The American merchant ship, the Wolfe, made the first direct voyage from Boston to St. Petersburg, inaugurating direct trade between Russia and the American colonies.

Adrian Poisson grew up studying science and math by day and art after hours beginning at the age of five

Christos

trois dispositions nécessiteraient des modifications législatives (sur l’article L.312-16 du code de l’éducation)

spending a few hours each week in close proximity to a certain kind of teacher changed something about their behavior.

eachers convey to their students deep messages — often implicitly or even subliminally — about belonging, connection, ability, and opportunity.

you can’t clearly identify and measure skills, it’s hard to convince people to take them seriousl

And perhaps what students need more than anything for these positive academic habits to flourish is to spend as much time as possible in environments where they feel a sense of belonging, independence, and growth

16. Assessment

Israel was forbidden to set up sacred stones, pillars: “you shall not set up a pillar (massebah), which the LORD your God hates” (Deuteronomy 16:22).

Water immobilization is a cool thing! The simplest way to accomplish it is by freezing. But can you think of how water might be immobilized (so to speak) at temperatures above freezing, say at 50°F (10°C)? Think Jell-O and a new process that mimics caviar and you have two methods that nearly stop water in its tracks.

Abomination of desolation

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Volume VIII: Freud, "The Interpretation of Dreams, Chapter VII" and Shaw, "Man and Superman"

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