Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp® DNA Mini Kit, Reischl et al.’s method and FTA® Elute. FTA® Elute significantly showed the highest median DNA extraction efficiency of 76.9% for E. coli and 108.9% for S. aureus. The Reischl et al. method and QIAamp® DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA® Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall, the FTA® Elute method was found to be the most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity, and transport and storage conditions

Interplay between Kinase Domain Autophosphorylation and F-Actin Binding Domain in Regulating Imatinib Sensitivity and Nuclear Import of BCR-ABL

BACKGROUND: The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood. METHODOLOGY/PRINCIPAL FINDINGS: By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib. CONCLUSIONS/SIGNIFICANCE: Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML

Outlier SNPs detect weak regional structure against a background of genetic homogeneity in the Eastern Rock Lobster, Sagmariasus verreauxi

Genetic differentiation is characteristically weak in marine species making assessments of population connectivity and structure difficult. However, the advent of genomic methods has increased genetic resolution, enabling studies to detect weak, but significant population differentiation within marine species. With an increasing number of studies employing high resolution genome-wide techniques, we are realising that the connectivity of marine populations is often complex and quantifying this complexity can provide an understanding of the processes shaping marine species genetic structure and to inform long-term, sustainable management strategies. This study aims to assess the genetic structure, connectivity, and local adaptation of the Eastern Rock Lobster (Sagmariasus verreauxi), which has a maximum pelagic larval duration of 12 months and inhabits both subtropical and temperate environments. We used 645 neutral and 15 outlier SNPs to genotype lobsters collected from the only two known breeding populations and a third episodic population—encompassing S. verreauxi's known range. Through examination of the neutral SNP panel, we detected genetic homogeneity across the three regions, which extended across the Tasman Sea encompassing both Australian and New Zealand populations. We discuss differences in neutral genetic signature of S. verreauxi and a closely related, co-distributed rock lobster, Jasus edwardsii, determining a regional pattern of genetic disparity between the species, which have largely similar life histories. Examination of the outlier SNP panel detected weak genetic differentiation between the three regions. Outlier SNPs showed promise in assigning individuals to their sampling origin and may prove useful as a management tool for species exhibiting genetic homogeneity

KRAB–Zinc Finger Proteins and KAP1 Can Mediate Long-Range Transcriptional Repression through Heterochromatin Spreading

Krüppel-associated box domain-zinc finger proteins (KRAB–ZFPs) are tetrapod-specific transcriptional repressors encoded in the hundreds by the human genome. In order to explore their as yet ill-defined impact on gene expression, we developed an ectopic repressor assay, allowing the study of KRAB–mediated transcriptional regulation at hundreds of different transcriptional units. By targeting a drug-controllable KRAB–containing repressor to gene-trapping lentiviral vectors, we demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases (kb) away from their DNA binding sites, with an efficiency which is generally higher for promoters located within 15 kb or less. Silenced promoters exhibit a loss of histone H3-acetylation, an increase in H3 lysine 9 trimethylation (H3K9me3), and a drop in RNA Pol II recruitment, consistent with a block of transcriptional initiation following the establishment of silencing marks. Furthermore, we reveal that KRAB–mediated repression is established by the long-range spreading of H3K9me3 and heterochromatin protein 1 β (HP1β) between the repressor binding site and the promoter. We confirm the biological relevance of this phenomenon by documenting KAP1–dependent transcriptional repression at an endogenous KRAB–ZFP gene cluster, where KAP1 binds to the 3′ end of genes and mediates propagation of H3K9me3 and HP1β towards their 5′ end. Together, our data support a model in which KRAB/KAP1 recruitment induces long-range repression through the spread of heterochromatin. This finding not only suggests auto-regulatory mechanisms in the control of KRAB–ZFP gene clusters, but also provides important cues for interpreting future genome-wide DNA binding data of KRAB–ZFPs and KAP1

Technology, ethics and religious language: early Anglophone Christian reactions to “cyberspace”

The very recent past has seen an upswing of scholarly interest not so much in the Internet and Web themselves but in the terms in which they have been discussed and understood. This article examines a remarkable effusion of writing in the 1990s that addressed the spiritual and ethical implications of “cyberspace”. Christian critics reacted in different ways to prophecies of technological revolution. Some saw ethical challenges in relation to economic and social exclusion and the nature of interpersonal relations. Others elaborated a semi-mystical evolutionary understanding of the Web as an ontologically concrete “space”. Others again revived older anxieties about the challenge apparently posed to human uniqueness and autonomy posed by computerisation more generally, which cyberspace threatened to magnify. However, this thinking did not occur in isolation from the sweep of Anglophone social thought. I suggest instead that the wider discourse about the ethics of the Internet and Web, both learned and popular, was infused at every level with religious imagery. As such, the article contributes to the ongoing debate on the extent to which the cultures of the UK and North America have been secularised: even if religious observance has declined, the English language still bears the marks of its Christian past