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The feasibility of substitute building materials (SBMs) in engineering applications was investigated within the project. A geogrid-reinforced soil structure (GRSS) was built using SBM as the fill material as well as vegetated soil for facing and on top of the construction. Four different SBMs were used as fill material, namely blast furnace slag (BFS), electric furnace slag (EFS), track ballast (TB), and recycled concrete (RC). For the vegetated soil facing, a mixture of either recycled brick (RB) material or crushed lightweight concrete (LC) mixed with organic soil was used. The soil mechanical and chemical parameters for all materials were determined and assessed. In the next step, a GRSS was built as a pilot application consisting of three geogrid layers with a total height of 1.5 m and a slope angle of 60°. The results of the soil mechanical tests indicate that the used fill materials are similar or even better than primary materials, such as gravel. The results of the chemical tests show that some materials are qualified to be used in engineering constructions without or with minor restrictions. Other materials need a special sealing layer to prevent the material from leakage. The vegetation on the mixed SBM material grew successfully. Several ruderal and pioneer plants could be found even in the first year of the construction. The porous material (RB and LC) provide additional water storage capacity for plants especially during summer and/or heat periods. With regard to the results of the chemical analyses of the greening layers, they are usable under restricted conditions. Here special treatment is necessary. Finally, it can be stated that SBMs are feasible in GRSS, particularly as fill material but also as a mixture for the greenable soil. more...

IntroductionHuman induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are being evaluated for their use in pharmacological and toxicological testing, particularly for electrophysiological side effects. However, little is known about the composition of the commercially available iCell cardiomyocyte (Fuijifilm Cellular Dynamics) cultures and the transcriptomic phenotype of individual cells.MethodsWe characterized iCell cardiomyocytes (assumed to be a mixture of nodal-, atrial-, and ventricular-like cardiomyocytes together with potential residual non-myocytes) using bulk RNA-sequencing, followed by investigation of cellular heterogeneity using two different single-cell RNA-sequencing platforms.ResultsBulk RNA-sequencing identified key cardiac markers (TNNT2, MYL7) as well as fibroblast associated genes (P4HB, VIM), and cardiac ion channels in the iCell cardiomyocyte culture. High-resolution single cell RNA-sequencing demonstrated that both, cardiac and fibroblast-related genes were co-expressed throughout the cell population. This approach resolved two cell clusters within iCell cardiomyocytes. Interestingly, these clusters could not be associated with known cardiac subtypes. However, transcripts of ion channels potentially useful as functional markers for cardiac subtypes were below the detection limits of the single-cell approaches used. Instead, one cluster (10.8% of the cells) is defined by co-expression of cardiac and cell cycle-related genes (e.g. TOP2A). Incorporation of bromodeoxyuridine further confirmed the capability of iCell cardiomyocytes to enter cell cycle.DiscussionThe co-expression of cardiac related genes with cell cycle or fibroblast related genes may be interpreted either as aberrant or as an immature feature. However, this excludes the presence of a non-cardiomyocyte sub-population and indicates that some cardiomyocytes themselves enter cell cycle. published more...

Combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has recently become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomic changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard – fresh cells – to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single-cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies. more...

Objective Insulin release from pancreatic islet β cells should be tightly controlled to avoid hypoglycemia and insulin resistance. The cortical actin cytoskeleton is a gate for regulated exocytosis of insulin secretory granules (SGs) by restricting their mobility and access to the plasma membrane. Prior studies suggest that SGs interact with F-actin through their transmembrane cargo islet cell autoantigen 512 (Ica512) (also known as islet antigen 2/Ptprn). Here we investigated how Ica512 modulates SG trafficking and exocytosis. Methods Transcriptomic changes in Ica512−/− mouse islets were analyzed. Imaging as well as biophysical and biochemical methods were used to validate if and how the Ica512-regulated gene villin modulates insulin secretion in mouse islets and insulinoma cells. Results The F-actin modifier villin was consistently downregulated in Ica512−/− mouse islets and in Ica512-depleted insulinoma cells. Villin was enriched at the cell cortex of β cells and dispersed villin−/− islet cells were less round and less deformable. Basal mobility of SGs in villin-depleted cells was enhanced. Moreover, in cells depleted either of villin or Ica512 F-actin cages restraining cortical SGs were enlarged, basal secretion was increased while glucose-stimulated insulin release was blunted. The latter changes were reverted by overexpressing villin in Ica512-depleted cells, but not vice versa. Conclusion Our findings show that villin controls the size of the F-actin cages restricting SGs and, thus, regulates their dynamics and availability for exocytosis. Evidence that villin acts downstream of Ica512 also indicates that SGs directly influence the remodeling properties of the cortical actin cytoskeleton for tight control of insulin secretion., Highlights • Ica512-depletion reduces the genetic expression of the F-actin modifier villin. • Villin-depletion enhances basal insulin granule mobility and exocytosis. • Villin regulates the size of actin cages restraining insulin granules. • Villin acts downstream of insulin granule cargo Ica512. • The Ica512-villin genetic link enables granules to control cytoskeleton plasticity. more...

Gene functionality is closely connected to its expression specificity across tissues and cell types. RNA-Seq is a powerful quantitative tool to explore genome wide expression. The aim of this study is to provide a comprehensive RNA-Seq dataset across the same 13 tissues for mouse and rat, two of the most relevant species for biomedical research. The dataset provides the transcriptome across tissues from three male C57BL6 mice and three male Han Wistar rats. We also describe our bioinformatics pipeline to process and technically validate the data. Principal component analysis shows that tissue samples from both species cluster similarly. We show by comparative genomics that many genes with high sequence identity with respect to their human orthologues also have a highly correlated tissue distribution profile and are in agreement with manually curated literature data for human. In summary, the present study provides a unique resource for comparative genomics and will facilitate the analysis of tissue specificity and cross-species conservation in higher organisms. Machine-accessible metadata file describing the reported data (ISA-Tab format) more...

The arrival of next-generation sequencing (NGS) technologies has led to novel opportunities for expression profiling and genome analysis by utilizing vast amounts of short read sequence data. Here, we demonstrate that expression profiling in organisms lacking any genome or transcriptome sequence information is feasible by combining Illumina's mRNA-seq technology with a novel bioinformatics pipeline that integrates assembled and annotated Chinese hamster ovary (CHO) sequences with information derived from related organisms. We applied this pipeline to the analysis of CHO cells which were chosen as a model system owing to its relevance in the production of therapeutic proteins. Specifically, we analysed CHO cells undergoing butyrate treatment which is known to affect cell cycle regulation and to increase the specific productivity of recombinant proteins. By this means, we identified sequences for >13,000 CHO genes which added sequence information of approximately 5000 novel genes to the CHO model. More than 6000 transcript sequences are predicted to be complete, as they covered >95% of the corresponding mouse orthologs. Detailed analysis of selected biological functions such as DNA replication and cell cycle control, demonstrated the potential of NGS expression profiling in organisms without extended genome sequence to improve both data quantity and quality. more...

In neuropsychiatry, animal studies demonstrating causal effects of environmental manipulations relevant to human aetiology on behaviours relevant to human psychopathologies are valuable. Such valid models can improve understanding of aetio-pathophysiology and preclinical discovery and development of new treatments. In depression, specific uncontrollable stressful life events are major aetiological factors, and subsequent generalized increases in fearfulness, helplessness and fatigue are core symptoms or features. Here we exposed adult male C57BL/6 mice to 15-day psychosocial stress with loss of social control but minimal physical wounding. One cohort was assessed in a 3-day test paradigm of motor activity, fear conditioning and 2-way avoid-escape behaviour on days 16-18, and a second cohort was assessed in a treadmill fatigue paradigm on days 19 and 29, followed by the 3-day paradigm on days 30-32. All tests used a physical aversive stimulus, namely mild, brief electroshocks. Socially stressed mice displayed decreased motor activity, increased fear acquisition, decreased 2-way avoid-escape responding (increased helplessness) and increased fatigue. They also displayed increased plasma TNF and spleen hypertrophy, and adrenal hypertrophy without hyper-corticoidism. In a third cohort, psychosocial stress effects on brain gene expression were assessed using next generation sequencing. Gene expression was altered in pathways of inflammation and G-protein coupled receptors in prefrontal cortex and amygdala; in the latter, expression of genes important in dopamine function were de-regulated including down-regulated Drd2, Adora2a and Darpp-32. This model can be applied to identify targets for treating psychopathologies such as helplessness or fatigue, and to screen compounds/biologics developed to act at these targets. more...

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder. Presently, there is no causal therapy available to slow down or halt disease progression. The presynaptic protein alpha-synuclein aggregates to form intraneuronal Lewy bodies in PD. It is generally believed that intermediates on the way from monomers to the large aggregates would mediate neurotoxicity, but the precise species and mechanism responsible for neuronal death are controversially debated. To study alpha-synuclein-mediated toxicity, we developed a new model in which moderate overexpression of wild-type alpha-synuclein led to gradual death of human postmitotic dopaminergic neurons. In accordance with findings in postmortem PD brains, small oligomeric species occurred and the autophagic flux was impaired in our model. The phenothiazine neuroleptic trifluoperazine, an activator of macroautophagy, selectively reduced one particular alpha-synuclein species and rescued cells. Inversely, blocking of autophagy led to an accumulation of this oligomeric species and increased cell death. These data show that activation of autophagy is a promising approach to protect against alpha-synuclein pathology and likely acts by targeting one specific alpha-synuclein species. more...

Background The aim of the present study was to evaluate the cardiovascular effects of the novel bradykinin B1 receptor antagonist BI-113823 following myocardial infarction (MI) and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin II type 1 (AT1) receptor antagonist after MI in rats. Methodology/Principal Findings Sprague Dawley rats were subjected to permanent occlusion of the left descending coronary artery. Cardiovascular function was determined at 7 days post MI. Treatment with either B1 receptor antagonist (BI-113823) or AT1 receptor antagonist (irbesartan) alone or in combination improved post-MI cardiac function as evidenced by attenuation of elevated left ventricular end diastolic pressure (LVEDP); greater first derivative of left ventricular pressure (± dp/dt max), left ventricle ejection fraction, fractional shorting, and better wall motion; as we as reductions in post-MI up-regulation of matrix metalloproteinases 2 (MMP-2) and collagen III. In addition, the cardiac up-regulation of B1 receptor and AT1 receptor mRNA were markedly reduced in animals treated with BI 113823, although bradykinin B2 receptor and angiotensin 1 converting enzyme (ACE1) mRNA expression were not significantly affected by B1 receptor blockade. Conclusions/Significance The present study demonstrates that treatment with the novel B1 receptor antagonist, BI-113823 improves post-MI cardiac function and does not influence the cardiovascular effects of AT1 receptor antagonist following MI. more...

As many users who are charged with process monitoring need to focus mainly on other work while performing monitoring as a secondary task, monitoring systems that purely rely on visual means are often not well suited for this purpose. Sonification, the presentation of data as (non-speech) sound, has proven in several studies that it can help in guiding the user's attention, especially in scenarios where process monitoring is performed in parallel with a different, main task. However, there are several aspects that have not been investigated in this area so far, for example if a continuous soundscape can guide the user's attention better than one that is based on auditory cues. We have developed a system that allows reproducible research to answer such questions. In this system, the participants' performance both for the main task (simulated by simple arithmetic problems) and for the secondary task (a simulation of a production process) can be measured in a more fine-grained manner than has been the case for existing research in this field. In a within-subject study (n=18), we compared three monitoring conditions - visual only, visual + auditory alerts and a condition combining the visual mode with continuous sonification of process events based on a forest soundscape. Participants showed significantly higher process monitoring performances in the continuous sonification condition, compared to the other two modes. The performance in the main task was at the same time not significantly affected by the continuous sonification. HighlightsWe present a system for reproducible research in sonification for process monitoring.We developed an experiment design to analyze effectiveness in monitoring as secondary task.We compared continuous sonification to visual-only and auditory alerts.Continuous sonification significantly enhances the adequacy of interactions.Participants find continuous sonification significantly more helpful and reassuring. more...