Toxicity assessment of single-walled carbon nanotubes

The objective of this study was to investigate the potential adverse health effects of occupational exposure to SWCNT dust using various in vitro approaches. Lung epithelium (represented by A549 and NHBE cells) was chosen as target cell model and exposed to minimally processed particle samples under submerse conditions and effects were compared to that of carbon black and crocidolite asbestos exposure. Cytotoxicity studies indicated low acute cytotoxicity of SWCNT. Morphological changes included decreases in microvilli and increased numbers of surfactant storing lamellar bodies and changes in cytoskeleton. Due to interactions of SWCNT and toxicity indicator dyes, the clonogenic assay was chosen as an alternative to quantify cytotoxicity and showed that long-term exposure lead to decreased cell proliferation. This effect was not only attributed to direct particle effects but also secondary SWCNT toxicity due to medium depletion. Intracellular reactive oxygen species (ROS) could only be detected in the absence of foetal bovine serum (FBS) supplements. An inflammatory mediator response was never observed. In contrast, suppression of lipid and inflammatory mediators was seen as characterised by the downregulation of interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) gene expression, inhibition of IL-8 and IL-6 promoter activation and decreased protein release of IL-8, IL-6 and macrophase chemoattractant protein-1 (MCP-1). The same response was found for cells immunologically induced by tumour-necrosis factor-a (TNF-a). Suppressive effects were partly due to TNF-a adsorption onto SWCNT surfaces. Dispersion of SWCNT in dipalmitoylphosphatidylcholine (DPPC) solution decreased particle agglomerate sizes and increased both cytotoxicity and oxidative potential

Molecular architecture of human polycomb repressive complex 2.

Polycomb Repressive Complex 2 (PRC2) is essential for gene silencing, establishing transcriptional repression of specific genes by tri-methylating Lysine 27 of histone H3, a process mediated by cofactors such as AEBP2. In spite of its biological importance, little is known about PRC2 architecture and subunit organization. Here, we present the first three-dimensional electron microscopy structure of the human PRC2 complex bound to its cofactor AEBP2. Using a novel internal protein tagging-method, in combination with isotopic chemical cross-linking and mass spectrometry, we have localized all the PRC2 subunits and their functional domains and generated a detailed map of interactions. The position and stabilization effect of AEBP2 suggests an allosteric role of this cofactor in regulating gene silencing. Regions in PRC2 that interact with modified histone tails are localized near the methyltransferase site, suggesting a molecular mechanism for the chromatin-based regulation of PRC2 activity.DOI:http://dx.doi.org/10.7554/eLife.00005.001

Computer simulations to optimize the design of marker-assisted backcrossing for high-throughput marker systems

Marker-assisted backcrossing (MABC) is to date the most successful application of DNA markers in plant breeding. Nevertheless, the large-scale implementation of genome-wide background selection with DNA markers has lagged behind expectations due to the high costs of marker analysis. It has been hypothesized that this problem will be overcome by high-throughput (HT) marker assays which enable genotyping a high number of marker loci at comparatively low cost per individual marker data point. The optimum backcross designs for HT assays have previously not been investigated. The objective of the study was therefore the development of novel selection strategies for the efficient use of HT assays in different applications of MABC. For this purpose, computer simulations were employed. The cost-efficiency of HT assays compared to SM assays was greatest in short, highly intense backcross programs, while it decreased with increasing marker fixation in advanced backcross generations. The optimum breeding designs for SM assays were characterized by increasing marker densities and population sizes. The optimum breeding designs for HT assays were in contrast characterized by few backcross generations, constant marker densities and constant or decreasing population sizes. Combining SM and HT assays at different stages of a backcross program reduced the cost of marker analysis compared to using only HT assays. Using HT assays with a combined selection index for foreground and background selection reduces the logistic efforts and allows more differentiated selection decisions. It can be concluded that HT assays have the potential to increase the relative efficiency of background selection for many applications of MABC.Markergestützte Rückkreuzung ist bislang die erfolgreichste Anwendung von DNA-Markern in der Pflanzenzüchtung. Dennoch ist die Anwendung der genomweiten Hintergrundselektion zur Wiederherstellung des Rezipientengenoms hinter den Erwartungen zurückgeblieben. Die Ursachen lagen in den hohen Kosten für die Vielzahl der benötigten Einzelmarkeranalysen begründet. Eine Lösung stellen Hochdurchsatzanalysen wie SNP-Chips dar, mit denen eine hohe Zahl von Markern zu vergleichsweise geringen Kosten pro Markerdatenpunkt genotypisiert werden kann. Die optimalen Selektionsstrategien für Hochdurchsatzmarkeranalysen wurden bislang noch nicht untersucht. Das Ziel der vorliegenden Arbeit war es daher, neue Strategien für den effizienten Einsatz von Hochdurchsatzmarkeranalysen in verschiedenen Anwendungen der markergestützten Rückkreuzung zu entwickeln. Zu diesem Zweck wurden Computersimulationen in verschiedenen Kulturarten durchgeführt. Die Kosteneffizienz der Hochdurchsatzsysteme war am größten in kurzen Rückkreuzungsprogrammen mit dem Ziel hohen Selektionsgewinns, nahm jedoch mit zunehmender Markerfixierung in fortgeschrittenen Rückkreuzungsgenerationen ab. Effiziente Zuchtschemata für Einzelmarkeranalysen waren durch ansteigende Markerdichten und Populationsgrößen gekennzeichnet. Im Gegensatz dazu waren optimale Zuchtschemata für Hochdurchsatzmarkeranalysen durch wenige Rückkreuzungsgenerationen, konstante Markerdichten und konstante oder abnehmende Populationsgrößen gekennzeichnet. Die Kombination von Einzelmarker- und Hochdurchsatzanalysen in verschiedenen Phasen eines Rückkreuzungsprogramms reduzierte die Kosten im Vergleich zur ausschließlichen Anwendung von Hochdurchsatzanalysen. Die Verwendung eines kombinierten Selektionsindexes für gleichzeitige Vorder- und Hintergrundselektion reduzierte en logistischen Aufwand und erlaubte differenzierte Selektionssentscheidungen. Hochdurchsatzmarkeranalysen können die Effizienz vieler Anwendungen der markergestützten Rückkreuzung im Vergleich zu Einzelmarkeranalysen erhöhen

Differential impact of dietary branched chain and aromatic amino acids on chronic kidney disease progression in rats

The metabolism of dietary proteins generates waste products that are excreted by the kidney, in particular nitrogen-containing urea, uric acid, ammonia, creatinine, and other metabolites such as phosphates, sulfates, and protons. Kidney adaptation includes an increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) and represents a burden for diseased kidneys increasing the progression rate of CKD. The present study aimed at identifying potential differences between amino acid (AA) groups constituting dietary proteins regarding their impact on RPF, GFR, and CKD progression. We utilized the well-established 5/6 nephrectomy (5/6 Nx) CKD model in rats and submitted the animals for 5 weeks to either the control diet (18% casein protein) or to diets containing 8% casein supplemented with 10% of a mix of free amino acids, representing all or only a subset of the amino acids contained in casein. Whereas the RPF and GFR measured in free moving animals remained stable during the course of the diet in rats receiving the control mix, these parameters decreased in animals receiving the branched chain amino acid (BCAA) supplementation and increased in the ones receiving the aromatic amino acids (AAAs). In animals receiving essential amino acids (EAAs) containing both BCAAs and AAAs, there was only a small increase in RPF. The kidneys of the 5/6 Nx rats receiving the BCAA diet showed the strongest increase in smooth muscle actin and collagen mRNA expression as a result of higher level of inflammation and fibrosis. These animals receiving BCAAs also showed an increase in plasma free fatty acids pointing to a problem at the level of energy metabolism. In contrast, the animals under AAA diet showed an activation of AMPK and STAT3. Taken together, our results demonstrate that subsets of EAAs contained in dietary proteins, specifically BCAAs and AAAs, exert contrasting effects on kidney functional parameters and CKD progression

High resolution tumor targeting in living mice by means of multispectral optoacoustic tomography

BACKGROUND: Tumor targeting is of high clinical and biological relevance, and major efforts have been made to develop molecular imaging technologies for visualization of the disease markers in tissue. Of particular interest is apoptosis which has a profound role within tumor development and has significant effect on cancer malignancy. METHODS: Herein, we report on targeting of phosphatidylserine-exposing cells within live tumor allograft models using a synthetic near infrared zinc(II)-dipicolylamine probe. Visualization of the probe biodistribution is performed with whole body multispectral optoacoustic tomography (MSOT) system and subsequently compared to results attained by planar and tomographic fluorescence imaging systems. RESULTS: Compared to whole body optical visualization methods, MSOT attains remarkably better imaging capacity by delivering high-resolution scans of both disease morphology and molecular function in real time. Enhanced resolution of MSOT clearly showed that the probe mainly localizes in the vessels surrounding the tumor, suggesting that its tumor selectivity is gained by targeting the phosphatidylserine exposed on the surface of tumor vessels. CONCLUSIONS: The current study demonstrates the high potential of MSOT to broadly impact the fields of tumor diagnostics and preclinical drug development

SWCNT Suppress Inflammatory Mediator Responses in Human Lung Epithelium in Vitro

Single walled carbon nanotubes have gained enormous popularity due to a variety of potential applications which will ultimately lead to increased human and environmental exposure to these nanoparticles. This study was carried out in order to evaluate the inflammatory response of immortalised and primary human lung epithelial cells (A549 and NHBE) to single walled carbon nanotube samples (SWCNT). Special focus was placed on the mediating role of lung surfactant on particle toxicity. The toxicity of SWCNT dispersed in cell culture medium was compared to that of nanotubes dispersed in dipalmitoylphosphatidylcholine (DPPC, the main component of lung lining fluid). Exposure was carried out for 6 to 48 hours with the latter time-point showing the most significant responses. Moreover, exposure was performed in the presence of the pro-inflammatory stimulus tumour necrosis factor-α (TNF-α) in order to mimic exposure of stimulated cells, as would occur during infection. Endpoints evaluated included cell viability, proliferation and the analysis of inflammatory mediators such as interleukin (IL)-8, IL-6, TNF-α and macrophage chemoattractant protein-1 (MCP-1). Crocidolite asbestos was included as a well characterised, toxic fibre control. The results of this study showed that HiPco SWCNT samples suppress inflammatory responses of A549 and NHBE cells. This was also true for TNF-α stimulated cells. The use of DPPC improved the degree of SWCNT dispersion in A549 medium and in turn, lead to increased particle toxicity, however, it was not shown to modify NHBE cell responses

Novel Mixed Reality Use Cases for Pilot Training

This study explored novel mixed reality (MR) use cases for pilot training using a mix of methods rooted in the general innovation theory of dynamic capabilities. The aim was to identify areas of improvement for various aspects of the flight training based on MR, in a socially and economically sustainable manner. Multiple surveys and workshops have been conducted with flight instructors, administrative staff, pilots and student pilots. The main result of this study is a systematic identification of the three most promising MR use cases: interactive theory training, cockpit procedure, and outside check training. These results are important because they inform the development of technical didactic tools for pilot training. The applicability of MR technologies to accommodate diverse user needs and preferences is addressed, while also considering aspects of economical sustainability

Biodistribution of the recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in rats

AbstractIntroductionThe recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) is undergoing clinical trials for prophylaxis and on-demand treatment of haemophilia B patients. The aim of this study was to investigate the pharmacokinetics, whole-body and knee joint distribution of rIX-FP following intravenous administration to rats, compared with a marketed, non-fused rFIX and recombinant human albumin.Material and Methods[3H]-rIX-FP, [3H]-rFIX or [3H]-albumin were administered to rats followed by quantitative whole-body autoradiography over 24 or 240hours, and the tissue distribution as well as elimination of radioactivity were measured.ResultsElimination of all radioactivity derived from the three proteins was shown to occur primarily via the urine. The tissue distribution of [3H]-rIX-FP and [3H]-rFIX (but not of [3H]-albumin) was comparable, both penetrating predominantly into bone, and well-perfused tissues, suggesting that the rIX moiety determines the distribution pattern of rIX-FP, while the albumin moity is responsible for the prolonged plasma and tissue retention. Detailed knee-joint analysis indicated rapid presence of [3H]-rIX-FP and [3H]-rFIX in synovial and mineralised bone tissue, mostly localised to the zone of calcified cartilage. Longest retention times were observed in the bone marrow and the endosteum of long bones. Intriguingly, [3H]-rIX-FP- and [3H]-albumin-derived radioactive signals were detectable up to 240hours, while [3H]-rFIX-derived radioactivity rapidly declined after 1hour post-dosing correlating to the extended plasma half-life of [3H]-rIX-FP.ConclusionThe prolonged plasma and tissue retention of rIX-FP achieved by albumin fusion may allow a reduction in dosing frequency leading to increased therapeutic compliance and convenience

Multispectral Optoacoustic Tomography of Matrix Metalloproteinase Activity in Vulnerable Human Carotid Plaques

Elevated expression of cathepsins, integrins and matrix metalloproteinases (MMPs) is typically associated with atherosclerotic plaque instability. While fluorescent tagging of such molecules has been amply demonstrated, no imaging method was so far shown capable of resolving these inflammation-associated tags with high fidelity and resolution beyond microscopic depths. This study is aimed at demonstrating a new method with high potential for noninvasive clinical cardiovascular diagnostics of vulnerable plaques using high-resolution deep-tissue multispectral optoacoustic tomography (MSOT) technology. MMP-sensitive activatable fluorescent probe (MMPSense (TM) 680) was applied to human carotid plaques from symptomatic patients. Atherosclerotic activity was detected by tuning MSOT wavelengths to activation-dependent absorption changes of the molecules, structurally modified in the presence of enzymes. MSOT analysis simultaneously provided morphology along with heterogeneous MMP activity with better than 200 micron resolution throughout the intact plaque tissue. The results corresponded well with epi-fluorescence images made from thin cryosections. Elevated MMP activity was further confirmed by zymography, accompanied by increased macrophage influx. We demonstrated, for the first time to our knowledge, the ability of MSOT to provide volumetric images of activatable molecular probe distribution deep within optically diffuse tissues. High-resolution mapping of MMP activity was achieved deep in the vulnerable plaque of intact human carotid specimens. This performance directly relates to pre-clinical screening applications in animal models and to clinical decision potential as it might eventually allow for highly specific visualization and staging of plaque vulnerability thus impacting therapeutic clinical decision making

Stacked ensembles on basis of parentage information can predict hybrid performance with an accuracy comparable to marker-based GBLUP

Testcross factorials in newly established hybrid breeding programs are often highly unbalanced, incomplete, and characterized by predominance of special combining ability (SCA) over general combining ability (GCA). This results in a low efficiency of GCA-based selection. Machine learning algorithms might improve prediction of hybrid performance in such testcross factorials, as they have been successfully applied to find complex underlying patterns in sparse data. Our objective was to compare the prediction accuracy of machine learning algorithms to that of GCA-based prediction and genomic best linear unbiased prediction (GBLUP) in six unbalanced incomplete factorials from hybrid breeding programs of rapeseed, wheat, and corn. We investigated a range of machine learning algorithms with three different types of predictor variables: (a) information on parentage of hybrids, (b) in addition hybrid performance of crosses of the parental lines with other crossing partners, and (c) genotypic marker data. In two highly incomplete and unbalanced factorials from rapeseed, in which the SCA variance contributed considerably to the genetic variance, stacked ensembles of gradient boosting machines based on parentage information outperformed GCA prediction. The stacked ensembles increased prediction accuracy from 0.39 to 0.45, and from 0.48 to 0.54 compared to GCA prediction. The prediction accuracy reached by stacked ensembles without marker data reached values comparable to those of GBLUP that requires marker data. We conclude that hybrid prediction with stacked ensembles of gradient boosting machines based on parentage information is a promising approach that is worth further investigations with other data sets in which SCA variance is high