2015). Transwell inserts (24 well, 8 μm pore size, Costar, Corning)were used to conduct migration assays. Inserts were equilibrated by adding 1% FBS containing media to the upper chamber, as well as lower chamber of the insert (companion plate) and placed in a 37ºC incubator with 5% CO2till use. Cells were harvested, counted using hemocytometer
    3. Transwell migration assay
  11. Apr 2019
  12. Dec 2018
    1. A geographical line has been drawn across the Union, and all the States north of that line have united in the election of a man to the high office of President of the United States, whose opinions and purposes are hostile to slavery. He is to be entrusted with the administration of the common Government, because he has declared that that “Government cannot endure permanently half slave, half free,” and that the public mind must rest in the belief that slavery is in the course of ultimate extinction.

      14 + 16 - The author expresses disdain towards the election of a Republican president, not elected in the South, is considered a hostile to slavery, and plans on abolishing it. These concerns imply that he should not be the president of the South.

  13. Nov 2018
    1. DISOLUCIÓN ANTICIPADA DE LAS CÁMARAS Cuya finalidad es reforzar la mayoría parlamentaria del gobierno a base de una nueva elección electoral. La iniciativa es que el Presidente tenga deliberación con el Consejo de Ministros. Sus aspectos son : 1 Que el decreto este firmado por el Rey y refrendado por el Presidente. art 64 2 Debe de haber una convocatoria electoral. art 68 3 Puede afectar a una o las dos Cámaras. Título III. Sus límites son: -No puede haber una disolución anticipada mediante una moción de censura. art 113. -No puede haber una nueva disolución sin haber pasado un año del anterior. art 115 y art 99 -No puede haber disolución en los supuestos del art.116.

  14. Oct 2018
    1. It is possible that a Federative union of the British North American Provinces would afford to the Canadian Government the – readiest mode of escape from the difficulties and embarrassments which now surround the settlement of the “seat of Government ” question, and I presume that I am right in supposing that, although the ostensible object of the proposed inquiry is the union by Federative bonds with Canada of the other British North American Provinces, the Canadian Government have no less in view the, severance of the bond which now joins the two Canadas in a Legislative Union, and the substitution for that bond of a more elastic tie of a Federal or a Federative character.

      §.16 of the Constitution Act, 1867.

  15. Jul 2018
    1. 11

      Step 15:

      Secure piece assembled in the previous step using the 4 102509 with a Flat-Head screwdriver. Consult the graph for proper alignment.

      Step 16:

      Insert the 8 101345 studs into the pre-drilled holes.

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    Annotators

  16. Mar 2018
    1. It was desirable the General Government should have the control of the medium through which the trade and commerce of the country was carried on, and that in the establishment of banks, the issue of paper money and in offering to the public the paper representative of their labor, in whatever part of the country, there should be the same legislative security for the people

      §§.91(2)(14)(15)(16) of the Constitution Act, 1867.

  17. Nov 2017
    1. To be safe, always pack all medications in your carry on—never in checked luggage. Airplanes don't have refrigerators. If you have a drug that must be kept cold, ask your pharmacist how to package it safely for travel.

      This is great information for travelers as they often have many questions about where to carry their medications and whether or not they need to be documented in the checked luggage.

    1. Under the ACAA(Section 382.57), the airlines are not allowed to charge you for your dialysis machine.iii

      This is great information to be sharing with clients when educating them about life style changes and considerations.

  18. Jul 2017
  19. Jun 2017
  20. Apr 2017
    1. The committee was moved by the willingness of Canadians to welcome Syrian refugees and the eagerness of Syrian refugees to become contributing members of Canadian society. It was concerned, however, that the Government of Canada is not allocating enough resources to help them integrate
    1. Syrian refugees who receive a 1 rating will be on a list of as many as 10,000 names that the UNHCR office in Jordan will offer to the Canadian government.  (Annie Sakkab for The Globe and Mail) Let’s help our own without forgetting to help the refugees
  21. Dec 2016
  22. Nov 2016
    1. Some blocks in my neighborhood are getting downright spooky – front yards are filling with spider webs and tombstones, and ghosts peek through the bushes. Along with the piles of pumpkins and inevitable candy corn appearing in the supermarket, they are a reminder that Halloween is just around the corner. Americans celebrate Halloween on October 31 by trick-or-treating, displaying jack-o’-lanterns (carved pumpkins) on their porches or windowsills, holding costume parties, and sharing scary stories

      american way of celebrating halloween

  23. Oct 2016
    1. In Wall-E and the Toy Storytrilogy, the pleasure is of the suspen-sion of knowledge—the pleasure of notknowing.
    2. One of the distinct pleasures in Pixar’s films is the pleasure of seeing the deepest of human struggles, timeless philosophical questions projected in and through remote forms of representation.
  24. Aug 2016
  25. Jul 2016
    1. Engage adult new writers with online communities of writers, as contributors, readers, and peers, to foster their self-directed learning, self-study, and persistence.

      I know it was not brought up, but when I have my young students peer review, there is always some tension because people are embarrassed to let their peers read their work? For adult learners, i wonder if that anxiety goes away, or if it is heightened because they are adults and sometimes as adults we like to appear as if we know the correct answers most of the time (or at least this is something I do).

    2. discuss, but do not debate

      This is a great tool but I feel like it is not being modeled enough in society. How can we shift this back into our world before it completely goes by the wayside? Do you think social media has caused a decline in the art of "discussion"?

    3. Know your students

      A lot of these strategies can be interlaced with educating youth as well.

    4. Because adults learn by doing, effective instruction focuses on tasksthat adults can perform, rather than on memorization of content. Be-cause adults are problem-solvers and learn best when the subject is of immediate use, effective instruction involves the learner in solving real-life problems.

      When we conduct PDs, these are good points to know to make our teaching more effective

    5. Because adults need to know whythey are learning something, effective teachers explain their reasons for teaching specific skills

      This is also emphasized at the high school level at times. Students often ask, "Why are we doing this?" "Am I ever going to use this?" Maybe instead of viewing that as whining, maybe it as actually more of an "adult" way of questioning.

    6. Is motivated to learn byinternal, rather than ex-ternal, factors

      I'm not too sure that this is always true. A raise, a degree, or a career choice are technically external factors.

  26. Jun 2016
    1. There are many appealing strengths to the idea that learning should be organized around authentic problems and projects that are frequently encountered in nonschool settings: in John Dewey’s vision, “School should be less about preparation for life and more like life itself.”

      I agree and attempt to incorporate "real life" problems as often as I can into my classroom. The problem I often come across is the textbook's idea of a "real life" problem is often much different from what the students relate with. Still, it always helps to add context, especially in math where the question "where are we ever going to use this?" is asked on a daily basis.

    2. there is an infinite number of numbers between any two rational numbers

      This is a fact that can be really unsettling for students to hear, but is also very important for them to actually think about. It not only helps them understand fractions, but also the concept of infinity. Many students, even in high school just simply do not understand fractions, or are not willing to spend the time to complete problems with fractions.

    3. Gradually, students come to ask self-regulatory questions themselves as the teacher fades out. At the end of each of the problem-solving sessions, students and teacher alternate in characterizing major themes by analyzing what they did and why. The recapitulations highlight the generalizable features of the critical decisions and actions and focus on strategic levels rather than on the specific solutions

      This is challenging for me to get the hang of as a teacher. It is very easy for a class to do an activity, a then have the discussion get taken over by a only a few select students that were faster at catching on to the concepts. You really have to be careful as the teacher to make sure you allow for enough time so that all students are able to finish and form ideas about what they just learned. Then having a system to allow all groups to share something is very helpful.

    4. Tests of transfer that use graduated prompting provide more fine-grained analysis of learning and its effects on transfer than simple one-shot assessments of whether or not transfer occurs

      I like to use this type of graduated prompting when doing discovery based tasks. It allows me to give the appearance of giving students hints, when really I am just trying to get them to remember or realize what they already know.

    5. For example, a group of Orange County homemakers did very well at making supermarket best-buy calculations despite doing poorly on equivalent school-like paper-and-pencil mathematics problems (Lave, 1988). Similarly, some Brazilian street children could perform mathematics when making sales in the street but were unable to answer similar problems presented in a school contex

      This could come back to the idea that students perceive themselves as "bad at math" so in a school setting, they automatically shut down and don't actually think about the problems. But when they are presented outside of an educational context, they don't think about it as math, they just think about it as part of their job or social obligation.

    6. e.g., a person may be performance oriented in mathematics but learning oriented in science and social studies or vice versa).

      I come across this issue often in math classes. I have to be very careful when assigning tasks to students, because it is a very fine line between too easy and too challenging. Especially in math, it seems that there a lot of students who have just accepted that they are "bad at math" so they are quick to give up until someone explicitly shows them the answer and how to do it. This is a big issue that I try to fix with students on a daily basis.

    7. A number of studies converge on the conclusion that transfer is enhanced by helping students see potential transfer implications of what they are learning

      I also like to assign problems where students have to identify the error in another student's work. It seems to help students see common mistakes, and helps prevent them from making them in the future.

    8. Understanding when, where, and why to use new knowledge can be enhanced through the use of “contrasting cases,” a concept from the field of perceptual learning (see, e.g., Gagné and Gibson, 1947; Garner, 1974; Gibson and Gibson, 1955). Appropriately arranged contrasts can help people notice new features that previously escaped their attention and learn which features are relevant or irrelevant to a particular concept

      I like using this in situations where a short-cut may be able to be used for one math problem, but cannot be applied in a similar looking problem. I am always sure to draw attention to the two problems so that students can see the slight differences and why they affect the outcome of the problem.

    9. In the rote method, students were taught to drop a perpendicular and then apply the memorized solution formula.Transfer Both groups performed well on typical problems asking for the area of parallelograms; however, only the understanding group could transfer to novel problems, such as finding the area of the figures below.or distinguishing between solvable and unsolvable problems such asThe response of the “rote” group to novel problems was, “We haven’t had that yet.”

      This is good, and also is the reason I hate teaching my students formulas. I almost always show them and make them practice problems first by figuring out what is going on, rather than giving them a formula. The second I give them a formula, they start blindly plugging numbers in, and cannot complete seemingly simple problems that look slightly different.

    10. The concept of adaptive expertise (Hatano and Inagaki, 1986) provides an important model of successful learning. Adaptive experts are able to approach new situations flexibly and to learn throughout their lifetimes. They not only use what they have learned, they are metacognitive and continually question their current levels of expertise and attempt to move beyond them. They don’t simply attempt to do the same things more efficiently; they attempt to do things better. A major challenge for theories of learning is to understand how particular kinds of learning experiences develop adaptive expertise or “virtuosos.”

      I like this, I know, and try to make it clear to my students, that I am not a complete expert in my field. It is ok for even me to make mistakes, or to admit that I am unsure about something (as long as I eventually find them the answer). When students see this, I believe they become less self-conscious about making mistakes in front of their classmates. It also helps the more advanced students understand that even they still have a lot to learn.

    11. Sometimes, however, students can solve sets of practice problems but fail to conditionalize their knowledge because they know which chapter the problems came from and so automatically use this information to decide which concepts and formulas are relevant. Practice problems that are organized into very structured worksheets can also cause this proble

      My textbooks often do this, but I try to combat it as much as possible by either assigning problems in review sections that are already mixed up, or at least providing practice where problems are all mixed up before a large assessment. I could never understand why books would organize so many problem sets in ways that allowed students to work through them thoughtlessly.

    12. Experts usually mentioned the major principle(s) or law(s) that were applicable to the problem, together with a rationale for why those laws applied to the problem and how one could apply them (Chi et al., 1981). In contrast, competent beginners rarely referred to major principles and laws in physics; instead, they typically described which equations they would use and how those equations would be manipulated

      I often notice this in my classroom. I will teach a concept in a way similar to the expert, where I try to focus on the large concept so that students are able to apply that concept in other situations. However, often times when I hear students explaining what I taught to the other students around them, they will only focus on the single method used to solve that exact problem.

    13. Research shows that students who think that intelligence is a fixed entity are more likely to be performance oriented than learning oriented—they want to look good rather than risk making mistakes while learning. These students are especially likely to bail out when tasks become difficult

      These types of students are always the most challenging for me to teach. I firmly believe that the best way to learn is to try something out, get it wrong, then figure out for yourself why it is wrong and how to fix it. Students that are afraid to be wrong cannot learn in this way until their bad habits are broken.

    14. There is no universal best teaching practice.

      Could not agree more. I have been most successful when I have blended teaching strategies, or have switched strategies based on the goal of the lesson.

    15. This will require active coordination of the curriculum across school years.

      I completely agree that this would be the best environment for students to learn, but it has seemed to me very difficult to actually make it happen. Even within school districts there is often little communication between the elementary, middle, and high schools. And even within each level there is not always a natural flow from one classroom to the next. Having teachers keep classes of students for more than a semester or year at a time would help, but that is generally not the case, especially in core subject areas.

    16. Experts are also able to fluently access relevant knowledge because their understanding of subject matter allows them to quickly identify what is relevant. Hence, their attention is not overtaxed by complex events.

      This is an important skill. So many students seem to think that google can solve all of their problems, therefore they don't have to know/learn as much on their own. But the ability to sift through information and actually determine what is relevant and useful comes from your own understanding of the material.

    17. A common misconception regarding “constructivist” theories of knowing (that existing knowledge is used to build new knowledge) is that teachers should never tell students anything directly but, instead, should always allow them to construct knowledge for themselves.

      It seems to me that there always has to be some material where it is very difficult to draw on prior knowledge as it is completely new to students. On these types of lessons, teaching through discovery can lead to more frustration than good. I love to allow my students to discover concepts on their own, but it simply does not work for everything.

    18. In the most general sense, the contemporary view of learning is that people construct new knowledge and understandings based on what they already know and believe (e.g., Cobb, 1994; Piaget, 1952, 1973a,b, 1977, 1978; Vygotsky, 1962, 1978). A classic children’s book illustrates this point; see Box 1.2. A logical extension of the view that new knowledge must be constructed from existing knowledge is that teachers need to pay attention to the incomplete understandings, the false beliefs, and the naive renditions of concepts that learners bring with them to a given subject.

      Assessing students' prior knowledge is one of the most important tools for a teacher. Being able to relate a difficult concept to a simpler concept that students already understand allows students to come to a much deeper understanding of the content.

    19. At the same time, students often have limited opportunities to understand or make sense of topics because many curricula have emphasized memory rather than under- Page 9 Share Cite Suggested Citation: "1 Learning: From Speculation to Science." National Research Council. How People Learn: Brain, Mind, Experience, and School: Expanded Edition. Washington, DC: The National Academies Press, 2000. doi:10.17226/9853. × Save Cancel standing.

      I see this as a hard line to walk, especially being a math teacher, it is expected for students to memorize certain properties/equations, where as a teacher your focus has to be on getting the students to understand how and when to actually use the equation, and why it works in the first place.

    20. Fundamental understanding about subjects, including how to frame and ask meaningful questions about various subject areas, contributes to individuals’ more basic understanding of principles of learning that can assist them in becoming self-sustaining, lifelong learners.

      I agree, I can generally tell more about what a student knows by the questions they ask, compared to anything else. It is also important as teachers to ask meaningful questions to the students, and avoiding simple yes/no type questions.

  27. Apr 2016
    1. The following autumn I ventured upon a college career against my mother's will.    I had written for her approval, but in her reply I found no encouragement.

      This passage exemplifies the theme of Zitkala Sa's despondent longing for her mother's encouragement and support. Zitkala Sa expresses this theme more as she moves farther away from her mother and continually discriminated against for being different.

  28. Mar 2016
    1. While I thus went on, filled with the thoughts of freedom, and resisting oppression as well as I was able, my life hung daily in suspense, particularly in the surfs I have formerly mentioned, as I could not swim. These are extremely violent throughout the West Indies, and I was ever exposed to their howling rage and devouring fury in all the islands.

      The use of imagery inspires fear and exemplifies Equiano's challenges.

    2. "—No peace is given To us enslav'd, but custody severe; And stripes and arbitrary punishment Inflicted—What peace can we return? But to our power, hostility and hate; Untam'd reluctance, and revenge, though slow, Yet ever plotting how the conqueror least May reap his conquest, and may least rejoice In doing what we most in suffering feel."

      This poem expresses the emotion and sensory experience of perseverance through brutality.

    3. those many instances of oppression, extortion, and cruelty, which I have been a witness to in the West Indies: but, were I to enumerate them all, the catalogue would be tedious and disgusting. The punishments of the slaves on every trifling occasion are so frequent, and so well known, together with the different instruments with which they are tortured, that it cannot any longer afford novelty to recite them; and they are too shocking to yield delight either to the writer or the reader. I shall therefore hereafter only mention such as incidentally befel myself in the course of my adventures.

      Equiano's use of emotion through the times that he is beaten is hard to read. Yet, the way he perseveres through his struggles is captivating.

    4. This speech of the captain was like life to the dead to me, and instantly my soul glorified God; and still more so on hearing my master immediately say that I was a sensible fellow, and he never did intend to use me as a common slave; and that but for the entreaties of the captain, and his character of me, he would not have let me go from the stores about as I had done; that also, in so doing, he thought by carrying one little thing or other to different places to sell I might make money.

      Equiano seems ecstatic that others are showing him compassion that most common slaves did not receive during this time period. This shift in tone could easily make the reader feel empathy and happiness for Equiano at the same time.

    5. Had I wished to run away I did not want opportunities, which frequently presented themselves; and particularly at one time, soon after this. When we were at the island of Gaurdeloupe there was a large fleet of merchantmen bound for Old France; and, seamen then being very scarce, they gave from fifteen to twenty pounds a man for the run. Our mate, and all the white sailors, left our vessel on this account, and went on board of the French ships. They would have had me also to go with them, for they regarded me; and they swore to protect me, if I would go: and, as the fleet was to sail the next day, I really believe I could have got safe to Europe at that time.

      Equiano is incredibly smart and he uses pathos to convince his readers of this. Even though there are a lot of opportunities for Equiano to leave and escape, he stays and through his servitude is able to gain freedom.

    6. in the year 1765; and during the time we were loading her, and getting ready for the voyage, I worked with redoubled alacrity, from the hope of getting money enough by these voyages to buy my freedom in time, if it should please God; and also to see the town of Philadelphia, which I had heard a great deal about for some years past; besides which, I had always longed to prove my master's promise the first day I came to him.

      During this time period, many Europeans feared that the bible would lead to higher educated slaves. In addition they believed that these educated slaves would in turn revolt against the abuse of power displayed by landowning whites.

    7. for one Sunday night, as I was with some negroes in their master's yard in the town of Savannah, it happened that their master, one Doctor Perkins, who was a very severe and cruel man, came in drunk; and, not liking to see any strange negroes in his yard, he and a ruffian of a white man he had in his service beset me in an instant, and both of them struck me with the first weapons they could get hold of. I cried out as long as I could for help and mercy; but, though I gave a good account of myself, and he knew my captain, who lodged hard by him, it was to no purpose. They beat and mangled me in a shameful manner, leaving me near dead. I lost so much blood from the wounds I received, that I lay quite motionless, and was so benumbed that I could not feel any thing for many hours. Early in the morning they took me away to the jail.

      The injustices committed by white landowning males demonstrates the destructiveness of the slave trade. Through abusing and dehumanizing slaves such as Equiano's slave owners and powerful white males were able to strip slaves of their identity and in Equiano's case even his family.

    8. When we were safe arrived at Montserrat, and I had got ashore, I forgot my former resolutions.—Alas! how prone is the heart to leave that God it wishes to love! and how strongly do the things of this world strike the senses and captivate the soul!—After our vessel was discharged, we soon got her ready, and took in, as usual, some of the poor oppressed natives of Africa, and other negroes; we then set off again for Georgia and Charlestown.

      Equiano sympathizes with African Americans as he was from Africa. Even though this is not proven, "Equiano must surely have known that the most intensive search would be made to discredit his work and, through it, the abolition movement. To invent a childhood that could at any time be publicly revealed as a fraud would be such a potentially damaging maneuver that Equiano, who was clearly deeply committed to the cause, would not have risked it. Under such circumstances, Equiano's account of his African origins must surely be reliable."

      Carey, Brycchan. "OLAUDAH EQUIANO African or American?" Ideas, iEsthetics, and Inquiries in the Early Modern Era: 229-46. Print.

    9. I had been told one evening of a wise woman, a Mrs. Davis, who revealed secrets, foretold events, &c. I put little faith in this story at first, as I could not conceive that any mortal could foresee the future disposals of Providence, nor did I believe in any other revelation than that of the Holy Scriptures; however, I was greatly astonished at seeing this woman in a dream that night, though a person I never before beheld in my life; this made such an impression on me, that I could not get the idea the next day out of my mind, and I then became as anxious to see her as I was before indifferent; accordingly in the evening, after we left off working, I inquired where she lived, and being directed to her, to my inexpressible surprise, beheld the very woman in the very same dress she appeared to me to wear in the vision. She immediately told me I had dreamed of her the preceding night; related to me many things that had happened with a correctness that astonished me; and finally told me I should not be long a slave: this was the more agreeable news, as I believed it the more readily from her having so faithfully related the past incidents of my life. She said I should be twice in very great danger of my life within eighteen months, which, if I escaped, I should afterwards go on well; so, giving me her blessing, we parted.

      This vision that Equiano encounters exemplifies his religious faith. Through this religious faith Equiano is able to think about the future in different settings. This particular vision foreshadows Equiano's plans and journey in the near future.

    10. When I left the room I immediately went, or rather flew, to the vessel, which being loaded, my master, as good as his word, trusted me with a tierce of rum, and another of sugar, when we sailed, and arrived safe at the elegant town of Philadelphia. I soon sold my goods here pretty well; and in this charming place I found every thing plentiful and cheap.

      Equiano continues to invest his good fortunes for greater profits in the future. His frugality and ability to understand a capitalistic system eventually leads Equiano to purchasing his freedom.

    11. but as I thought that if it were God's will I ever should be freed it would be so, and, on the contrary, if it was not his will it would not happen; so I hoped, if ever I were freed, whilst I was used well, it should be by honest means; but, as I could not help myself, he must do as he pleased; I could only hope and trust to the God of Heaven; and at that instant my mind was big with inventions and full of schemes to escape.

      In the scholarly article titled, "The Poetics of Belonging in the Age of Enlightenment: Spiritual Metaphors of Being in Olaudah Equiano's Interesting Narrative," author Rebecka Fisher argues that, "Equiano uses onto-theological metaphors of being to resist the realities and politics of empire, a politics that ensured that polities could never smoothly transition from empires to states, that upheaval and shifting allegiances would consistently characterize an individual’s existence, and that the character of modern human being would always be of a fractured but navigable nature."

      Fisher, Rebecka Rutledge. "The Poetics of Belonging in the Age of Enlightenment: Spiritual Metaphors of Being in Olaudah Equiano's Interesting Narrative." Early American Studies: An Interdisciplinary Journal: 72-97. MLA International Bibliography. Web. 7 Mar. 2016.

    12. 'I must sell you again: you cost me a great deal of money, no less than forty pounds sterling; and it will not do to lose so much. You are a valuable fellow,' continued he; 'and I can get any day for you one hundred guineas, from many gentlemen in this island.' And then he told me of Captain Doran's brother-in-law, a severe master, who ever wanted to buy me to make me his overseer. My captain also said he could get much more than a hundred guineas for me in Carolina.

      This is a common theme of separation from friends and family. Most slaves were dehumanized through being separated from their loved ones.

    13. I went with him into this vessel, and we took a load of new slaves for Georgia and Charles Town. My master now left me entirely to the captain, though he still wished for me to be with him; but I, who always much wished to lose sight of the West Indies, was not a little rejoiced at the thoughts of seeing any other country.

      This quote exemplifies Equiano's servitude but it also shows that he is partaking in some of the same cruelties that brought him to America. Instead of practicing civil disobedience Equiano is looking out for himself instead of a group of mistreated slaves.

    14. is it surprising that slaves, when mildly treated, should prefer even the misery of slavery to such a mockery of freedom? I was now completely disgusted with the West Indies, and thought I never should be entirely free until I had left them.

      Equiano expresses his opinions about slavery and being free. He laments that supposedly free blacks could be mistreated just as slaves are. In most cases legal documents that free blacks obtained were ignored and their liberties were basically non existent. This mockery of freedom is basically another form of slavery and with freedom came a lack of protection from one's master.

    15. These things opened my mind to a new scene of horror to which I had been before a stranger. Hitherto I had thought only slavery dreadful; but the state of a free negro appeared to me now equally so at least, and in some respects even worse, for they live in constant alarm for their liberty; and even this is but nominal, for they are universally insulted and plundered without the possibility of redress; for such is the equity of the West Indian laws, that no free negro's evidence will be admitted in their courts of justice.

      In the article titled,"Equiano's Nativity: Negative Birthright, Indigenous Ethic, and Universal Human Rights," author Yael Ben-Zvi argues that ""Equiano’s decision is not bound by English law but is enabled by a foundational indigenous liberty. Within a couple of weeks, Equiano experiences Blackstone’s qualifications of the “liberty” of the “free negro,” which, especially in the colonies. By contrast, his “original free African state” is an unambiguous alternative that had failed him only when the logic of affirmative communal attachment on which it rests was violated, at his initial kidnapping in Africa."

      Ben-Zvi, Yael. "Equiano's Nativity: Negative Birthright, Indigenous Ethic, and Universal Human Rights." Early American Literature: 399-423. MasterFILE Premier. Web. 7 Mar. 2016.

    16. Joseph Clipson, he told him he was not free, and that he had orders from his master to bring him to Bermudas. The poor man could not believe the captain to be in earnest; but he was very soon undeceived, his men laying violent hands on him: and although he shewed a certificate of his being born free in St. Kitt's, and most people on board knew that he served his time to boat building, and always passed for a free man, yet he was taken forcibly out of our vessel. He then asked to be carried ashore before the secretary or magistrates, and these infernal invaders of human rights promised him he should; but, instead of that, they carried him on board of the other vessel: and the next day, without giving the poor man any hearing on shore, or suffering him even to see his wife or child, he was carried away, and probably doomed never more in this world to see them again.

      This exemplifies the common theme of being separated from family. Not only does this dehumanize someone but it also represents that lack of rights of supposedly free blacks. In 1623 the English conquered St. Kitts and used slaves there. Since, St, Kitts was a starting destination for slavery it makes since that slavery and the injustices that come with it were common place at St.Kitts.

    17. In the midst of these thoughts I therefore looked up with prayers anxiously to God for my liberty; and at the same time I used every honest means, and endeavoured all that was possible on my part to obtain it.

      This is a common theme of Christianity in which Equiano uses as a resource to remain formidable. This quote also represents how Equiano's humility and servitude are gained through his religious faith.

    18. At one of our trips to St. Kitt's I had eleven bits of my own; and my friendly captain lent me five bits more, with which I bought a Bible. I was very glad to get this book, which I scarcely could meet with any where.

      In the article titled, "Cutting Edge: CHRISTIAN IMPERIALISM AND THE TRANSATLANTIC SLAVE TRADE," author Katie Cannon claims that Europeans during the 18th century "Used the Bible as the world’s constitution, regime apologists used legalistic literalist hermeneutics to convince themselves that African people are cursed with perpetual servitude. In turn, Europeans are ordained by God to control inferior people and exhorted to deal with such primitive pagans as usable, disposable functionaries"

      Cannon, Katie Geneva. "Cutting Edge: CHRISTIAN IMPERIALISM AND THE TRANSATLANTIC SLAVE TRADE." Journal of Feminist Studies in Religion (Indiana University Press): 127-34. Publisher Provided Full Text Searching File. Web. 7 Mar. 2016.

    19. We then proceeded to the markets to sell them; and Providence was more favourable to us than we could have expected, for we sold our fruits uncommonly well; I got for mine about thirty-seven bits. Such a surprising reverse of fortune in so short a space of time seemed like a dream to me, and proved no small encouragement for me to trust the Lord in any situation.

      Equiano is frugal and is incredibly smart. Through smart investments and good trading Equiano was able take advantage of a capitalistic system that was not supposed to favor slaves. This frugality and ability to save eventually led to Equiano purchasing his freedom.

    20. I now, in the agony of distress and indignation, wished that the ire of God in his forked lightning might transfix these cruel oppressors among the dead.

      Equiano tries to control his pride and suppress his anger through relying on God. Through his reliance on God Equiano is able to display the virtue of patience and fortitude. This particular text brings up the common theme of Christianity.

    21. When we came there, in some little convenient time he and I went ashore with our fruits to sell them; but we had scarcely landed when we were met by two white men, who presently took our three bags from us. We could not at first guess what they meant to do; and for some time we thought they were jesting with us; but they too soon let us know otherwise, for they took our ventures immediately to a house hard by, and adjoining the fort, while we followed all the way begging of them to give us our fruits, but in vain. They not only refused to return them, but swore at us, and threatened if we did not immediately depart they would flog us well.

      The cruelty of the rich exemplifies the abuse of power that the rich inflicted upon the poor in an effort to maintain complete control.

    22. Thus was I going all about the islands upwards of four years, and ever trading as I went, during which I experienced many instances of ill usage, and have seen many injuries done to other negroes in our dealings with Europeans: and, amidst our recreations, when we have been dancing and merry-making, they, without cause, have molested and insulted us. Indeed I was more than once obliged to look up to God on high, as I had advised the poor fisherman some time before.

      Equiano conveys hopelessness and vulnerability through the use of emotion. his description of inhumane treatment pulls at the heartstrings of the readers through sympathizing with them.

    23. But the captain liked me also very much, and I was entirely his right-hand man. I did all I could to deserve his favour, and in return I received better treatment from him than any other I believe ever met with in the West Indies in my situation.

      Equiano works diligently and adheres to legitimate authority. Equiano's hard work represents the dignity of Africans. This quote also exemplifies the high moral code that Equiano exemplifies through his servitude.

    24. I immediately thought I might in time stand some chance by being on board to get a little money, or possibly make my escape if I should be used ill: I also expected to get better food, and in greater abundance; for I had felt much hunger oftentimes, though my master treated his slaves, as I have observed, uncommonly well.

      even though Equiano and other slaves were treated well by his master, this was rarely the case. Around the mid 1400s when the Portuguese started using slave labor, Europeans realized that the use of slaves was a great economic opportunity. Thus, the Triangular trade started in full swing with the growth of sugar plantations where European manufactured goods were sent to Africa, the African Slaves were purchased and sent to America, and then the cash crops purchased in Americas returned to Europe. Since Slaves were seen as products they were often overworked and received little to no education, which led to many slaves having a strong reliance in God.

    25. Some time in the year 1763 kind Providence seemed to appear rather more favourable to me.

      In the article titled "The Trans Atlantic Slave Trade and the U.S. Underground Railroad," author Jones Hood described the horrors that were inflicted upon slaves during the transatlantic slave trade, "between 1441 and 1888, Europeans and African nations engaged in an economic practice that enslaved many millions of Africans. This enslavement caused terrible misery and wreaked untold havoc upon their cultural foundation."

      Hood, Jones Lottie. "The Trans Atlantic Slave Trade and the U.S. Underground Railroad." International Congregational Journal: 47-57. EBSCO eBook Collection. Web. 8 Mar. 2016.

    1. But over his heart there seemed to be a warm spot, where those young hands had placed that precious dollar. Tom put up his hand, and held it close to his heart.

      This is an incredible example of Stowe's attempt to create an emotional response within the reader. It truly sums up the love that Tom and George have for each other, which, in many ways, mirrors a father-son relationship.

    2. "O! Mas'r George, ye mustn't talk so 'bout yer father!"

      This mirrors the conversation that Chloe and Tom had earlier in the chapter. As previously stressed, the characterization of master George is critical. He is one of the few characters appearing in the novel thus far that is not only unhappy with slavery, but is attempting to do something about it. In many ways, he is the model character in the story. It is interesting to note that his age is never mentioned in the story...perhaps to create a broader sense of sympathy from the audience.

    3. It was in vain that he said to himself that he had a right to do it,—that everybody did it,—and that some did it without even the excuse of necessity;—he could not satisfy his own feelings; and that he might not witness the unpleasant scenes of the consummation, he had gone on a short business tour up the country, hoping that all would be over before he returned.

      This scene is incredibly powerful and again, plays an important role in creating an audience connection and provoking emotion. Here, we see Mr. Shelby desperately trying to convince himself that he made the right decision - that it was something that everybody did. However, in his attempt to do so, it is revealed that he is truly deeply troubled by the situation so much so that he cannot bear to witness Tom being taken away. This is one of Stowe's attempts to prove that the slave trade is immoral - that people who are involved in it are not ignorant to what may (and probably will) happen to these slaves.

    4. "I'm sorry," said Tom, "that Mas'r George happened to be away." George had gone to spend two or three days with a companion on a neighboring estate, and having departed early in the morning, before Tom's misfortune had been made public, had left without hearing of it. "Give my love to Mas'r George," he said, earnestly.

      The bond that Stowe has created between Master George and Tom in such a short amount of interaction is incredible. Tom says nothing about Mr. Shelby, who he passionately defended. It appears that Tom and Master George have a strong relationship that is introduced in a previous chapter in which George is teaching Tom how to properly write. It is an interesting that Stowe chose to create this strong relationship between a slave and a white child. Perhaps she did so to emphasize the idea that children cannot see color.

    5. Tom got in, and Haley, drawing out from under the wagon seat a heavy pair of shackles, made them fast around each ankle.

      It is interesting to note how passive Tom is during this situation. While the reader is receiving incredible levels of emotion from others - Aunt Chloe, Mrs. Shelby, etc - is seems that there is actually an incredibly minute focus on Tom, as he is being portrayed as emotionless and passive. This truly underlines his faith.

    6. "Tom," she said, "I come to—" and stopping suddenly, and regarding the silent group, she sat down in the chair, and, covering her face with her handkerchief, began to sob. "Lor, now, Missis, don't—don't!" said Aunt Chloe, bursting out in her turn; and for a few moments they all wept in company. And in those tears they all shed together, the high and the lowly, melted away all the heart-burnings and anger of the oppressed.

      This is devastating but also an incredible example of racial unity. This is an absolutely critical scene for Stowe to allow the audience to witness the pain that slavery causes for both white and black individuals. It is also a strategic technique. Throughout the text, Stowe characterizes Mrs. Shelby as the "perfectly right and just white woman". With her intended audience of women for this piece, this really allows women readers to sympathize with both black and white, as they yearn to by the "perfect" woman that Mrs. Shelby is.

    7. The boys, having eaten everything there was on the breakfast-table, began now to take some thought of the case; and, seeing their mother crying, and their father looking very sad, began to whimper and put their hands to their eyes. Uncle Tom had the baby on his knee, and was letting her enjoy herself to the utmost extent, scratching his face and pulling his hair, and occasionally breaking out into clamorous explosions of delight, evidently arising out of her own internal reflections.

      This is also an emotional scene for the reader. We are presented with two young children who seemingly don't understand what is going on - the children that, like Chloe, were raised in slavery and know nothing bad of it. It's interesting the way in which Stowe has created this "complex hierarchy of knowledge" here. There is Chloe, who understands the situation and is devastated, the two boys who understand that there is a bad situation occurring but know no details, and the baby, who understands nothing of it and is filled with happiness. Perhaps Stowe is utilizing this family to symbolize the white response to slavery.

    8. "Lor, Pete," said Mose, triumphantly,

      It is interesting to note Stowe's choice of names for these children - both of whom share commonalities with common biblical names: Moses and Peter. Perhaps Stowe chose these names as a reflection of Chloe and Tom's religious beliefs. On the other hand, they could have simply been a reflection of common names at the time.

    9. This nerves the African, naturally patient, timid and unenterprising, with heroic courage, and leads him to suffer hunger, cold, pain, the perils of the wilderness, and the more dread penalties of recapture.

      Stowe really plays with the audience's emotions in this entire description of the African American race. She describes them in a way that one may describe either a young child or a loyal pet- naturally innocent and naive. Here, she is attempting to provoke sympathy and/or empathy from the audience, who is receiving this image that African Americans are, in many ways, helpless. This really emphasizes Stowe's views that she does truly see many differences between the black and white race.

    10. I can't jest make out whar 't is, but thar's wrong somewhar, I'm clar o' that.

      This is incredibly sad. Aunt Chloe has spent her entire life in slavery and, knowing nothing else, she struggles to pinpoint the fault of the situation (though she knows there is one). At the time, millions of African American children were born into slavery and, for these children/adults, it was the norm. I wonder how that played a role in the abolitionist movement because many slaves were (so they thought) completely dependent on their white masters.

    11. it must be remembered that all the instinctive affections of that race are peculiarly strong

      This is interesting in the way that she is recognizing that there is a defined difference between the two races (this is introduced in her 1852 preface to her text where she describes African Americans as "...a character so essentially unlike the hard and dominant Anglo-Saxon race"). This text was met with much criticism, as many claimed that Stowe possessed prejudicial ideas. This paragraph truly underlines that view. The novel "The Concept of "Race" in Natural and Social Science" delves more into Stowe's incorporation of this, particularly focusing on this paragraph in the novel.

      Gates, E. Nathaniel. The Concept of Race "in Natural and Social Science." New York: Garland Publishing Inc, 1997. Print.

    12. thar don't a sparrow fall without him

      This is also a reference to biblical literature taken from Matthew 10:29, which essentially explains that one should not have fear because God always takes care of the situation. Again, this not only shows Tom's deep sense of religion, but Stowe's as well. She has inserted numerous references to the Bible and is evidently well educated in religion.

      Wong, Cliff, and Andrew Kwong. A Biblical Perspective on How to Handle Worry and Fear. N.p.: n.p., 2010. Print.

    13. "Chloe! now, if ye love me, ye won't talk so, when perhaps jest the last time we'll ever have together! And I'll tell ye, Chloe, it goes agin me to hear one word agin Mas'r. Wan't he put in my arms a baby?—it's natur I should think a heap of him. And he couldn't be spected to think so much of poor Tom. Mas'rs is used to havin' all these yer things done for 'em, and nat'lly they don't think so much on 't. They can't be spected to, no way. Set him 'longside of other Mas'rs—who's had the treatment and livin' I've had? And he never would have let this yer come on me, if he could have seed it aforehand. I know he wouldn't."

      It's incredibly interesting how much love that Tom has for his owner, Mr. Shelby, and how willing he is to stand up for him after he had just sold Tom. In many ways, Stowe is shifting the focus with this defense. He seems to be, in a way, idolizing his white master and creating a hierarchy that is illustrating himself as a lesser (ie. referring to himself as "poor Tom"). However, I think is also does show his intelligence via his recognition of the slaveholders being blinded by their own privilege.

    14. Ah, brave, manly heart,—smothering thine own sorrow, to comfort thy beloved ones!

      I found this to be strange. Throughout the text, we have seen a "distant narrator" who essentially exists only for the purpose of explaining what is happening between character dialogue. However, here we see something very different. For the first time, Stowe seems to be breaking out of that emotionless narrator position. Perhaps she does so to appeal to the readers' emotions on a deeper level by expressing a level of admiration for Tom and to truly emphasize the sadness of the situation. I found this change of narration to be incredibly interesting and quite effective.

    15. "There'll be the same God there, Chloe, that there is here."

      Again, this is an incredible claim by Tom. As his wife is slowly losing faith, he is putting complete faith in his God to carry him through the situation. This really shows the type of religious character that Tom is.

    16. "lifted up her voice and wept."

      This is from Genesis 21:16 (English Standard Version). In this section of the Book of Genesis, the word "slave" is frequently used. Many individuals at the time used the fact that slavery appeared in biblical texts to justify it. This is an interesting choice for Stowe to include as it truly underlines her value of religion.

    17. "So long as your grand folks wants to buy men and women, I'm as good as they is," said Haley; "'tan't any meaner sellin' on 'em, that 't is buyin'!"

      This is a really important point that Stowe is making. Since the first chapter, the audience is persuaded to dislike Haley. Here, he is making an incredible point: he is no worse than Mr. Shelby, who is seen as a "good guy". This really emphasizes the idea that no matter your role in slavery, you are equally wrong. With this quote, Haley is putting everyone on the same playing field.

    18. only rubbed away over and over on the coarse shirt, already as smooth as hands could make it

      This really aids in enhancing the emotional connection for the readers. Here, Aunt Chloe is trying desperately to distract herself from the immense pain of the fact that her husband is about to be taken away. Stowe does an incredible job at encouraging emotion from the reader, which is an incredibly critical part of this story. At the time, African Americans were seen as non-humans - they were thought not to be able to feel human emotions such a sadness and sorrow. Stowe forces the audience to feel sympathy throughout this entire text. This technique is incredibly clever.

    19. "I'll be real good, Uncle Tom, I tell you," said George. "I'm going to be a first-rater; and don't you be discouraged. I'll have you back to the place, yet. As I told Aunt Chloe this morning, I'll build our house all over, and you shall have a room for a parlor with a carpet on it, when I'm a man. O, you'll have good times yet!"

      The characterization of George was set up to allow the audience to build an emotional connection and to prove a point. He, in many ways, resembles the idea that nobody is born racist (also seen in previous chapters when he is teaching Tom how to write). He possesses an incredible amount of innocence (which is also seen a few lines down where he is telling Haley that he should be ashamed of himself) that offers him the idea that when he's "a man", he will make things perfect and good. He also appears not to see skin color and is one of the few individuals in the text who refers to black individuals as "men and women" as opposed to "creatures", going as far as to compare their treatment to the ways in which cattle are treated.

    20. Tom sat by, with his Testament open on his knee, and his head leaning upon his hand

      At this point in the text, we have already been introduced to religion as a theme. Tom turning to religion during a time like this - hours before he is about to be traded and torn from his family - speaks incredible volumes about the way that religion played a role in society. Stowe creates a wide range of individuals in this text who all can seemingly be connected through their beliefs. We have been introduced to slaves that have lost their faith because of their enslavement. This instance truly emphasizes the important role of religion in all individuals at the time.

  29. Jan 2016
    1. Agriculture arose sometime between nine- and five-thousand years ago, almost simultaneously in the Eastern and Western Hemispheres. Mesoamericans in modern-day Mexico and Central America first domesticated maize and and developed perhaps the hemisphere’s first settled population around 1,200 BCE.

      How did they get the maize?