Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5′-end. In qPCR, the 5′-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5′-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5′-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5′-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5′-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5′-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions

”New” POPs in marine mammals in Nordic Arctic and NE Atlantic areas during three decades

The report describes the findings of a Nordic study aiming to depict possible trends in “new” contaminants in marine mammals in Nordic Arctic waters over three decennia. The “new” contaminants in focus are the brominated flame retardants, BFRs, methoxylated PBDEs, perfluorinated compounds including the PFOS family, and polychlorinated naphthalenes, PCNs. In addition, brominated dioxins and dibenzofurans were analysed in a subset of the samples. The study aims at giving a wide scope of the presence of a selection of these “new” contaminants in marine mammals in recent time and so far back as is possible with extracting samples from specimen banks. The marine mammal species analysed were fin whale, minke whale, pilot whale, white-sided dolphins, harbour porpoise, ringed seal and hooded seal. The study is the result of collaboration between Norway, Denmark/Greenland, Faroe Island, Iceland and Sweden. The funding for large parts of the project has been made available by the Nordic Council of Ministers via the working group on Akvatiska Ekosystemer

Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin

<p>Abstract</p> <p>Background</p> <p>Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs) delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B) of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs.</p> <p>Methods</p> <p>Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels.</p> <p>Results</p> <p>Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimick the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by immunohistological examination.</p> <p>Conclusions</p> <p>Our studies consolidate the properties of lentiviral vectors as a tool for potent gene delivery and for evaluation of mRNA targets for anti-inflammatory therapy. However, in contrast to local anti-TNFα treatment, the therapeutic potential of targeting IL-12B at the RNA level in psoriasis is questioned.</p

Genome-wide assessment of differential roles for p300 and CBP in transcription regulation

Despite high levels of homology, transcription coactivators p300 and CREB binding protein (CBP) are both indispensable during embryogenesis. They are largely known to regulate the same genes. To identify genes preferentially regulated by p300 or CBP, we performed an extensive genome-wide survey using the ChIP-seq on cell-cycle synchronized cells. We found that 57% of the tags were within genes or proximal promoters, with an overall preference for binding to transcription start and end sites. The heterogeneous binding patterns possibly reflect the divergent roles of CBP and p300 in transcriptional regulation. Most of the 16 103 genes were bound by both CBP and p300. However, after stimulation 89 and 1944 genes were preferentially bound by CBP or p300, respectively. Target genes were found to be primarily involved in the regulation of metabolic and developmental processes, and transcription, with CBP showing a stronger preference than p300 for genes active in negative regulation of transcription. Analysis of transcription factor binding sites suggest that CBP and p300 have many partners in common, but AP-1 and Serum Response Factor (SRF) appear to be more prominent in CBP-specific sequences, whereas AP-2 and SP1 are enriched in p300-specific targets. Taken together, our findings further elucidate the distinct roles of coactivators p300 and CBP in transcriptional regulation

Jasmonic Acid-Induced Changes in Brassica oleracea Affect Oviposition Preference of Two Specialist Herbivores

Jasmonic acid (JA) is a key hormone involved in plant defense responses. The effect of JA treatment of cabbage plants on their acceptability for oviposition by two species of cabbage white butterflies, Pieris rapae and P. brassicae, was investigated. Both butterfly species laid fewer eggs on leaves of JA-treated plants compared to control plants. We show that this is due to processes in the plant after JA treatment rather than an effect of JA itself. The oviposition preference for control plants is adaptive, as development time from larval hatch until pupation of P. rapae caterpillars was longer on JA-treated plants. Total glucosinolate content in leaf surface extracts was similar for control and treated plants; however, two of the five glucosinolates were present in lower amounts in leaf surface extracts of JA-treated plants. When the butterflies were offered a choice between the purified glucosinolate fraction isolated from leaf surface extracts of JA-treated plants and that from control plants, they did not discriminate. Changes in leaf surface glucosinolate profile, therefore, do not seem to explain the change in oviposition preference of the butterflies after JA treatment, suggesting that as yet unknown infochemicals are involved

A competence-based and multidimensional operationalization and measurement of employability

Employability is a critical requirement for enabling both sustained competitive advantage at the firm level and career success at the individual level. We propose a competence-based approach to employability derived from an expansion of the resource-based view of the firm. In this contribution, we present a reliable and valid instrument for measuring employability. This measure is based on a five-dimensional conceptualization of employability, in which occupational expertise is complemented with generic competences. Two sources of raters (employees and their immediate supervisors) are involved in developing and testing the measure. Since the five dimensions of employability explain a significant amount of variance in both objective and subjective career success, the predictive validity of the tool is promising. This instrument facilitates further scientific HRM research and is of practical value in light of job and career assessments, recruitment, staffing, career mobility, and development practice

Characterization of the role of ribonucleases in Salmonella small RNA decay

In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. To better understand the turnover of regulatory sRNAs in the model pathogen, Salmonella typhimurium, we have constructed mutants for several ribonucleases (RNase E, RNase G, RNase III, PNPase) and Poly(A) Polymerase I. The expression profiles of four sRNAs conserved among many enterobacteria, CsrB, CsrC, MicA and SraL, were analysed and the processing and stability of these sRNAs was studied in the constructed strains. The degradosome was a common feature involved in the turnover of these four sRNAs. PAPI-mediated polyadenylation was the major factor governing SraL degradation. RNase III was revealed to strongly affect MicA decay. PNPase was shown to be important in the decay of these four sRNAs. The stability of CsrB and CsrC seemed to be independent of the RNA chaperone, Hfq, whereas the decay of SraL and MicA was Hfq-dependent. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs

Topical Application of Activity-based Probes for Visualization of Brain Tumor Tissue

Several investigators have shown the utility of systemically delivered optical imaging probes to image tumors in small animal models of cancer. Here we demonstrate an innovative method for imaging tumors and tumor margins during surgery. Specifically, we show that optical imaging probes topically applied to tumors and surrounding normal tissue rapidly differentiate between tissues. In contrast to systemic delivery of optical imaging probes which label tumors uniformly over time, topical probe application results in rapid and robust probe activation that is detectable as early as 5 minutes following application. Importantly, labeling is primarily associated with peri-tumor spaces. This methodology provides a means for rapid visualization of tumor and potentially infiltrating tumor cells and has potential applications for directed surgical excision of tumor tissues. Furthermore, this technology could find use in surgical resections for any tumors having differential regulation of cysteine cathepsin activity