Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

Solid organ transplantation across the ABO histo-blood group barrier: a case report

The ABO blood group system until recently constituted an insuperable barrier for solid organ transplantation, but cases of heart transplantation in infants and kidney transplantation in adults have been reported, wherein ABO-incompatible grafts have been successful. In 1990, the molecular genetic basis of three major alleles at the ABO locus was elucidated; A and B glycosyltransferases are specified by a variety of functional alleles at this locus. The antibody response to ABH antigens, namely, naturally occurring anti-A/B IgM and IgG isotype agglutinins, are controlled preoperatively by recipient conditioning using plasma exchange, immunoadsorption, and immunosuppressive regimens. We report an O-type patient who accidentally received a B-type cardiac allograft in 1997 who survived for 5 years, dying for an unrelated reason. Over a period of 45 months semiquantitatively we monitored the expression of ABO-type antigens in graft heart vessels using monoclonal antibodies on sections of formalin-fixed, paraffin-embedded biopsies. We observed a progressive change in the antigenic profile of graft endothelial cells from B- to O-type, which was first detected at 1 year posttransplant and most prominent 3 years later, the end of the observation period. No temporal relationship was observed between the transition from B to O expression, the anti-B antibody levels or the immunosuppressive regimen.

MHP Oriented Interactive Augmented Reality System for Sports Broadcasting Environments

Television and movie images have been altered ever since it was technically possible. Nowadays embedding advertisements, or incorporating text and graphics in TV scenes, are common practice, but they can not be considered as integrated part of the scene. The introduction of new services for interactive augmented television is discussed in this paper. We analyse the main aspects related with the whole chain of augmented reality production. Interactivity is one of the most important added values of the digital television: This paper aims to break the model where all TV viewers receive the same final image. Thus, we introduce and discuss the new concept of interactive augmented television, i. e. real time composition of video and computer graphics - e.g. a real scene and freely selectable images or spatial rendered objects - edited and customized by the end user within the context of the user's set top box and TV receiver.

The E-learning Circle – a holistic software design tool for e-learning

The article introduces the E-learning Circle, a tool developed to assure the quality of the software design process of e-learning systems, considering pedagogical principles as well as technology. The E-learning Circle consists of a number of concentric circles which are divided into three sectors. The content of the inner circles is based on pedagogical principles, while the outer circle specifies how the pedagogical principles may be implemented with technology. The circle’s centre is dedicated to the subject taught, ensuring focus on the specific subject’s properties. The three sectors represent the student, the teacher and the learning objectives. The strengths of the E-learning Circle are the compact presentation combined with the overview it provides, as well as the usefulness of a design tool dealing with complexity, providing a common language and embedding best practice. The E-learning Circle is not a prescriptive method, but is useful in several design models and processes. The article presents two projects where the E-learning Circle was used as a design tool.

Mathe-Üben in Studium und Abitur

Fehlende Grundkenntnisse in der Mathematik zählen zu den größten Hindernissen für einen erfolgreichen Start in ein Hochschulstudium. Studienanfänger in einem MINT-Studium bringen inzwischen deutlich unterschiedliche Vorrausetzungen mit: „Mathe-Angst“ gilt als typisches Phänomen und der Übergang in ein selbstbestimmtes Lernverhalten stellt eine große Herausforderung dar. Diese Fall-Studie beschreibt, wie mit Hilfe einer Mathe-App bereits zu Beginn des Studiums aktives Lernen unterstützt und selbstbestimmtes Lernen eingeübt werden kann. Das neue Kurskonzept mit App-Unterstützung stößt an der Hochschule Offenburg auf breite Akzeptanz. Der mobile BYOD-Ansatz ermöglicht Lern-Szenarien, die über PC- bzw.- Laptop-gebundene eLearning-Lösungen nicht realisierbar sind. Der Inhalt des MassMatics-Vorbereitungskurs orientiert sich am Mindestanforderungskatalog des cosh-Arbeitskreises für den Übergang Schule-Hochschule. In der Zwischenzeit wurde der App-gestützte Kurs mit seinen über 500 Aufgaben von mehr als 1000 Studierenden besucht.

Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium ("Pan-Flavo") and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum.

Large deviations for heavy-tailed random elements in convex cones

We prove large deviation results for sums of heavy-tailed random elements in rather general convex cones being semigroups equipped with a rescaling operation by positive real numbers. In difference to previous results for the cone of convex sets, our technique does not use the embedding of cones in linear spaces. Examples include the cone of convex sets with the Minkowski addition, positive half-line with maximum operation and the family of square integrable functions with arithmetic addition and argument rescaling.

Lions-type compactness and Rubik actions on the Heisenberg group

In this paper we prove a Lions-type compactness embedding result for symmetric unbounded domains of the Heisenberg group. The natural group action on the Heisenberg group TeX is provided by the unitary group U(n) × {1} and its appropriate subgroups, which will be used to construct subspaces with specific symmetry and compactness properties in the Folland-Stein’s horizontal Sobolev space TeX. As an application, we study the multiplicity of solutions for a singular subelliptic problem by exploiting a technique of solving the Rubik-cube applied to subgroups of U(n) × {1}. In our approach we employ concentration compactness, group-theoretical arguments, and variational methods.

Space, diffusion and mobility

Given its origins in traditional dialectology, and given advances in our understanding of the social embedding of language variation, it is paradoxical that space should be one of the categories that has received least attention of all in variationist sociolinguistics. Until recently, space has largely been treated as an empty stage on which sociolinguistic processes are enacted. It has been unexamined, untheorized, and its role in shaping and being shaped by variation and change untested. One function of this chapter, therefore, is to assert that space makes a difference, and to begin, in a very hesitant way, to map out what a geographically informed variation analysis might need to address. It also examines variationist interactions with the related concept of mobility. It might be reasonable to think that human geographers would provide some clues on how to proceed. As we will see, they have engaged in a great deal of soul searching about the goals of their discipline, its very existence as a separate field of enquiry, and the directions it should take. Indeed there are remarkable parallels between the recent history of human geographic thought, and interest in language variation across space. Although space has been undertheorized in variation studies, a number of researchers, from the traditional dialectologists through to those interested in the dialectology of mobility and contact, have, of course, been actively engaged in research on geographical variation and language use. Their work will be contextualized here to highlight both the parallels with theory-building in human geography, but also some of the criticisms of earlier approaches which have fed through to human geography, but remain largely unquestioned in variationist practice. The chapter therefore presents a brief theoretical background to space and mobility, before exemplifying these concepts in variationist research through an examination of, for example, the spatial diffusion of linguistic innovations, the spatial configuration of linguistic boundaries and initial steps to examine the consequences of mobility for variationist research.

On new maximal supergravity and its BPS domain-walls

We revise the SU(3)-invariant sector of N  = 8 supergravity with dyonic SO(8) gaugings. By using the embedding tensor formalism, analytic expressions for the scalar potential, superpotential(s) and fermion mass terms are obtained as a function of the electromagnetic phase ω and the scalars in the theory. Equipped with these results, we explore non-supersymmetric AdS critical points at ω ≠ 0 for which perturbative stability could not be analysed before. The ω-dependent superpotential is then used to derive first-order flow equations and obtain new BPS domain-wall solutions at ω ≠ 0. We numerically look at steepest-descent paths motivated by the (conjectured) RG flows.

Triality, periodicity and stability of SO(8) gauged supergravity

While electromagnetic duality is a symmetry of many supergravity theories, this is not the case for the N = 8 gauged theory. It was recently shown that this rotation leads to a one-parameter family of SO(8) supergravities. It is an open question what the period of this parameter is. This issue is investigated in the SO(4) invariant sectors of the theory. We classify such critical points and find a novel branch of non-supersymmetric and unstable solutions, whose embedding is related via triality to the two known ones. Secondly, we show that the three branches of solutions lead to a π/4 periodicity of the vacuum structure. The general interrelations between triality and periodicity are discussed. Finally, we comment on the connection to other gauge groups as well as the possibility to achieve (non-)perturbative stability around AdS/Mkw/dS transitions.

From language maintenance to bilingual parenting: Negotiating behavior and language choice at the dinner table in binational-bilingual families

This article contributes to the study of language maintenance as an everyday activity in binational-bilingual families. By embedding the question of language maintenance into a language socialization framework and adopting a conversation-analytic approach to language alternation, three excerpts of mealtime interactions in Russian–French speaking families are analyzed. Their analysis shows that in bilingual families situations focusing on the interactional definition and negotiation of children’s behavior simultaneously involve the negotiation of language choice. It reveals how parents in binational-bilingual families accomplish bilingual (language) socialization in daily practice while dealing with the complex task of combining educational goals with language maintenance.

Next-generation tissue microarray (ngTMA) increases the quality of biomarker studies: an example using CD3, CD8, and CD45RO in the tumor microenvironment of six different solid tumor types

Background Tissue microarray (TMA) technology revolutionized the investigation of potential biomarkers from paraffin-embedded tissues. However, conventional TMA construction is laborious, time-consuming and imprecise. Next-generation tissue microarrays (ngTMA) combine histological expertise with digital pathology and automated tissue microarraying. The aim of this study was to test the feasibility of ngTMA for the investigation of biomarkers within the tumor microenvironment (tumor center and invasion front) of six tumor types, using CD3, CD8 and CD45RO as an example. Methods Ten cases each of malignant melanoma, lung, breast, gastric, prostate and colorectal cancers were reviewed. The most representative H&E slide was scanned and uploaded onto a digital slide management platform. Slides were viewed and seven TMA annotations of 1 mm in diameter were placed directly onto the digital slide. Different colors were used to identify the exact regions in normal tissue (n = 1), tumor center (n = 2), tumor front (n = 2), and tumor microenvironment at invasion front (n = 2) for subsequent punching. Donor blocks were loaded into an automated tissue microarrayer. Images of the donor block were superimposed with annotated digital slides. Exact annotated regions were punched out of each donor block and transferred into a TMA block. 420 tissue cores created two ngTMA blocks. H&E staining and immunohistochemistry for CD3, CD8 and CD45RO were performed. Results All 60 slides were scanned automatically (total time < 10 hours), uploaded and viewed. Annotation time was 1 hour. The 60 donor blocks were loaded into the tissue microarrayer, simultaneously. Alignment of donor block images and digital slides was possible in less than 2 minutes/case. Automated punching of tissue cores and transfer took 12 seconds/core. Total ngTMA construction time was 1.4 hours. Stains for H&E and CD3, CD8 and CD45RO highlighted the precision with which ngTMA could capture regions of tumor-stroma interaction of each cancer and the T-lymphocytic immune reaction within the tumor microenvironment. Conclusion Based on a manual selection criteria, ngTMA is able to precisely capture histological zones or cell types of interest in a precise and accurate way, aiding the pathological study of the tumor microenvironment. This approach would be advantageous for visualizing proteins, DNA, mRNA and microRNAs in specific cell types using in situ hybridization techniques.

Imported human rabies in Switzerland, 2012: a diagnostic conundrum

Human rabies is rare in Western Europe. It is not easily recognized in the absence of a history of exposure. We describe the clinical course, diagnosis and follow-up of an imported human rabies case in Switzerland. The patient, a U.S. citizen, presented at an outpatient clinic in Iraq with pain in his right shoulder on July 5, 2012. On July 8 he was transferred to a hospital in the United Arab Emirates, where he exhibited progressive encephalitis with coma. On July 29, he was transferred to a hospital in Switzerland, where he died on July 31, 2012. The autopsy showed severe encephalitis. Rabies was diagnosed by the rapid fluorescent focus inhibition test (RFFIT) and confirmed by fluorescence antibody testing (FAT) in brain smears and immunohistochemistry on paraffin-embedded brain sections. The viral strain was characterized by RT-PCR followed by sequencing and phylogenetic analysis as an American bat rabies strain associated with Tadarida brasiliensis. Close contacts and exposed health care workers received postexposure prophylaxis (PEP).

Jatropha mahafalensis oil from Madagascar: properties and suitability as liquid biofuel

Access to affordable and renewable sources of energy is crucial to reducing poverty and enhancing rural development in countries of the global South. Straight vegetable oil was recently identified as a possible alternative to conventional biomass for rural energy supply. In this context, the Jatropha curcas Linn. species has been extensively investigated with regard to its potential as a biofuel feedstock. In contrast, only little is known about Jatropha mahafalensis Jum. & H. Perrier, which is an indigenous and endemic representative of the Jatropha genus in Madagascar. This paper explores the potential and suitability of J. mahafalensis as a biofuel feedstock. Seed samples were collected in the area of Soalara in south-western Madagascar in February and September 2011. Two agro-ecological zones (coastal area and calcareous plateau) and two plant age groups (below and above 10 years) were considered. These four sample groups were analyzed with regard to oil properties, element contents, and fatty acid profiles. Measured values differed greatly between the two harvests, probably owing to different climatic or storage conditions. No direct relation between age of trees or location and oil quality could be established. The analyses indicate that J. mahafalensis oil can be used in oil lamps, cooking stoves and stationary combustion engines for electrification or for biodiesel production. However, modifications in storage and extraction methods, as well as further processing steps are necessary to enable its utilization as a straight vegetable oil and feedstock for biodiesel production. If these technical requirements can be met, and if it turns out that J. mahafalensis oil is economically competitive in comparison with firewood, charcoal, paraffin and petroleum, it can be considered as a promising feedstock for rural energy supply.