Expression levels of heat shock protein 60 in human endothelial cells in vitro are unaffected by exposure to 50 Hz magnetic fields

Magnetic fields (MFs) from domestic power sources have been implicated as being a potential risk to human health. A number of epidemiological studies have found a significant link between exposure to MFs and increased rates of cancers. There have also been a number of in vivo and in vitro studies reporting effects of MFs in animal disease models and on the expression or activity of a range of proteins. In the past decade, our group proposed that atherosclerosis may have an autoimmune component, with heat shock protein 60 (Hsp60) expressed in endothelial cells as the dominant autoantigen. A number of stressors have been shown to induce the expression of Hsp60, including the classical risk factors for atherosclerosis. We were interested to see if the exposure of endothelial cells to an MF elicited increased expression of Hsp60, as has been reported previously for Hsp70. The present work describes the exposure of endothelial cells to domestic power supply (50 Hz) MFs at an intensity of 700 microT. The results from our system indicate that cultured endothelial cells exposed to a high intensity of MF either alone or in combination with classical heat stress show no effects on the expression of Hsp60 at either the messenger ribonucleic acid or the protein level. As such, there is no evidence that exposure to extremely low-frequency MF would be expected to increase the expression of Hsp60 and therefore the initiation or progression of atherosclerosis.     

Elevated expression of p53 in early colon polyps in a pig model of human familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is a hereditary predisposition to formation of colon polyps that can progress to colorectal cancer (CRC). The severity of polyposis varies substantially within families bearing the same germline mutation in the adenomatous polyposis coli (APC) tumour suppressor gene. The progressive step-wise accumulation of genetic events in tumour suppressor genes and oncogenes leads to oncogenic transformation, with driver alterations in the tumour protein p53 (TP53) gene playing a key role in advanced stage CRC. We analysed groups of pigs carrying a truncating mutation in APC (APC^1311/+; orthologous to human APC^1309/+) to study the influence of TP53 polymorphisms and expression on the frequency of polyp formation and polyp progression in early-stage FAP. Five generations of APC^1311/+ pigs were examined by colonoscopy for polyposis severity and development. A total of 19 polymorphisms were found in 5′-flanking, coding, and 3′ untranslated regions of TP53. The distribution of TP53 genotypes did not differ between APC^1311/+ pigs with low (LP) and high (HP) number of colon polyps. p53 mRNA expression was analysed in distally located normal mucosa samples of wild-type pigs, APC^1311/+ LP and HP pigs, and also in distally located polyp samples histologically classified as low-grade (LG-IEN) and high-grade intraepithelial dysplastic (HG-IEN) from APC^1311/+ pigs. p53 mRNA expression was found to be significantly elevated in HG-IEN compared to LG-IEN samples (p?= 0.012), suggesting a role for p53 in the early precancerous stages of polyp development.     

The new pig on the block: modelling cancer in pigs

The molecular mechanisms underlying many human cancers are now reasonably well understood. The challenge now is to bridge the gap between laboratory and clinical oncology, so these accomplishments can be translated into practical benefits for human patients. While genetically modified mice have played a prominent role in basic research, they are less suitable for many preclinical studies. Other animals can provide important complementary resources to aid the development, validation and application of new medicines and procedures. Powerful methods of genetic engineering have now been extended to physiologically more relevant species, particularly the pig, opening the prospect of more representative, genetically defined, cancer models at human scale. We briefly review the field and outline our program to generate gene-targeted pigs carrying mutations in tumour suppressor genes and proto-oncogenes that replicate key lesions responsible for a variety of human cancers. We also highlight some important issues for the future development and usefulness of porcine cancer models.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Efficient Generation of Rat Induced Pluripotent Stem Cells Using a Non-Viral Inducible Vector

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.     

An X-ray diffraction analysis of crystallised whey and whey-permeate powders

Amorphous whey, whey-permeate and lactose powders have been crystallised at various air temperatures and humidities, and these crystallised powders have been examined using X-ray diffraction. The most stable lactose crystal under normal storage conditions, alpha-lactose monohydrate, forms preferentially in whey and whey-permeate powders at 50 degrees C, provided sufficient moisture is available, whereas anhydrous beta-lactose and mixed anhydrous lactose crystals, which are unstable under normal storage conditions, form preferentially at 90 degrees C. Thus, faster crystallisation at higher temperatures is offset by the formation of lactose-crystal forms that are less stable under normal storage conditions. Very little alpha-lactose monohydrate crystallised in the pure lactose powders over the range of temperatures and humidities tested, because the crystallisation of alpha- and beta-lactose is considerably more rapid than the mutarotation of beta- to alpha-lactose in the amorphous phase and the hydration of alpha-lactose during crystallisation. Protein and salts hinder the crystallisation process, which provides more time for mutarotation and crystal hydration in the whey and whey-permeate powders.     

Crystallization kinetics of amorphous lactose, whey-permeate and whey powders

Amorphous lactose, whey-permeate and whey powders have been converted to their crystalline forms by exposure to air at various temperatures and relative humidities. The total time required for sorption, induction and crystallization of these powders was observed by following the time-dependent mass change of the powders during treatment. These experiments have shown that higher temperatures and relative humidities lead to shorter crystallization times. Lactose crystallizes within 1 min at an air temperature of 100 degrees C and relative air humidity of 80%, whereas whey-permeate and whey powders requires up to 5 min at the same set of conditions. Thus, as previously described, the presence of proteins and salts in the whey-permeate and whey powders reduces the crystallization rate. The rate constants and activation energies have been determined over a range of temperatures and humidities to enable the calculation of crystallization times for the design of an industrial process that crystallizes whey and whey-permeate powders. Finally, the crystallization rates found in this work are sufficiently fast to be applicable in an industrial process that crystallizes whey and whey-permeate powders.     

Dual Fluorescent Reporter Pig for Cre Recombination: Transgene Placement at the ROSA26 Locus

We are extending the Cre/loxP site-specific recombination system to pigs, focussing on conditional and tissue-specific expression of oncogenic mutations to model human cancers. Identifying the location, pattern and extent of Cre recombination in vivo is an important aspect of this technology. Here we report pigs with a dual fluorochrome cassette under the control of the strong CAG promoter that switches expression after Cre-recombination, from membrane-targeted tandem dimer Tomato to membrane-targeted green fluorescent protein. The reporter cassette was placed at the porcine ROSA26 locus by conventional gene targeting using primary mesenchymal stem cells, and animals generated by nuclear transfer. Gene targeting efficiency was high, and analysis of foetal organs and primary cells indicated that the reporter is highly expressed and functional. Cre reporter pigs will provide a multipurpose indicator of Cre recombinase activity, an important new tool for the rapidly expanding field of porcine genetic modification.     

Viunalikeviruses are environmentally common agents of horizontal gene transfer in pathogens and biocontrol bacteria

Bacteriophages have been used as natural biocontrol and therapeutic agents, but also as biotechnological tools for bacterial engineering. We showed recently that the transducing bacteriophage ϕMAM1 is a ViI-like phage and a member of the new genus, ‘Viunalikevirus''. Here, we show that four additional ViI-like phages and three new environmentally isolated viunalikeviruses, all infecting plant and human pathogens, are very efficient generalised transducers capable of transducing chromosomal markers at frequencies of up to 10⁻⁴ transductants per plaque-forming unit. We also demonstrate the interstrain transduction of plasmids and chromosomal markers, including genes involved in anabolism, genes for virulence and genes encoding secondary metabolites involved in biocontrol. We propose that all viunalikeviruses are likely to perform efficient horizontal gene transfer. Viunalikeviruses therefore represent useful agents for functional genomics and bacterial engineering, and for chemical and synthetic biology studies, but could be viewed as inappropriate choices for phage therapy.Combined morphological, genomic and phylogenetic analyses have recently led to the proposed creation of a new phage genus, ‘Viunalikevirus'', within the Myoviridae family (Adriaenssens et al., 2012a). The first member of this proposed genus, Salmonella phage ViI, was isolated in the 1930s (Craigie and Yen, 1938) and multiple viunalikeviruses have been sequenced and characterised since 2010 (Pickard et al., 2010; Anany et al., 2011; Hooton et al., 2011; Kutter et al., 2011; Matilla and Salmond, 2012; Park et al., 2012; Adriaenssens et al., 2012a, 2012b; Hsu et al., 2013; Luna et al., 2013; Shahrbabak et al., 2013). Viunalikeviruses are characterised as virulent (lytic) phages showing similar genome size, extensive DNA homology, strong gene synteny and a complex adsorption apparatus, which uses tail spike proteins as host-recognition determinants (Adriaenssens et al., 2012a).We recently isolated the ViI-like phage, ϕMAM1, that infects several environmental and clinical isolates belonging to Serratia and Kluyvera genera (Matilla and Salmond, 2012). During the characterisation of ϕMAM1, we showed that it mediates highly efficient generalised transduction (Matilla and Salmond, submitted for publication). These observations were consistent with a previous report, that the Salmonella phage ViI was also capable of transduction (Cerquetti and Hooke, 1993) and we have confirmed that phage ViI can transduce chromosomal markers and plasmids at frequencies of up to 4.6 × 10⁻⁵ transductants per plaque-forming unit (p.f.u.; Figure 1a; Supplementary Table 1).Open in a separate windowFigure 1Transduction capabilities of viunalikeviruses. (a) Transduction frequencies of LIMEstone1, LIMEstone2, ViI and CBA120 phages. The graph also shows transduction efficiencies of LIMEstone phages within and between Dickeya solani strains. Transduction efficiency was defined as the number of transductants obtained per p.f.u. In all cases, error bars represent the standard deviations (n=3). (b) Skimmed milk agar plates showing protease production in the wild-type (wt) Dickeya solani strains MK10, MK16 and IPO 2222. LIMEstone1- (LS1) and LIMEstone2- (LS2) mediated transduction of the spp::Km marker from the protease negative mutant strain MK10P1 to the wild-type strains MK10, MK16 and IPO 2222 result in a protease-negative phenotype. (c–e) LIMEstone-mediated transduction of the oocN::Km marker from the oocydin A-negative mutant strain MK10oocN to the wild-type strains MK10 (c), MK16 (d) and IPO 2222 (e) results in an oocydin A-negative phenotype and, consequently, in the generation of strains defective in their antimicrobial activity against the plant pathogenic oomycete, Pythium ultimum. The anti-oomycete assays were performed as described previously (Matilla et al., 2012).Most generalised transducers utilise a headful packing strategy where phage terminases recognise specific sequences (pac sites) in the DNA and perform cycles of packing that result in mature phage particles (Fineran et al., 2009a). Indeed, phage terminases with reduced specificity for pac sequences may lead to the evolution of efficient transducing phages (Schmeiger, 1972). Based on the high similarity between the terminases of ϕMAM1, ViI and those of other previously sequenced viunalikeviruses, we hypothesised that all of these ViI-like phages should be capable of transduction in their respective bacterial hosts. To test this hypothesis, we investigated three additional viunalikeviruses, Escherichia coli phage CBA120 (Kutter et al., 2011), and Dickeya phages LIMEstone1 and LIMEstone2 (Adriaenssens et al., 2012b). All the bacteriophages, bacterial strains, plasmids and primers used in this study are listed in the Supplementary Tables 2 and 3. Experimental procedures are presented as Supplementary Material.The LIMEstone phages specifically infect some strains of the emerging plant pathogen, Dickeya solani (Adriaenssens et al., 2012b), and here we showed that they also infect the recently sequenced D. solani strains MK10, MK16 and IPO 2222. As predicted, we confirmed that the LIMEstone phages effected efficient transduction of various auxotrophic markers between Dickeya solani strains (Figure 1a; Supplementary Table 4). To our knowledge, only one Dickeya transducing phage, ϕEC2, has been isolated previously (Resibois et al., 1984). Additional mutant strains were constructed and the generalised nature of the transduction was confirmed by transfer of multiple chromosomal markers, including mutations in the gene cluster encoding biosynthesis of the anti-oomycete haterumalide, oocydin A (Matilla et al., 2012) and in the locus for synthesis and secretion of protease virulence factors. Transduction frequency was higher at an multiplicity of infection (m.o.i.) of 0.1 and 0.01 with efficiencies of up to 10⁻⁴ transductants per p.f.u. (Figure 1a; Supplementary Tables 4 and 5).We also demonstrated transduction of a kanamycin resistance-marked plasmid pECA1039-Km3 between strains MK10, MK16 and IPO 2222 at frequencies of up to 8.6 × 10⁻⁵ (Supplementary Table 4). Plasmid pECA1039 (originally isolated from the phytopathogen, Pectobacterium atrosepticum) encodes a bifunctional type III Toxin-Antitoxin (TA) system, ToxIN, with abortive infection capacity. Although ToxIN aborts infection of various enterobacteria by diverse phages (Fineran et al., 2009b) it did not protect against infection by the tested viunalikeviruses, ϕMAM1, ViI, CBA120, LIMEstone1 or LIMEstone2 (not shown). Furthermore, another type III TA system, TenpIN, from the insect pathogen, Photorhabdus luminescens (Blower et al., 2012), failed to protect against any of the five ViI-like phages (not shown).In addition, we also tested the transduction capacity of the E. coli phage, CBA120, and confirmed transduction of plasmid-borne antibiotic resistances at a frequency of up to 10⁻⁴ transductants per p.f.u. (Figure 1a; Supplementary Table 6).We decided to test our hypothesis that the viunalikeviruses may all be generalised transducers by first isolating new viunalikeviruses from the environment. From treated sewage effluent, we isolated three new bacteriophages infecting Dickeya solani, ϕXF1, ϕXF3 and ϕXF4, as defined initially by their very characteristic ViI-like morphology in electron microscopy (Figures 2a–c). As predicted, all of these new phages were able to transduce chromosomal markers and plasmids at frequencies of up to 3 × 10⁻⁶ transductants per p.f.u. (Figure 2e; Supplementary Table 7). Sequencing of structural and non-structural protein-encoding genes of ϕXF1, ϕXF3 and ϕXF4 showed high nucleotide homology (between 80% and 100%) with the corresponding orthologs in LIMEstone1 (Supplementary Figure 1), indicating that these virgin environmental isolates also clade within the Viunalikevirus genus.Open in a separate windowFigure 2Environmental isolation and characterisation of new viunalikeviruses with generalised transduction functionality. Transmission electron micrographs of phages ϕXF1 (a), ϕXF3 (b), ϕXF4 (c) and ϕXF28 (d) are shown. As an internal control, ϕXF28 was an example of a new lytic phage isolated from the same environment but showing no transduction capabilities. Bars, 50 nm. (e) Transduction frequencies of the new viunalikeviruses ϕXF1, ϕXF3 and ϕXF4. Transduction experiments were performed using 10⁹ cells with ϕXF1, ϕXF3, ϕXF4 at an m.o.i. of 0.01. Transduction efficiency was defined as the number of transductants obtained per p.f.u. Error bars represent the standard deviations (n=3).Although we did not have access to other ViI-like Salmonella phages SFP10 (Park et al., 2012), ϕSH19 (Hooton et al., 2011) and Marshall (Luna et al., 2013), Escherichia phage PhaxI (Shahrbabak et al., 2013), Shigella phage ϕSboM-AG3 (Anany et al., 2011) and Klebsiella phage 0507-KN2-1 (Hsu et al., 2013), our results allow us to predict that all of these phages will mediate generalised transduction. Importantly, these phages would be expected to contribute to the horizontal gene transfer of virulence factors and antimicrobial-resistance determinants in diverse environments.Viunalikeviruses do not seem to be limited to the enterobacteria as bacteriophages showing ViI-like morphology have been isolated in Acinetobacter (Ackermann et al., 1994), Bordetella (Adriaenssens et al., 2012b) and Sinorhizobium (Werquin et al., 1988). Furthermore, another ViI-like morphotype phage (ϕM12 of Sinorhizobium meliloti) has also been shown to be an efficient transducer (Finan et al., 1984). Taken together, these results suggest that, even in the absence of strongly predictive comparative genomic detail, a characteristically discrete ViI-like morphology in electron microscopy may be sufficient to identify new phages as strong candidates for possession of generalised transduction capacity.The emergence and dissemination of antibiotic-resistant pathogens coupled with low discovery rates for new antimicrobials, plus increasing legal constraints on the use of chemical pesticides, have (re)focussed attention on the potential use of bacteriophages for ‘natural biocontrol'' of human, animal and plant pathogens. Several viunalikeviruses have been proposed as candidate therapeutic agents for the control of bacterial infections (Anany et al., 2011; Hooton et al., 2011; Park et al., 2012; Hsu et al., 2013; Shahrbabak et al., 2013) and the LIMEstone phages have been used in successful field trials for biocontrol of D. solani infections (Adriaenssens et al., 2012b). However, their efficient transduction capacities could provide a route for dissemination of virulence factors, such as proteases (Marits et al., 1999). In fact, we have demonstrated the interstrain transduction of plasmids and oocydin A, auxotrophy and protease markers between three different D. solani strains, at high frequencies (Figures 1 and ​and2;2; Supplementary Tables 4 and 7). Also, the irregular distribution of the oocydin A gene cluster within the Dickeya genus and the fact that its genomic context varies between strains raises the possibility of phage-mediated horizontal gene transfer between bacterial strains. These results emphasize strongly that when considering the genomics of phages for ‘phage therapy'' the absence of genes readily defined as playing roles in lysogeny or bacterial virulence may be insufficient to inspire confidence that use of a particular therapeutic phage presents no risk–particularly among the high efficiency-transducing viunalikeviruses.