RepTAGs: universal tags for isolation and labeling of proteins, for labeling live mammalian cells and for drug discovery

Methods for specific immobilization, isolation and labeling of proteins are central to the elucidation of cellular functions. Based on bacterial repressor proteins, which bind to specific target sequences in response to small molecules (macrolide and tetracycline antibiotics) or environmental parameters (temperature), we have developed a set of protein tags (RepTAGs), which enable reversible immobilization of the protein of interest on a solid support for the isolation and quantification as well as for the specific labeling of target proteins with fluorescent dyes for tracking them within a complex protein mixture. Similarly, live mammalian cells were specifically labeled with a fluorescent operator sequence bound to RepTAGs, which were directed towards the cell surface for easy discrimination between transfected and untransfected cell populations. Based on the drug-responsive RepTAG-DNA interactions, it was also possible to quantify or discover antibiotics in environmental samples or compound libraries by means of rapid, sensitive detection methods involving fluorescence polarization and bioluminescence. We believe that the universally applicable RepTAGs will become essential for the analysis and manipulation of proteins in the most diverse areas of protein chemistry and cell biology.     

Molecular chaperones regulate p53 and suppress senescence programs

Many types of cancer cells constitutively express major molecular chaperones at high levels. Recent findings demonstrate that specific depletion of individual chaperones, including various members of the Hsp70 family, small heat shock proteins, or VCP/p97, leads to activation of p53 pathway and subsequently triggers cellular senescence. Here, we discuss a possibility that in cancer cells high levels of chaperones serve to keep the p53 signaling under control, thus allowing cancer cells to evade the default senescence and form tumors.     

Collagen biomaterial doped with colominic acid for cell culture applications with regard to peripheral nerve repair

Colominic acid (CA) is a homopolymer of sialic acid residues and is solely composed of polymerised units of alpha-2,8-linked N-acetylneuraminic acid. CA is a specific derivative of polysialic acid (PSA), produced as the capsular polysaccharide of Escherichia coli K1 derived molecule of PSA. PSA in vivo plays a significant role in synaptic plasticity and neural development. The use of collagen materials doped with defined CA is presented for the cultivation of various cell lines relevant for possible applications in Tissue Engineering. First, the release behaviour under culture conditions of the collagen-based (C-CA) materials was investigated by thiobarbituric acid assay. Additionally, the established cell lines, PC-12 and immortalised Schwann cells (ISC), used for neurobiological and neurochemical studies and the model liver cell line Hep-G2 as indicator for biocompatibility testing, were cultured on the C-CA matrix. Cell proliferation (MTT-test) and cell adhesion (DAPI-staining) of the cell lines on the matrices were observed. Likewise, gene expression of the marker genes thyrosine hydroxylase for the PC-12 cells, and albumin, transferrin and CYP3A4 for the Hep-G2 cells was evaluated via RT-PCR. The results indicate that CA integration in established biomaterial constructs enhances cell proliferation and offers promising features as conduits additive in regarding peripheral nerve regeneration.     

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.     

Aging Schwann cells in vitro

Schwann cells (SCs) can support the regeneration of lesioned fiber tracts of the peripheral and central nervous system and have been transplanted alone or in combination with synthetic nerve guides. For neuronal tissue engineering purposes, the cells must be isolated from small biopsies and expanded in vitro. In this study we analyze the impact of cell expansion on 9 different cell parameters, comparing short- and long-term cultured rat SCs, which we refer to as 'young' and 'old' or 'aged' cells, respectively. In comparison to young SCs, old SCs doubled the axonal outgrowth from dorsal root ganglion explants and displayed only one-third as much adhesion to the gray and white matter of spinal cord cryosections. In a 3-dimensional extracellular matrix the two cell populations showed very different cellular responses with regard to cell morphology and cell-cell adhesion. Cell proliferation of old SCs was independent of serum components and was not hampered by contact inhibition. In addition, population doubling times were reduced by a factor of almost three compared to those of young SCs. Despite considerable karyotype changes, with an average of 68.7 chromosomes versus 42 in native rat cells, old SCs did not show any increase in telomerase activity and loss of anchorage dependence--characteristics that are typical of tumor cells. The data also provide biological insights into which cell characteristics (proliferation and adhesion, for example) are functionally clustered and either change or remain constant with aging in vitro. Though the data indicate a lack of tumorigenic transformation coupled with increased neurite outgrowth-promoting activity after extensive SC expansion in vitro, thus suggesting better regeneration qualities, we strongly recommend that in vitro aged rat SCs (>11 passages) should not be employed for tissue engineering.     

Calpain-10 variants and haplotypes are associated with polycystic ovary syndrome in Caucasians

PCOS is known to be associated with an increased risk of T2DM and has been proposed to share a common genetic background with T2DM. Recent studies suggest that the Calpain-10 gene (CAPN10) is an interesting candidate gene for PCOS susceptibility. However, contradictory results were reported concerning the contribution of certain CAPN10 variants, especially of UCSNP-44, to genetic predisposition to T2DM, hirsutism, and PCOS. By means of MALDI-TOF MS technique, we genotyped an expanded single nucleotide polymorphism panel, including the CAPN10 UCSNP-44, -43, -56, ins/del-19, -110, -58, -63, and -22 in a sample of 146 German PCOS women and 606 population-based controls. Statistical analysis revealed an association between UCSNP-56 and susceptibility to PCOS with an odds ratio (OR) of 2.91 (95% CI=1.51-5.61) for women carrying an AA genotype compared with GG. As expected, the 22-genotype of the ins/del-19 variant, which is in high linkage disequilibrium (r2=0.98) with UCSNP-56, was also significantly associated (OR=2.98, 95% CI=1.55-5.73). None of the additionally tested variants alone showed any significant association with PCOS. A meta-analysis including our study (altogether 623 PCOS cases and 1,224 controls) also showed significant association only with ins/del-19. The most common haplotype TGG3AGCA was significantly associated with a lower risk for PCOS (OR=0.487, P=0.0057). In contrast, the TGA2AGCA haplotype was associated with an increased risk for PCOS (OR=3.557, P=0.0011). By investigating a broad panel of CAPN10 variants, our results pointed to an allele dose-dependent association of UCSNP-56 and ins/del-19 with PCOS.     

A systematic strategy for large-scale analysis of genotype phenotype correlations: identification of candidate genes involved in African trypanosomiasis

It is increasingly common to combine Microarray and Quantitative Trait Loci data to aid the search for candidate genes responsible for phenotypic variation. Workflows provide a means of systematically processing these large datasets and also represent a framework for the re-use and the explicit declaration of experimental methods. In this article, we highlight the issues facing the manual analysis of microarray and QTL data for the discovery of candidate genes underlying complex phenotypes. We show how automated approaches provide a systematic means to investigate genotype-phenotype correlations. This methodology was applied to a use case of resistance to African trypanosomiasis in the mouse. Pathways represented in the results identified Daxx as one of the candidate genes within the Tir1 QTL region. Subsequent re-sequencing in Daxx identified a deletion of an amino acid, identified in susceptible mouse strains, in the Daxx-p53 protein-binding region. This supports recent experimental evidence that apoptosis could be playing a role in the trypanosomiasis resistance phenotype. Workflows developed in this investigation, including a guide to loading and executing them with example data, are available at http://workflows.mygrid.org.uk/repository/myGrid/PaulFisher/.     

Rapid conditional knock-down-knock-in system for mammalian cells

RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down-knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein.