Recommandation 4. Introduire les notions d’éducation à la sexualité dans les programmes officiels decertaines disciplines concernées, au-delà des disciplines liées aux aspects biologiques et sanitaires et del’enseignement moral et civique.Le cadre actuel pose des difficultés liées l’absence de précisions sur l’organisation des séances d’éducation àla sexualité dans l’article L. 312-16 du code de l’éducation, qui a rendu obligatoires au moins trois séancesannuelles en matière d’éducation à la sexualité en 2001. Les circulaires successives ont défini un cadreopérationnel qui a été modifié à plusieurs reprises. Le cadre actuel décrit par la dernière circulaire, en datedu 12 septembre 2018, n’a pas repris deux points sur les supports horaires et les modalités précises sur laprise en charge concrète des séances qui figuraient dans les circulaires de 1998 et 2003. Les modificationssuivantes − sur le modèle de l’article L. 542-3 du code de l’éduction sur l’organisation de la séance annuelled’information et de sensibilisation sur l’enfance maltraitée95 − permettraient de clarifier ces questionsimportantes sur la mise en œuvre concrète de l’EAS :Recommandation 5. Inscrire au moins trois séances annuelles dédiées dans l’emploi du temps des élèves desécoles, des collèges et des lycées (disposition complétant l’article L. 312-16 du code de l’éducation).Recommandation 6. Attribuer la mission d’organisation des séances annuelles aux chefs d’établissement, enlien avec les comités d’éducation à la santé et la citoyenneté (disposition complétant l’article L. 312-16 ducode de l’éducation).

Recommandation 6. Attribuer la mission d’organisation des séances annuelles aux chefs d’établissement, enlien avec les comités d’éducation à la santé et la citoyenneté (disposition complétant l’article L. 312-16 ducode de l’éducation)

Recommandation 5. Inscrire au moins trois séances annuelles dédiées dans l’emploi du temps des élèves desécoles, des collèges et des lycées (disposition complétant l’article L. 312-16 du code de l’éducation)

Samson, last of the Judges, who kills thelion and obtains honey from its corpse, attaches burning torches to the tailsof foxes, and carries the heavy gates of Gaza upon his shoulders

Cette intuition est juste, puisque c’estégalement une approche par la négative queretient la Convention internationale des droitsde l’enfant (CIDE) pour reconnaître, dans sonarticle 16, le droit au respect de la vie privée :« Nul enfant ne fera l’objet d’immixtionsarbitraires ou illégales dans sa vie privée, safamille, son domicile ou sa correspondance,ni d’atteintes illégales à son honneur ou à saréputation »

Cette intuition est juste, puisque c’estégalement une approche par la négative queretient la Convention internationale des droitsde l’enfant (CIDE) pour reconnaître, dans sonarticle 16, le droit au respect de la vie privée :« Nul enfant ne fera l’objet d’immixtionsarbitraires ou illégales dans sa vie privée, safamille, son domicile ou sa correspondance,ni d’atteintes illégales à son honneur ou à saréputation »

16 - droit del’enfant à la protection contre les immixtionsarbitraires ou illégales dans sa vie privée, safamille, son domicile, sa correspondance etcontre les atteintes illégales à son honneur et àsa réputation.

Cette intuition est juste, puisque c’estégalement une approche par la négative queretient la Convention internationale des droitsde l’enfant (CIDE) pour reconnaître, dans sonarticle 16, le droit au respect de la vie privée :« Nul enfant ne fera l’objet d’immixtionsarbitraires ou illégales dans sa vie privée, safamille, son domicile ou sa correspondance,ni d’atteintes illégales à son honneur ou à saréputation ».

The btoa() function takes a JavaScript string as a parameter. In JavaScript strings are represented using the UTF-16 character encoding: in this encoding, strings are represented as a sequence of 16-bit (2 byte) units. Every ASCII character fits into the first byte of one of these units, but many other characters don't. Base64, by design, expects binary data as its input. In terms of JavaScript strings, this means strings in which each character occupies only one byte. So if you pass a string into btoa() containing characters that occupy more than one byte, you will get an error, because this is not considered binary data:

Tutubox installation guide for iPhone and iPad Method 1 – install Tutubox directly to the device As the first step, you need to clear the cache and history in your Safari browser by navigating to the Safari settings.Now you need to go to the general settings and make sure that there are no certificates available in the certificate list.Then go to Wi-Fi settings and then you need to click configure the proxy. Under the configure proxy you need to paste this URL http://ffapple.com in the automatic field. Turn off Wi-Fi for about 10 seconds and enable it again and check if your proxy is still enabled, if not just go and enable it.The next step is to get the latest Tutubox version to your iDevice. You can use the official Tutubox website (tutubox.io). Visit this website from your Safari browser and then download Tutubx iOS for your device and install it.When the installation process is done, you need to go to Settings, General, Device manager, and then click the certificate and trust it. If you do not do this step, you will get a notification when you are trying to open Tutubox that says the term Untrusted Enterprise Developer.Now you have successfully installed the Tutubox latest version on your iPhone or iPad. You are good to go with installing apps, games, and other tools with Tutubox.

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Sympathy for the Devil

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Tg(u22122.4shha-ABC:GFP)

We explore them in other chapters and in particular in Macroeconomic Policy Around the World.

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).

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

SUPPLEMENTARY DATA

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).

Source Data

Source Data

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.

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

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

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Supplemental material

Supplemental material

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.

you may proceed with standard verification

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vous

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NiSO4. 6H2O stock soloution

Anti-Stm and anti-E coli /gG subclass assay

Total Anti-Stm and anti-E coli antibody assay

Enzyme linked Immunosorbant Assays (ELISA .... )

Statistical Analysis

Transwell experiment

RNA extraction/c-DNA synthesis

Reverse Zymograph

Somatotyping Children

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

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

Molecular dynamics (MD) simulatio

temperature for 2h. The nitrocellulose membrane was washed extensively with PBST and developed using chemiluminescence substrate from Pierce (USA).

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

Western Blot

bacteria. The cells were washed once with ice-cold PBS and the uptake of labeled bacteria was analyzed by flow-cytometry

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

Measurement of phagocytic ability of macrophages

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).

Assay for Inhibition of in Vitro Protein Synthesis

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.

Isolation of stable clones.

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.

RAPID FLUORESCENT FOCUS INHIBITION TEST (RFFIT)

Immediatelyafterisolation,thetissueswereweighedandsubjectedtolipidextractionthatwascarriedoutinduplicateaccordingtoFolchetal.(1957)

Totallipids

Salineextracts(0.89%)oftissueswerepreparedinTeflonglasshomogenizer.ThecarbohydratecontentoftheextractwasdoneaccordingtothemethodofShibkoetal.(1967)

Carbohydrates

TotalproteincontentwasdeterminedbytheFolin-CiocalteaumethodofLowryetal.(1951)asmodifiedbyZakandCohen(1961).Bovinecrystallinealbuminwasusedasa referencestandard

Totalproteins

Aftereffluentexposure,thecontrolandexperimentalfisheswerekilledbyhammeringonheadanddissectedimmediately.Excisedbrain,liver,muscle,gill,kidneyandair-breathingorgans werepooled incoldcondition andusedforbiochemicalestimations.

BiochemicalAnalysis

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

LBON(Ts) phenotype

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

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)

SMG resistance

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

Lac phenotype

Scoring for phenotypes

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).

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

GC-MS analysis of ergosterol

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).

Generation of pXG-GFP+2-Ld27 (CYP5122Al)

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

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

Measurement of vacuole pH

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

Ligation

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

Mutant rescue

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

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

Radioactive labelling of oligonucleotides

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

Post-hybridization washes

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

In vitroubiquitylation assay

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

For reporter assay, GUS and GFP marked Xanthomonas oryzaepv. oryzicolastrains and control strains were grown overnight in PS medium. 0.2

Reporter assays with β-Glucuronidase (GUS) and green fluorescent protein (GFP)

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

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

Transwell migration assay

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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.

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.

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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

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.

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

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

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

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

In Wall-E and the Toy Storytrilogy, the pleasure is of the suspen-sion of knowledge—the pleasure of notknowing.

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.

October 13-16, 2016

Engage adult new writers with online communities of writers, as contributors, readers, and peers, to foster their self-directed learning, self-study, and persistence.

discuss, but do not debate

Know your students

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.

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

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

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.”

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

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

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

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

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

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

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

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.”

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.”

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

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

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

There is no universal best teaching practice.

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

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.

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.

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.

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.

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.

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.

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.

"—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."

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.

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.

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.

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.

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.

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.

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.

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.

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.

'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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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