Sustainable Innovation in a Multi-University Master Course

Mobility, multi-locality, and transnational migration are current social developments among the population of the European Union. These social developments in society and companies, linked to the challenges of sustainability, lead to new requirements for working in the European Union. Teaching and learning in higher education needs to adapt to these requirements. As a result, new and innovative teaching and learning practices in higher education should provide competencies for transnational teamwork in the curriculum of tomorrow's engineers in order to ensure their competitiveness in the job market and advantage in their future careers. Thirteen European students from four countries participated in a new project-based course, called the "European Engineering Team". Students focused on the development of two innovative and sustainable products. The goal of this paper is to present the thermal pallet cover, which is the result of the first one-year transnational and sustainability-oriented project. This paper also aims to present the process of performing the project. It provides the overview and discussion of engineering and management tasks that students completed in the transnational environment, working remotely at their own campuses between scheduled transnational meetings. The work contributes to project-oriented learning that may constitute a basis for teaching holistic engineering courses at mechanical and industrial engineering departments

DNA Methylation Mediated Control of Gene Expression Is Critical for Development of Crown Gall Tumors

Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA–encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA–mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors

Tumor growth is enhanced in DNA methylation mutants.

<p>Tumor weights were determined four weeks after infection of methylation mutants (<i>ddc</i> and <i>ago4</i>) and the <i>rdd</i> demethylation mutants. Plants were inoculated with the virulent <i>A. tumefaciens</i> strain C58 at the base of the inflorenscence stem. Error bars represent mean values (± SEM) of at least 45 plants per <i>A. thaliana</i> genotype. Statistical analysis was performed using student's t-test: p-value<0.05 (*); p-value<0.01 (**).</p

Intergenic regions (IGRs) of the T-DNA–encoded oncogenes become methylated upon an siRNA trigger.

<p>(A) IGRs upstream of the oncogenes <i>IaaH</i>, <i>IaaM</i> (IGR1) and <i>Ipt</i> (IGR2) from the T-DNA of pTiC58 were analyzed by applying bisulte sequencing (model not drawn to scale). Coding sequences for <i>IaaH</i>, <i>IaaM</i> and <i>Ipt</i> are depicted as arrows, colored bars illustrate the two IGRs. (B) Detailed map of all methylated cytosines within IGR1 and IGR2 in the crown gall tumor induced by the wildtype <i>A. tumefaciens</i> strain C58 (C58 WT) and (C) of plant material inoculated with the same strain C58, but in addition harboring a binary vector with a hairpin construct directed against both IGRs (siRNA-IGR1/2). Percentages of methylation at each position are visualized by pie charts filled with different colors for the three methylation motifs (mCG brown, mCHG blue, mCHH red). Numbers below pie charts indicate nucleotide positions from the start of the analyzed region. Cytosines outside of the displayed regions were unmethylated. Percentages were calculated from bisulfite sequencing results of multiple independent clones.</p

Distribution of differentially methylated regions (DMRs) within four gene types.

<p>(A) Percentages of DMRs between crown gall tumors and mock-inoculated stems in four types of annotated loci (protein coding, transposable elements, pseudogene and non-coding (nc)RNA). (B) Percentages of unchanged, hyper- and hypomethylated regions within each of the four gene types. The calculation is based on 2876 DMRs out of which 2052 belong to the group of protein coding genes, 729 to transposable elements, 53 to pseudogenes and 42 to ncRNAs.</p

ABA induces methylation and reduces transcript levels.

<p>(A) The difference in the levels of transcription (Transcription logFC) and DNA methylation (Methylation logFC) were determined from samples treated with ABA versus samples not treated with ABA for the <i>A. thaliana</i> genes <i>NDF4</i> (At3g16250), <i>SIG5</i> (At5g24120) and <i>F12A4.4</i> (At1g35420). Transcript levels were determined by qRT-PCR and methylation profiles of upstream regions by bisulfite sequencing. For these experiments <i>A. thaliana</i> seedlings were treated with ABA for two days according to the protocol of Nishimura <i>et al</i>. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003267#pgen.1003267-Nishimura1" target="_blank">[34]</a>. (B) Changes in methylation and transcription in the crown gall tumor compared to mock-iocculated stems according to methylome (Methylation logFC) and transcriptome data (Transcription logFC) of a previous study <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003267#pgen.1003267-Deeken1" target="_blank">[8]</a>. Each data set is based on at least three independent biological replicates and the logarithmic of fold changes (logFC) were calculated from the mean values.</p

Differences in the degree of methylation in upstream regions correlate with differential transcription.

<p>Differential transcription between crown galls and mock-inoculated stem tissue (control) and the DNA methylation status are depicted for selected genes listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003267#pgen.1003267.s009" target="_blank">Table S2</a>. The selected genes belong to the following functional categories of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003267#pgen-1003267-t001" target="_blank">Table 1</a>: (A) Development (At2g22870), (B) cell (At1g63640), (C) signaling (At4g11480), and (D) biotic stress (At1g78780). Transcriptional differences were calculated as logarithmic fold changes (logFC, grey bars in the top row). Regions of DNA methylation in tumor and control tissue (grey bars in the 2^nd and 3^rd row from the top) are shown as smoothed signal log ratios (sSLRs, mCIP versus input). The gene models (green) are displayed according to their corresponding genomic positions at the bottom of each subfigure.</p

Nimodipine fosters remyelination in a mouse model of multiple sclerosis and induces microglia-specific apoptosis

Despite continuous interest in multiple sclerosis (MS) research, there is still a lack of neuroprotective strategies, because the main focus has remained on modulating the immune response. Here we performed in-depth analysis of neurodegeneration in experimental autoimmune encephalomyelitis (EAE) and in in vitro studies regarding the effect of the well-established L-type calcium channel antagonist nimodipine. Nimodipine treatment attenuated clinical EAE and spinal cord degeneration and promoted remyelination. Surprisingly, we observed calcium channel-independent effects on microglia, resulting in apoptosis. These effects were cell-type specific and irrespective of microglia polarization. Apoptosis was accompanied by decreased levels of nitric oxide (NO) and inducible NO synthase (iNOS) in cell culture as well as decreased iNOS and reactive oxygen species levels in EAE. In addition, increased numbers of Olig2(+) APC(+) oligodendrocytes were detected. Overall, nimodipine application seems to generate a favorable environment for regenerative processes and therefore could be a treatment option for MS, because it combines features of immunomodulation with beneficial effects on neuroregeneration

Tumor development and oncogene transcription are inhibited by RNAi–mediated DNA methylation.

<p>(A) Crown gall tumor weights were determined four weeks after injection of recombinant virulent <i>A. tumefaciens</i> strains of C58 into <i>A. thaliana</i> stems (ecotype WS-2). The recombinant strain C58 harbored either an empty pHellsgate12 vector (C58) or a hairpin construct directed against IGR1 (siRNA-IGR1) or IGR2 (siRNA-IGR2) or against both IGRs (siRNA-IGR1/2). Bars represent mean values (± SEM) of at least 38 inoculated plants per <i>A. tumefaciens</i> genotype. Statistical analysis was performed using student's t-test: p-value<0.01 (**). (B) Relative transcript numbers of the oncogenes of strain C58 in <i>A. thaliana</i> stem tissue six days after inoculation with the <i>A. tumefaciens</i> genotypes used for the crown gall tumor growth assay in (A). Relative transcript numbers were quantified by real time qRT-PCR and normalized to 10.000 molecules of <i>ACTIN2/8</i>. Bars represent mean values (± SD) of three independent experiments.</p