Site specific insertion of a transgene into the murine α-casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk.

Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. The α-casein (CSN1S1) gene has assessed as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA, and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.

Anatomically Integrated In-Place Visualization of Patient Data for Cooperative Tasks with a Case Study on a Neurosurgical Ward.

The workflow in modern hospitals entails that the medical treatment of a patient is distributed between several physicians and nurses. This leads to intensive cooperation, which takes place under particular time pressure and requires efficient conveyance of relevant patient-related medical data to colleagues. This requirement is difficult to achieve with traditional data representation approaches. In this paper, we introduce a novel concept of anatomically integrated in-place visualization designed to engage with cooperative tasks on a neurosurgical ward by using a virtual patient's body as spatial representation of visually encoded abstract medical data. Based on the findings of our field studies, we provide a set of formal requirements and procedures for this kind of visual encoding. Moreover, we implemented a prototype on a mobile device that supports the diagnosis of spinal disc herniation and has been evaluated by 10 neurosurgeons. The physicians have assessed the proposed concept as beneficial, especially emphasizing the advantages of the anatomical integration such as intuitiveness and a better data availability due to providing all information at a glance. Particularly, four of nine respondents have stressed solely benefits of the concept, other four have mentioned benefits with some limitations and only one person has seen no benefits.

Perception and Quantization Model for Periodic Contour Modifications.

Periodic, wave-like modifications of 2D shape contours are often applied to convey quantitative data via images. However, to the best of our knowledge, there has been no in-depth investigation of the perceptual uniformity and legibility of these kind of approaches. In this paper, we design and perform a user study to evaluate the perception of periodic contour modifications related to their geometry and colour. Based on the study results, we statistically derive a perceptual model, which demonstrates a mainly linear stimulus-to-perception relationship for geometric and colour amplitude and a close-to-quadratic relationship for the respective frequencies, with a rather negligible dependency on the waveform. Furthermore, analyzing the distribution of perceived magnitudes and the overlapping of the respective 50% confidence intervals, we extract distinguishable, visually equidistant quantization levels for each contour-related visual variable. Moreover, we give first insights into the perceptual dependency between amplitude and frequency, and propose a scheme for transferring our model to glyphs with different size, which preserves the distinguishability and the visual equidistance. This work is seen as a first step towards a comprehensive understanding of the perception of periodic contour modifications in image-based visualizations.

A Generic Framework for Depth Reconstruction Enhancement.

We propose a generic depth-refinement scheme based on GeoNet, a recent deep-learning approach for predicting depth and normals from a single color image, and extend it to be applied to any depth reconstruction task such as super resolution, denoising and deblurring, as long as the task includes a depth output. Our approach utilizes a tight coupling of the inherent geometric relationship between depth and normal maps to guide a neural network. In contrast to GeoNet, we do not utilize the original input information to the backbone reconstruction task, which leads to a generic application of our network structure. Our approach first learns a high-quality normal map from the depth image generated by the backbone method and then uses this normal map to refine the initial depth image jointly with the learned normal map. This is motivated by the fact that it is hard for neural networks to learn direct mapping between depth and normal maps without explicit geometric constraints. We show the efficiency of our method on the exemplary inverse depth-image reconstruction tasks of denoising, super resolution and removal of motion blur.

A Generative Model for Generic Light Field Reconstruction.

Recently deep generative models have achieved impressive progress in modeling the distribution of training data. In this work, we present for the first time a generative model for 4D light field patches using variational autoencoders to capture the data distribution of light field patches. We develop a generative model conditioned on the central view of the light field and incorporate this as a prior in an energy minimization framework to address diverse light field reconstruction tasks. While pure learning-based approaches do achieve excellent results on each instance of such a problem, their applicability is limited to the specific observation model they have been trained on. On the contrary, our trained light field generative model can be incorporated as a prior into any model-based optimization approach and therefore extend to diverse reconstruction tasks including light field view synthesis, spatial-angular super resolution and reconstruction from coded projections. Our proposed method demonstrates good reconstruction, with performance approaching end-to-end trained networks, while outperforming traditional model-based approaches on both synthetic and real scenes. Furthermore, we show that our approach enables reliable light field recovery despite distortions in the input.

A cell-based assay system for activators of the environmental cell stress response.

Improved health span and lifespan extension in a wide phylogenetic range of species is associated with the induction of the environmental cell stress response through a signalling pathway regulated by the transcription factor Nrf2. Phytochemicals which stimulate this response may form part of therapeutic interventions which stimulate endogenous cytoprotective mechanisms, thereby delaying the onset of age-related diseases and promoting healthy ageing in humans. In order to identify compounds that activate the Nrf2 pathway, a cell-based reporter system was established in HepG2 cells using a luciferase reporter gene under the control of the Nqo1 promoter. Sulforaphane, an isothiocyanate derived from cruciferous vegetables and a known activator of the Nrf2 pathway, was used to validate the reporter system. The transfected cell line HepG2 C1 was subsequently used to screen natural product libraries. Five compounds were identified as activating the bioluminescent reporter by greater than 5-fold. The two most potent compounds, MBC20 and MBC37, were further characterised and shown to stimulate endogenous cytoprotective gene and protein expression. The bioluminescent reporter system will allow rapid, in vitro identification of novel compounds that have the potential to improve health span through activation of the environmental stress response.

Folate deficiency promotes differentiation of vascular smooth muscle cells without affecting the methylation status of regulated genes.

Elevated serum homocysteine, an intermediate of cellular one-carbon metabolism, is an independent risk factor for cardiovascular disease (CVD). Folate deficiency increases serum homocysteine and may contribute to CVD progression. Vascular smooth muscle cells (VSMCs) regulate vascular contractility, but also contribute to repair processes in response to vascular injury. Nutritional deficiencies, like folate deficiency, are thought to impact on this phenotypic plasticity, possibly by epigenetic mechanisms. We have investigated the effect of folate deficiency on VSMCs in two cell culture systems representing early and late stages of smooth muscle cells differentiation. We find that folate deficiency promotes differentiation towards a more contractile phenotype as indicated by increased expression of respective marker genes. However, microarray analysis identified markers of striated muscle as the predominant gene expression change elicited by folate deficiency. These changes are not merely a reflection of cell cycle arrest, as foetal calf serum restriction or iron deficiency do not replicate the gene expression changes observed in response to folate deficiency. Folate deficiency only has a marginal effect on global DNA methylation. DNA methylation of CpG islands associated with genes regulated by folate deficiency remains unaffected. This supports our earlier findings in a mouse model system which also did not show any changes in global DNA methylation in response to folate and vitamin B6/B12 deficiency. These data suggest that folate deficiency enhances the expression of smooth muscle marker gene expression, promotes a shift towards a skeletal muscle phenotype, and does not regulate gene expression via DNA methylation.

Pulse Based Time-of-Flight Range Sensing.

Pulse-based Time-of-Flight (PB-ToF) cameras are an attractive alternative range imaging approach, compared to the widely commercialized Amplitude Modulated Continuous-Wave Time-of-Flight (AMCW-ToF) approach. This paper presents an in-depth evaluation of a PB-ToF camera prototype based on the Hamamatsu area sensor S11963-01CR. We evaluate different ToF-related effects, i.e., temperature drift, systematic error, depth inhomogeneity, multi-path effects, and motion artefacts. Furthermore, we evaluate the systematic error of the system in more detail, and introduce novel concepts to improve the quality of range measurements by modifying the mode of operation of the PB-ToF camera. Finally, we describe the means of measuring the gate response of the PB-ToF sensor and using this information for PB-ToF sensor simulation.

Quantified, Interactive Simulation of AMCW ToF Camera Including Multipath Effects.

In the last decade, Time-of-Flight (ToF) range cameras have gained increasing popularity in robotics, automotive industry, and home entertainment. Despite technological developments, ToF cameras still suffer from error sources such as multipath interference or motion artifacts. Thus, simulation of ToF cameras, including these artifacts, is important to improve camera and algorithm development. This paper presents a physically-based, interactive simulation technique for amplitude modulated continuous wave (AMCW) ToF cameras, which, among other error sources, includes single bounce indirect multipath interference based on an enhanced image-space approach. The simulation accounts for physical units down to the charge level accumulated in sensor pixels. Furthermore, we present the first quantified comparison for ToF camera simulators. We present bidirectional reference distribution function (BRDF) measurements for selected, purchasable materials in the near-infrared (NIR) range, craft real and synthetic scenes out of these materials and quantitatively compare the range sensor data.

Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors.

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

Using the CRISPR/Cas9 system to understand neuropeptide biology and regulation.

Neuropeptides and their receptors play a role in physiological responses such as appetite, stress and inflammatory pain. With neuropeptides having such diverse and important physiological roles, knocking-out the genes encoding them, their receptors, parts of their regulatory sequences, or reproducing disease associated polymorphic variants are important steps in studying neuropeptides and how they may contribute to disease. Previously, knock-outs were generated using methods such as targeted homologous recombination in embryonic stem cells but this method is costly and time-consuming. The CRISPR/Cas9 system has rapidly taken over the genome editing field and will advance our understanding of neuropeptide genes and their regulation. With CRISPR/Cas9 technology, the time and costs involved in producing transgenic animal models, is greatly reduced. In this review, we describe how the system can be used to manipulate genomic sequences by "knock-out" or "knock-in" mutations in cell lines or in animal models. We also discuss the specificity of the system and methods to limit off-target effects. When combined with the availability of genome sequences, CRISPR/Cas9 directed genome editing in vitro and in vivo, promises to provide a deeper understanding of the biology of the neuropeptides in health and disease than has ever been available before.

Comprehensive Use of Curvature for Robust and Accurate Online Surface Reconstruction.

Interactive real-time scene acquisition from hand-held depth cameras has recently developed much momentum, enabling applications in ad-hoc object acquisition, augmented reality and other fields. A key challenge to online reconstruction remains error accumulation in the reconstructed camera trajectory, due to drift-inducing instabilities in the range scan alignments of the underlying iterative-closest-point (ICP) algorithm. Various strategies have been proposed to mitigate that drift, including SIFT-based pre-alignment, color-based weighting of ICP pairs, stronger weighting of edge features, and so on. In our work, we focus on surface curvature as a feature that is detectable on range scans alone and hence does not depend on accurate multi-sensor alignment. In contrast to previous work that took curvature into consideration, however, we treat curvature as an independent quantity that we consistently incorporate into every stage of the real-time reconstruction pipeline, including densely curvature-weighted ICP, range image fusion, local surface reconstruction, and rendering. Using multiple benchmark sequences, and in direct comparison to other state-of-the-art online acquisition systems, we show that our approach significantly reduces drift, both when analyzing individual pipeline stages in isolation, as well as seen across the online reconstruction pipeline as a whole.

CD13/aminopeptidase N is a negative regulator of mast cell activation.

Antigen-induced mast cell (MC) activation via cross-linking of IgE-bound high-affinity receptors for IgE (FcεRI) underlies type I allergy and anaphylactic shock. Comprehensive knowledge of FcεRI regulation is thus required. We have identified a functional interaction between FcεRI and CD13 in murine MCs. Antigen-triggered activation of IgE-loaded FcεRI results in cocapping and cointernalization of CD13 and equivalent internalization rates of up to 40%. Cointernalization is not unspecific, because ligand-driven KIT internalization is not accompanied by CD13 internalization. Moreover, antibody-mediated cross-linking of CD13 causes IL-6 production in an FcεRI-dependent manner. These data are indicative of a functional interaction between FcεRI and CD13 on MCs. To determine the role of this interaction, CD13-deficient bone marrow-derived MCs (BMMCs) were analyzed. Intriguingly, antigen stimulation of CD13-deficient BMMCs results in significantly increased degranulation and proinflammatory cytokine production compared to wild-type cells. Furthermore, in a low-dose model of passive systemic anaphylaxis, antigen-dependent decrease in body temperature, reflecting the anaphylactic reaction, is substantially enhanced by the CD13 inhibitor bestatin (-5.9 ± 0.6°C) and by CD13 deficiency (-8.8 ± 0.6°C) in contrast to controls (-1.2 ± 1.97°C). Importantly, bestatin does not aggravate anaphylaxis in CD13-deficient mice. Thus, we have identified CD13 as a novel negative regulator of MC activation in vitro and in vivo-Zotz, J. S., Wölbing, F., Lassnig, C., Kauffmann, M., Schulte, U., Kolb, A., Whitelaw, B., Müller, M., Biedermann, T., Huber, M. CD13/aminopeptidase N is a negative regulator of mast cell activation.

Imaging in scattering media using correlation image sensors and sparse convolutional coding.

Correlation image sensors have recently become popular low-cost devices for time-of-flight, or range cameras. They usually operate under the assumption of a single light path contributing to each pixel. We show that a more thorough analysis of the sensor data from correlation sensors can be used can be used to analyze the light transport in much more complex environments, including applications for imaging through scattering and turbid media. The key of our method is a new convolutional sparse coding approach for recovering transient (light-in-flight) images from correlation image sensors. This approach is enabled by an analysis of sparsity in complex transient images, and the derivation of a new physically-motivated model for transient images with drastically improved sparsity.

Rhodium-catalyzed enantioselective addition of organoaluminum reagents to N-tosyl ketimines.

Rhodium(I)/Binap complexes catalyze highly enantioselective additions of methyl- and arylaluminum reagents to cyclic α,ß-unsaturated N-tosyl ketimines. Depending on the solvent and substituents at the ring, the reaction occurs either in a 1,2-manner to deliver α-tertiary allylic amines or in a 1,4-manner to yield, after subsequent reduction, 3-substituted cycloalkyl amines. Well known in the case of the respective cycloalkenones, these first transformations of the aza-analogues enable the synthesis of amine structures of pharmaceutical and biochemical interest.

Improved synthesis of cyclic tertiary allylic alcohols by asymmetric 1,2-addition of AlMe3 to enones.

The development of an improved protocol for the enantioselective Rh(I) /binap-catalysed 1,2-addition of AlMe3 to cyclic enones is reported. (31)P NMR analysis of the reaction revealed that the catalyst in its resting state is a chloride-bridged dimer. This insight led to the use of AgBF4 as an additive for in situ activation of the dimeric precatalyst. Thus, the catalyst loading can now be reduced to only 1 mol% with respect to rhodium. Various 5-7-membered cyclic enones can be transformed into tertiary allylic alcohols with excellent levels of enantioselectivity and high yields. The obtained products are versatile synthetic building blocks, shown by a highly enantioselective formal total synthesis of the pheromone (-)-frontalin as well as formation of a bicyclic lactone that has the core structure of the natural flavour component "wine lactone".

Folate deficiency enhances the inflammatory response of macrophages.

B-vitamin deficiency is a risk factor for vascular disease. The mechanism by which the deficiency impacts on disease risk is unclear. We have analysed whether the inflammatory response of mononuclear cells can be modified by cellular folate status in vitro. We show that the mouse monocyte cell line RAW264.7 grown under folate restriction displays a decrease in intracellular folate levels and a reduced growth rate. The cells also show a 2- to 3-fold increase in expression of the inflammatory mediators, IL1ß, IL6, TNFα and MCP1 at the RNA and protein level (p<0.01) under conditions of folate deficiency. In contrast the production of the vaso-protective mediator nitric oxide is significantly reduced under these conditions. These metabolic changes are independent of the concentration of homocysteine in the medium and occur in the absence of significant changes in global DNA methylation. Folate deficiency may therefore exacerbate cardiovascular disease by augmenting pro-inflammatory signals in the monocyte-macrophage lineage.

Behaviour of postnatally growth-impaired mice during malnutrition and after partial weight recovery.

OBJECTIVES: Early malnutrition is a highly prevalent condition in developing countries. Different rodent models of postnatal early malnutrition have been used to approach the subject experimentally, inducing early malnutrition by maternal malnutrition, temporal maternal separation, manipulation of litter size or the surgical nipple ligation to impair lactation. Studies on the behaviour of (previously) malnourished animals using animal models have produced sometimes contradictory results regarding the effects of early postnatal malnutrition and have been criticized for introducing potential confounding factors. The present paper is a first report on the behavioural effects of early malnutrition induced by an alternative approach: mice nursed by α-casein-deficient knockout dams showed a severe growth delay during early development and substantial catch-up growth after weaning when compared with animals nursed by wild-type females. METHODS: Established behavioural tests were used to study the consequences of early postnatal malnutrition on mouse pups at weaning and after partial weight recovery. RESULTS: Despite the impaired growth, the only behavioural difference between malnourished and normally growing animals was found in exploratory behaviour during acute malnutrition at the time of weaning. After partial catch-up in weight early protein malnourished animals showed no indication of lasting effects on general activity, emotionality and exploration, memory, and pain reactivity. DISCUSSION: These results suggest that the role of early nutrition on behavioural development after recovery in animal models may have been overestimated. Further careful examination of this animal model in terms of maternal care and offspring behaviour will be necessary to confirm if mice nursed by α-casein-deficient dams offer an alternative to existing models while eliminating potential confounding factors.

Segmentation of pituitary adenoma: a graph-based method vs. a balloon inflation method.

Among all abnormal growths inside the skull, the percentage of tumors in sellar region is approximately 10-15%, and the pituitary adenoma is the most common sellar lesion. A time-consuming process that can be shortened by using adequate algorithms is the manual segmentation of pituitary adenomas. In this contribution, two methods for pituitary adenoma segmentation in the human brain are presented and compared using magnetic resonance imaging (MRI) patient data from the clinical routine: Method A is a graph-based method that sets up a directed and weighted graph and performs a min-cut for optimal segmentation results: Method B is a balloon inflation method that uses balloon inflation forces to detect the pituitary adenoma boundaries. The ground truth of the pituitary adenoma boundaries - for the evaluation of the methods - are manually extracted by neurosurgeons. Comparison is done using the Dice Similarity Coefficient (DSC), a measure for spatial overlap of different segmentation results. The average DSC for all data sets is 77.5±4.5% for the graph-based method and 75.9±7.2% for the balloon inflation method showing no significant difference. The overall segmentation time of the implemented approaches was less than 4s - compared with a manual segmentation that took, on the average, 3.9±0.5min.

Influence of rubber dam on objective and subjective parameters of stress during dental treatment of children and adolescents - a randomized controlled clinical pilot study.

BACKGROUND: Rubber dam is recommended for isolating the working field during adhesive dentistry procedures; however, dentists often omit rubber dam, particularly in paediatric dentistry, supposing that it would stress the patient. AIM: The aim of this study was to evaluate stress parameters during a standardized dental treatment procedure performed with or without rubber dam. The treatment time was measured as a secondary outcome variable. DESIGN: This study was designed as a randomized, controlled, clinical study with 72 patients (6-16 years; mean age, 11.1). During standardized fissure sealing procedures, objective parameters of stress (e.g., skin resistance, breath rate) were recorded. The operator's stress level was measured by pulse rate. Subjective pain (patients) and stress perception (operator) were evaluated by an interview. RESULTS: The breath rate was significantly (P<0.05) lower and the skin resistance level was significantly higher during treatment with rubber dam compared to the control group. Subjective pain perception was significantly lower for the test group. The treatment time needed for the fissure sealing procedure was 12.4% less in the test group. CONCLUSION: Isolation with rubber dam caused less stress in children and adolescents compared to relative isolation with cotton rolls if applied by an experienced dentist.