A soft matter computer for soft robots.

Despite the growing interest in soft robotics, little attention has been paid to the development of soft matter computational mechanisms. Embedding computation directly into soft materials is not only necessary for the next generation of fully soft robots but also for smart materials to move beyond stimulus-response relationships and toward the intelligent behaviors seen in biological systems. This article describes soft matter computers (SMCs), low-cost, and easily fabricated computational mechanisms for soft robots. The building block of an SMC is a conductive fluid receptor (CFR), which maps a fluidic input signal to an electrical output signal via electrodes embedded into a soft tube. SMCs could perform both analog and digital computation. The potential of SMCs is demonstrated by integrating them into three soft robots: (i) a Softworm robot was controlled by an SMC that generated the control signals necessary for three distinct gaits; (ii) a soft gripper was given a set of reflexes that could be programmed by adjusting the parameters of the CFR; and (iii) a two-degree of freedom bending actuator was switched between three distinct behaviors by varying only one input parameter. SMCs are a low-cost way to integrate computation directly into soft materials and an important step toward entirely soft autonomous robots.

Laparoscopic versus open appendectomy for complicated appendicitis in high risk patients.

INTRODUCTION: Laparoscopic appendectomy is widely used for the treatment of complicated appendicitis. Its use in patients with high operative risk is still on debate. The aim of the presented study was to investigate the benefits of laparoscopic appendectomy in patients with high peri- and postoperative risk factors. METHODS: We performed a retrospective analysis of all patients who underwent appendectomy in our center between 2006 and 2013. Patients were classified according to their preoperative risk (classification of the American Society of Anesthesia--ASA score). Only patients with ASA 3 and 4 were included and were divided into two groups--open appendectomy (OA group) and laparoscopic appendectomy (LA group). RESULTS: The operation time was slightly longer in the LA group (p = 0.05), but hospital stay was shorter (p = 0.05). Complications graded according to the Clavien Dindo classification were slightly more frequent in patients after LA, whereas severe complications occurred more frequently in patients after OA (p = 0.01). The postoperative WBC decreased steadily and significantly in patients after OA, whereas the decrease in patients after LA was delayed (p = 0.03). CRP slightly increased after OA and decreased thereafter, whereas it steadily decreased after LA (p = 0.05). CONCLUSION: Laparoscopic appendectomy can be recommended for patients with complicated appendicitis even with higher risk categories.

Exploiting short-term memory in soft body dynamics as a computational resource.

Soft materials are not only highly deformable, but they also possess rich and diverse body dynamics. Soft body dynamics exhibit a variety of properties, including nonlinearity, elasticity and potentially infinitely many degrees of freedom. Here, we demonstrate that such soft body dynamics can be employed to conduct certain types of computation. Using body dynamics generated from a soft silicone arm, we show that they can be exploited to emulate functions that require memory and to embed robust closed-loop control into the arm. Our results suggest that soft body dynamics have a short-term memory and can serve as a computational resource. This finding paves the way towards exploiting passive body dynamics for control of a large class of underactuated systems.

Representative Factor Generation for the Interactive Visual Analysis of High-Dimensional Data.

Datasets with a large number of dimensions per data item (hundreds or more) are challenging both for computational and visual analysis. Moreover, these dimensions have different characteristics and relations that result in sub-groups and/or hierarchies over the set of dimensions. Such structures lead to heterogeneity within the dimensions. Although the consideration of these structures is crucial for the analysis, most of the available analysis methods discard the heterogeneous relations among the dimensions. In this paper, we introduce the construction and utilization of representative factors for the interactive visual analysis of structures in high-dimensional datasets. First, we present a selection of methods to investigate the sub-groups in the dimension set and associate representative factors with those groups of dimensions. Second, we introduce how these factors are included in the interactive visual analysis cycle together with the original dimensions. We then provide the steps of an analytical procedure that iteratively analyzes the datasets through the use of representative factors. We discuss how our methods improve the reliability and interpretability of the analysis process by enabling more informed selections of computational tools. Finally, we demonstrate our techniques on the analysis of brain imaging study results that are performed over a large group of subjects.

Genetic aspects of cell line development from a synthetic biology perspective.

Animal cells can be regarded as factories for the production of relevant proteins. The advances described in this chapter towards the development of cell lines with higher productivity capacities, certain metabolic and proliferation properties, reduced apoptosis and other features must be regarded in an integrative perspective. The systematic application of systems biology approaches in combination with a synthetic arsenal for targeted modification of endogenous networks are proposed to lead towards the achievement of a predictable and technologically advanced cell system with high biotechnological impact.

Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells.

Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreER(T2), and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreER(T2). The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines.

The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.

A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration.

The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN gamma-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinase-mediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN gamma-retroviral vectors for clinical trials.

Toxin-antitoxin based transgene expression in mammalian cells.

Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs.

GARP: a key receptor controlling FOXP3 in human regulatory T cells.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

[CT coronary angiogram: state of the art in 2009]. / Coronarographie par CT-scan en 2009.

Thanks to 64 multi-detector CT-scan it is nowadays possible to visualise non invasively the coronary arteries: the recently published series have shown excellent sensitivity and specificity in the detection of coronary artery disease. Actually, the principal interest of the technique is the excellent negative predictive value which is very useful to rule out a significant coronary artery stenosis. For the time being, the angio CT is recommended for symptomatic patients with low to intermediate probability of coronary artery disease with inconclusive functional test. Despite some technical ameliorations, the irradiation doses delivered by multi-detector CT are significant and should restrict its indications specifically in women and young patients, in whom a late radio-induced cancer may be a concern.

The genome of the simian and human malaria parasite Plasmodium knowlesi.

Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors.

We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange.

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.

[Diagnostic imaging of the chest in intensive care patients]. / Bildgebende Diagnostik des Thorax bei Intensivpatienten.

Medical progress in intensive care has led to a modification of management in diagnostic imaging. All radiological imaging methods are available for intensive care patients. Thus, it is necessary to correlate the risk of patient transport with diagnostic and therapeutic benefit. This article describes the currently available imaging methods for the chest, and radiological findings of the most frequent pathologies.