External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data

The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.     

Pharmacokinetics of proline-rich tripeptides in the pig

Tripeptides may possess bioactive properties. For instance, blood pressure lowering is attributed to the proline-rich tripeptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP), and Val-Pro-Pro (VPP). However, little is known about their absorption, distribution, and elimination characteristics. The aim of this study was to characterize the pharmacokinetic behavior of IPP, LPP, and VPP in a conscious pig model. Synthetic IPP, LPP, and VPP were administered intravenously or intragastrically (4.0 mg kg⁻¹ BW in saline) to 10 piglets (approximately 25 kg body weight) in the postabsorptive state. After intravenous dosing, the elimination half-life for IPP was significantly higher (P < 0.001) than for LPP and VPP (2.5 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 min, respectively). After intragastric dosing, however, the elimination half-lives were not significantly different between the peptides (9 ± 1, 15 ± 4, and 12 ± 6 min, respectively). Maximum plasma concentrations were about 10 nmol l⁻¹ for the three tripeptides. The fraction dose absorbed was 0.077 ± 0.010, 0.059 ± 0.009, and 0.073 ± 0.015%, for IPP, LPP, and VPP, respectively. Proline-rich tripeptides reach the blood circulation intact, with an absolute bioavailability of about 0.1% when administered via a saline solution. Because half-lives of absorption and elimination were maximally about 5 and 15 min, respectively, this suggests that under these conditions a bioactive effect of these tripeptides would be rather acute.     

Rer1p maintains ciliary length and signaling by regulating γ-secretase activity and Foxj1a levels

Cilia project from the surface of most vertebrate cells and are important for several physiological and developmental processes. Ciliary defects are linked to a variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. In this paper, we identified Rer1p as the first endoplasmic reticulum/cis-Golgi–localized membrane protein involved in ciliogenesis. Rer1p, a protein quality control receptor, was highly expressed in zebrafish ciliated organs and regulated ciliary structure and function. Both in zebrafish and mammalian cells, loss of Rer1p resulted in the shortening of cilium and impairment of its motile or sensory function, which was reflected by hearing, vision, and left–right asymmetry defects as well as decreased Hedgehog signaling. We further demonstrate that Rer1p depletion reduced ciliary length and function by increasing γ-secretase complex assembly and activity and, consequently, enhancing Notch signaling as well as reducing Foxj1a expression.     

N-acyl-L-homoserine lactone quorum sensing controls butanediol fermentation in Serratia plymuthica RVH1 and Serratia marcescens MG1

Butanediol fermentation in two Serratia species is shown to be affected by N-acyl-L-homoserine lactone-dependent quorum sensing. Knockout of quorum-sensing signal production caused a shift towards enhanced acid production, resulting in early growth arrest, which was reversible by the addition of synthetic signal molecules.     

Gene Expression Analysis of Peripheral Cells for Subclassification of Pediatric Inflammatory Bowel Disease in Remission

Objective

In current clinical practice, optimal treatment of inflammatory bowel disease (IBD) aims at the induction and maintenance of clinical remission. Clinical remission is apparent when laboratory markers of inflammation are normal and clinical symptoms are absent. However, sub-clinical inflammation can still be present. A detailed analysis of the immune status during this inactive state of disease may provide a useful tool to categorize patients with clinical remission into subsets with variable states of immune activation.

Design

By using Affymetrix GeneChips, we analysed RNA gene expression profiles of peripheral blood leukocytes from pediatric IBD patients in clinical remission and controls. We performed (un)supervised clustering analysis of IBD-associated genes and applied Ingenuity® pathway software to identify specific molecular profiles between patients.

Results

Pediatric IBD patients with disease in clinical remission display heterogeneously distributed gene expression profiles that are significantly distinct from controls. We identified three clusters of IBD patients, each displaying specific expression profiles of IBD-associated genes.

Conclusion

The expression of immune- and IBD-associated genes in peripheral blood leukocytes from pediatric IBD patients in clinical remission was different from healthy controls, indicating that sub-clinical immune mechanisms are still active during remission. As such, RNA profiling of peripheral blood may allow for non-invasive patient subclassification and new perspectives in treatment regimes of IBD patients in the future.     

Differential role of basal keratinocytes in UV-induced immunosuppression and skin cancer

Cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) comprise major UV-induced photolesions. If left unrepaired, these lesions can induce mutations and skin cancer, which is facilitated by UV-induced immunosuppression. Yet the contribution of lesion and cell type specificity to the harmful biological effects of UV exposure remains currently unclear. Using a series of photolyase-transgenic mice to ubiquitously remove either CPDs or 6-4PPs from all cells in the mouse skin or selectively from basal keratinocytes, we show that the majority of UV-induced acute effects to require the presence of CPDs in basal keratinocytes in the mouse skin. At the fundamental level of gene expression, CPDs induce the expression of genes associated with repair and recombinational processing of DNA damage, as well as apoptosis and a response to stress. At the organismal level, photolyase-mediated removal of CPDs, but not 6-4PPs, from the genome of only basal keratinocytes substantially diminishes the incidence of skin tumors; however, it does not affect the UVB-mediated immunosuppression. Taken together, these findings reveal a differential role of basal keratinocytes in these processes, providing novel insights into the skin's acute and chronic responses to UV in a lesion- and cell-type-specific manner.     

Ascospore release from bottle-shaped asci in Dipodascus albidus

Yeasts utilize different mechanisms to release ascospores of different lengths from bottle-shaped asci. Using electron microscopy, confocal laser scanning microscopy, gas chromatography-mass spectrometry and digital live imaging, the individual release of oval ascospores from tight-fitting narrow bottle-necks, is reported in the yeast Dipodascus albidus. These ascospores are surrounded by compressible, oxylipin-coated sheaths enabling ascospores to slide past each other when forced by turgor pressure and by possible sheath contractions towards the narrowing ascus-neck. In this paper, the release mechanisms of ascospores of various lengths from bottle-shaped asci and produced by different yeasts are compared. We suggest that different release mechanisms, utilizing compressible sheaths or geared-alignment, have possibly evolved to compensate for variation in ascospore length. Alternatively, sheaths and ridges might be two evolutionary solutions to the same biomechanical problem, i.e. to release ascospores irrespective of length from bottle-shaped asci.     

BAC clones generated from sheared DNA

BAC libraries generated from restriction-digested genomic DNA display representational bias and lack some sequences. To facilitate completion of genome projects, procedures have been developed to create BACs from DNA physically sheared to create fragments extending up to 200 kb. The DNA fragments were repaired to create blunt ends and ligated to a new BAC vector. This approach has been tested by generating BAC libraries from Drosophila DNA with insert lengths between 50 and 150 kb. The libraries lack chimeric clone problems as determined by mapping paired BAC-end sequences to the assembled fly genome sequence. The utility of "sheared" libraries was demonstrated by closure of a previous clone gap and by isolation of clones from telomeric regions, which were notably absent from previous Drosophila BAC libraries.