Molecular chaperones in biology and medicine at Obernai

No Abstract Available     

Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry

Objective

Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression.

Methods

The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes.

Results

Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABA_A) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes.

Conclusions

Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

     

Meta-Fish-Lib: A generalised,dynamic DNA reference library pipeline for metabarcoding of fishes

The accuracy and reliability of DNA metabarcoding analyses depend on the breadth and quality of the reference libraries that underpin them. However, there are limited options available to obtain and curate the huge volumes of sequence data that are available on public repositories such as NCBI and BOLD. Here, we provide a pipeline to download, clean and annotate mitochondrial DNA sequence data for a given list of fish species. Features of this pipeline include (a) support for multiple metabarcode markers; (b) searches on species synonyms and taxonomic name validation; (c) phylogeny assisted quality control for identification and removal of misannotated sequences; (d) automatically generated coverage reports for each new GenBank release update; and (e) citable, versioned DOIs. As an example we provide a ready-to-use curated reference library for the marine and freshwater fishes of the U.K. To augment this reference library for environmental DNA metabarcoding specifically, we generated 241 new MiFish-12S sequences for 88 U.K. marine species, and make available new primer sets useful for sequencing these. This brings the coverage of common U.K. species for the MiFish-12S fragment to 93%, opening new avenues for scaling up fish metabarcoding across wide spatial gradients. The Meta-Fish-Lib reference library and pipeline is hosted at https://github.com/genner-lab/meta-fish-lib .     

Influence of steam and dry heat pretreatment on fibre properties and cellulase degradation of cellulosic fibres

Cellulosic fabric samples of cotton, viscose, lyocell and modal were pretreated with steam and dry heat in the range of 100–190°C. The samples were then treated with a Trichoderma reesei cellulase preparation (total culture filtrate – TC), with mechanical agitation, at a high enzyme dosage of 75% by weight of fabric, for 8 hours. Generally, viscose proved to be easily degradable, followed by cotton and modal. The degree of hydrolysis was the least for Lyocell. Dry heat pretreatments exerted a lower influence on degradation rate than steam pretreatments which showed a distinct maximum at a steam temperature of 130°C. The hydrolysis rate varied strongly depending on treatment conditions and fibre type. Water retention values in treated substrates were changed by up to 20% of initial values.     

Bacterially Produced Recombinant Influenza Vaccines Based on Virus-Like Particles

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein''s neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.