Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein Effects on hydrolysis by HIV-1 proteinase

AbstractMutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a β-branched (He) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a β-branched (Val) residue in the P1 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed

A robust coregistration method for in vivo studies using a first generation simultaneous PET/MR scanner

Purpose: Hybrid positron emission tomography (PET)/magnetic resonance (MR) imaging systems have recently been built that allow functional and anatomical information obtained from PET and MR to be acquired simultaneously. The authors have developed a robust coregistration scheme for a first generation small animal PET/MR imaging system and illustrated the potential of this system to study intratumoral heterogeneity in a mouse model. Methods: An alignment strategy to fuse simultaneously acquired PET and MR data, using the MR imaging gradient coordinate system as the reference basis, was developed. The fidelity of the alignment was evaluated over multiple study sessions. In order to explore its robustness in vivo, the alignment strategy was applied to explore the heterogeneity of glucose metabolism in a xenograft tumor model, using ^(18)F-FDG-PET to guide the acquisition of localized ^1H MR spectra within a single imaging session. Results: The alignment method consistently fused the PET/MR data sets with subvoxel accuracy (registration error mean=0.55 voxels, <0.28 mm); this was independent of location within the field of view. When the system was used to study intratumoral heterogeneity within xenograft tumors, a correlation of high ^(18)F-FDG-PET signal with high choline/creatine ratio was observed. Conclusions: The authors present an implementation of an efficient and robust coregistration scheme for multimodal noninvasive imaging using PET and MR. This setup allows time-sensitive, multimodal studies of physiology to be conducted in an efficient manner

Making sense of microarray data distributions

Motivation: Typical analysis of microarray data has focused on spot by spot comparisons within a single organism. Less analysis has been done on the comparison of the entire distribution of spot intensities between experiments and between organisms

In-cell intrabody selection from a diverse human library identifies C12orf4 protein as a new player in rodent mast cell degranulation.

The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in FcεRI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4), with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following FcεRI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody

Anti-C12orf4 intrabody inhibits mast cell degranulation.

<p>Analysis of stable clone 5H4: a) measurement of Annexin-V staining; b) β-hexosaminidase release; c) calcium flux and d) TNFα secretion. T-: Irrelevant intrabody. S: IgE/DNP stimulated. NS: unstimulated. Boxplot whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range. e) Measure of β-hexosaminidase release by retroviral infected populations. Clone R_8 is identical to the intrabody expressed by the plasmid clone 5H4. Sequences of the clones are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998.s006" target="_blank">Fig. S6</a>. Boxplot whiskers extend to the most extreme data point. f) Specific binding of 5H4-VH to C12orf4. Top panel: pull-down assay using 5H4-VH as capture agent and a commercial anti-C12orf4 polyclonal serum to reveal the protein. Irr: Irrelevant VH fragment, differing from 5H4 VH only by its CDR3 sequence. Low panel: subcellular localization of C12orf4 analyzed by confocal laser microscopy after double staining. Top left: Hoechst; top right: 5H4-VH-Fc fusion; bottom left: anti-C12orf4 commercial antibody; bottom right: merge. *: p<0.05; **: p<0.01; ***: p<0.001 (Student t-test).</p

Selection of intrabodies that inhibit mast cell degranulation.

<p>a) Schematic view of the selection method. The scFv/intrabody library previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998-Philibert1" target="_blank">[13]</a> was cloned in plasmid and retroviral vectors and used to transfect the RBL-2H3 cell line in order to induce a phenotypic diversity in a collection of cells. Clones displaying the desired phenotype, measured by inhibition of degranulation, were selected and the couple constituted by the inhibitory intrabody and its target antigen was identified and characterized. b) Annexin-V staining of cell populations from the library selection rounds is illustrated as the ratio of the geometric mean (MFI) of the FcεRI-stimulated (S) to the unstimulated (NS) cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998.s001" target="_blank">Fig. S1</a>).</p