7 research outputs found

    Sequence Defined Disulfide-Linked Shuttle for Strongly Enhanced Intracellular Protein Delivery

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    Intracellular protein transduction technology is opening the door for a promising alternative to gene therapy. Techniques have to address all critical steps, like efficient cell uptake, endolysosomal escape, low toxicity, while maintaining full functional activity of the delivered protein. Here, we present the use of a chemically precise, structure defined three-arm cationic oligomer carrier molecule for protein delivery. This carrier of exact and low molecular weight combines good cellular uptake with efficient endosomal escape and low toxicity. The protein cargo is covalently attached by a bioreversible disulfide linkage. Murine 3T3 fibroblasts could be transduced very efficiently with cargo nlsEGFP, which was tagged with a nuclear localization signal. We could show subcellular delivery of the nlsEGFP to the nucleus, confirming cytosolic delivery and expected subsequent subcellular trafficking. Transfection efficiency was concentration-dependent in a directly linear mode and 20-fold higher in comparison with HIV-TAT-nlsEGFP containing a functional TAT transduction domain. Furthermore, β-galactosidase as a model enzyme cargo, modified with the carrier oligomer, was transduced into neuroblastoma cells in enzymatically active form

    <i>cps</i> sequence alignments.

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    <p>Alignment of <i>cps</i> loci of serotype 29 isolate (A), serotype 7F isolate (B), serotype 6D isolate (C) and serotype 18B isolate (D) with reference <i>cps</i> sequence and best hit cps sequence according to WGS identification. Alignment was generated with Artemis Comparison Tool (<a href="http://www.sanger.ac.uk/science/tools/artemis-comparison-tool-act" target="_blank">http://www.sanger.ac.uk/science/tools/artemis-comparison-tool-act</a>).</p

    Comparison of sequential multiplex PCR, sequetyping and whole genome sequencing for serotyping of <i>Streptococcus pneumoniae</i>

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    <div><p><i>Streptococcus pneumoniae</i> is one of the major causes of pneumonia, meningitis and other pneumococcal infections in young children and elders. Determination of circulating <i>S</i>. <i>pneumoniae</i> serotypes is an essential service by public health laboratories for the monitoring of putative serotype replacement following the introduction of pneumococcal conjugate vaccines (PCVs) and of the efficacy of the immunization program. The Quellung method remains the gold standard for typing <i>S</i>. <i>pneumoniae</i>. Although this method is very effective, it is also costly, time consuming and not totally reliable due to its subjective nature. The objectives of this study were to test and evaluate the efficiency of 3 different molecular methods compared to the Quellung method. Sequential multiplex PCR, sequetyping and whole genome sequencing (WGS) were chosen and tested using a set of diverse <i>S</i>. <i>pneumoniae</i>. One-hundred and eighteen isolates covering 83 serotypes were subjected to multiplex PCR and sequetyping while 88 isolates covering 53 serotypes were subjected to WGS. Sequential multiplex PCR allowed the identification of a significant proportion (49%) of serotypes at the serogroup or subset level but only 27% were identified at the serotype level. Using WGS, 55% to 60% of isolates were identified at the serotype level depending on the analysis strategy used. Finally, sequetyping demonstrated the lowest performance, with 17% of misidentified serotypes. The use of Jin <i>cpsB</i> database instead of the GenBank database slightly improved results but did not significantly impact the efficiency of sequetyping. Although none of these molecular methods may currently replace the Quellung method, WGS remains the most promising molecular pneumococcal serotyping method.</p></div

    Whole genome core SNV maximum likelihood phylogenetic tree of 137 <i>Streptococcus pneumoniae</i> serotype 22F isolates collected in Canada from 2005–2015.

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    <p>The maximum likelihood phylogenetic tree is rooted on the reference genome of <i>S</i>. <i>pneumoniae</i> R6 (GenBank accession no. NC_003098.1) and the scale bar represents the estimated evolutionary divergence between isolates on the basis of average genetic distance between strains (estimated number of substitutions in the sample / total number of high quality SNVs). Clades A through F identified by cluster analysis are denoted with shading. Coloured columns in the right side heatmap represent: year of isolation (Year); province or territory isolated (Prov); patient age group (Age); clinical isolation source (Source); multi-locus sequence type (MLST); and antimicrobial susceptibilities to erythromycin (ERY), clindamycin (CLI), penicillin (PEN), cefepime (CFM), cefotaxime (CEF), ceftriaxone (CRO), meropenem (MER), trimethoprim/sulfamethoxazole (SXT) and tetracycline (TET).</p

    Core SNV phylogenetic comparison of genetic relatedness of <i>Streptococcus pneumoniae</i> serotype 22F.

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    <p>Node labels indicate serotype, an asterisk indicates USA strains and blue nodes indicate Pneumococcal Molecular Epidemiology Network (PMEN) clones. The length of the scale bar represents the estimated evolutionary divergence between isolates on the basis of average genetic distance between strains (estimated number of substitutions in the sample / total number of high quality SNVs).</p

    Multi-locus sequence type (MLST) comparison of the genetic relatedness of <i>Streptococcus pneumoniae</i> serotype 22F.

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    <p>Node colour indicates serotype and diameters are proportional to the number of isolates. Major MLST types of serotype 22F isolates are displayed in bold text and Pneumococcal Molecular Epidemiology Network (PMEN) clones are indicated. Branch labels are the number of allelic variations between sequence types; branch lengths are not to scale.</p
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