Luminescence Properties of Mesoporous Silica Nanoparticles Encapsulating Different Europium Complexes: Application for Biolabelling

In this work we have synthesized and characterized new hybrid nanoplatforms for luminescent biolabeling based on the concept of Eu3+ complexes encapsulation in mesoporous silica nanoparticles (≈100 nm). Eu complexes have been selected on the basis of their capability to be excited at 365 nm which is a currently available wavelength, on routine epifluorescence microscope. For Eu complexes encapsulation, two different routes have been used: the first route consists in grafting the transition metal complex into the silica wall surface. The second way deals with impregnation of the mesoporous silica NPs with the Eu complex. Using the second route, a silica shell coating is realized, to prevent any dye release, and the best result has been obtained using Eu-BHHCT complex. However, the best solution appears to be the grafting of Eu(TTA)3-Phen-Si to mesoporous silica NPs. For this hybrid, mSiO2-Eu(TTA)3(Phen-Si) full characterization of the nanoplatforms is also presented

Design of an improved set of oligonucleotide primers for genotyping MeCP2tm1.1Bird KO mice by PCR

BACKGROUND: The strain of MeCP2(tm1.1Bird )mice is a broadly used model for Rett syndrome. Because males carrying the invalidated MeCP2 locus are sterile, this strain has to be maintained in a heterozygous state. All animals therefore have to be genotyped at every generation to discriminate those carrying the invalidated allele (+/- females and y/- males) from those that do not. This is conveniently carried out by PCR on tail genomic DNA but because the primer pairs described initially for this purpose yield very similar size DNA bands on the WT and the KO alleles, this requires to carry out two independent PCR reactions on tail DNA preparations from all animals. RESULTS: After cloning and sequencing the PCR fragment amplified on the KO allele, we tested several sets of primers that were designed to yield PCR fragments of different sizes on the KO and WT alleles. CONCLUSION: We have thus identified a set of three primers that allows for efficient genotyping of the animals by a single PCR reaction. Furthermore, using of this set of primers also resolves a recurrent problem related to the tendency of one of the initial primers to give rise to a non specific band because of its capacity to anneal at both ends of a repeated genomic element which we have identified as MurvyLTR

Design of an improved set of oligonucleotide primers for genotyping MeCP2KO mice by PCR-0

<p><b>Copyright information:</b></p><p>Taken from "Design of an improved set of oligonucleotide primers for genotyping MeCP2KO mice by PCR"</p><p>http://www.molecularneurodegeneration.com/content/2/1/16</p><p>Molecular Neurodegeneration 2007;2():16-16.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2018688.</p><p></p>NA preparations of the four genotypes (KO male: y/- ; WT male: y/+; Heterozygote female: +/- ; WT female: +/+) were submitted to the different PCR amplification protocols detailed in the methods section, before loading on a 1.5% agarose gel

Design of an improved set of oligonucleotide primers for genotyping MeCP2KO mice by PCR-1

<p><b>Copyright information:</b></p><p>Taken from "Design of an improved set of oligonucleotide primers for genotyping MeCP2KO mice by PCR"</p><p>http://www.molecularneurodegeneration.com/content/2/1/16</p><p>Molecular Neurodegeneration 2007;2():16-16.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2018688.</p><p></p>NA preparations of the four genotypes (KO male: y/- ; WT male: y/+; Heterozygote female: +/- ; WT female: +/+) were submitted to the different PCR amplification protocols detailed in the methods section, before loading on a 1.5% agarose gel

Ligand Binding but Undetected Functional Response of FcR after Their Capture by T Cells via Trogocytosis

International audienceIntercellular transfer of cell surface proteins by trogocytosis is common and could affect T cell responses. Yet, the role of trogocytosis in T cell function is still elusive, and it is unknown whether a molecule, once captured by T cells, harbors the same biological properties as in donor APC. In this study, we showed that FcgammaR as well as the associated FcRgamma subunit could be detected at high levels on murine and human T cells after their intercellular transfer from FcgammaR-expressing APC. Capture of FcgammaR occurred during coculture of T cells with FcgammaR-expressing APC upon Ab- or Ag-mediated T cell stimulation. Once captured by T cells, FcgammaR were expressed in a conformation compatible with physiological function and conferred upon T cells the ability to bind immune complexes and to provision B cells with this source of Ag. However, we were unable to detect downstream signal or signaling-dependent function following the stimulation of FcgammaR captured by T cells, and biochemical studies suggested the improper integration of FcgammaR in the recipient T cell membrane. Thus, our study demonstrates that T cells capture FcgammaR that can efficiently exert ligand-binding activity, which, per se, could have functional consequences in T cell-B cell cooperation

MeCP2 overexpression diminishes MHC class I expression.

<p>N2A cells transfected with pCMX vectors expressing either murine or human MeCP2 were immunostained for one of the three MHC class I molecules (K^k, L^d or D^d), β2-microglobulin or the transferrin receptor on the cell surface as well as for intracellular MeCP2. The level of staining was then analysed by fluorescence-activated cell sorting. Panel A: Dot plots of the amount of surface antigen (x-axis) against the amount of intracellular MeCP2 (y-axis) in cells transiently transfected with pCMX expressing human MeCP2 and analysed 48 hrs later. Panel B: For each kind of staining, the variation in expression level was calculated as the ratio of MFI of MeCP2 overexpressing cells over MFI of mock-transfected cells. The histograms summarise the mean (±SEM) of the variation in cell surface levels from 15 independent transfections with vectors expressing mouse MeCP2<i>A</i> (grey fill) and 12 independent transfections with vectors expressing human MeCP2<i>A</i> (black fill). Panel C: N2A and NIH3T3 cells were transfected with empty pcDNA3.1(+) (mock cells) or expressing Myc-tagged human MeCP2<i>A</i> or <i>B</i> isoforms. 48 h after transfection, cells were subjected to double staining against cell surface MHC class I molecules and intracellular Myc-tagged MeCP2, then analyzed by flow cytometry. The variation in expression level of MHC class I molecules was calculated as the ratio of MFI of MeCP2 over-expressing cells over the MFI of mock cells. The histograms represent the mean (±SEM) of the cell surface level variation from 4 independent transfections with each of the vectors. Panel D: A representative example of dot-plots obtained for double immunostaining of transiently transfected N2A cells with anti-Myc 9E10 and rat-anti-mouse-MHC I M1/42 monoclonal antibodies. Dotted and continuous circles indicate the different populations expressing high and intermediate levels of MeCP2, respectively. Statistical significance of difference between groups was analysed by using an unpaired t-test (**, <i>p</i><0.01 ; ***, <i>p</i><0.001).</p

Transient overexpression of MeCP2 inhibits MHC class I induction by IFN-γ.

<p>N2A cells transfected with pCMX vectors expressing murine or human MeCP2 were treated or not with IFN-γ for 48 hrs and then double immunostained for cell surface β2-microglobulin, transferrin receptor or MHC class I and intracellular MeCP2. Panel A: Dot-plots of transfected cells analysed by flow cytometry showing the cell surface level of the MHC class I molecule L^d or β2-microglobulin (x-axis) plotted against the level of intracellular MeCP2 (y-axis). Panel B: The induction factor was calculated as the ratio of MFI of treated cells (over-expressing MeCP2 or untransfected cells) on MFI of untreated N2A cells. Values used for the histogram are the mean (±SEM) of induction factors obtained in seven independent transfection experiments. Statistical significance of difference between groups was analysed by using an unpaired t-test (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001).</p