Distributed LoRa based CO2 monitoring network - A standalone open source system for contagion prevention by controlled ventilation.

In the face of a global pandemic, such as that caused by the SARS-CoV-2 virus, the prevention of new infections is essential to stop the spread and ultimately return to normality. In addition to wearing masks and maintaining safe distances, regular ventilation in enclosed spaces where several people are gathered has proven to be an effective protective measure as advised by the World Health Organization. Additionally, as has been shown in a recent study of other airborne viruses, there is a strong correlation between the CO2level and aerosol content in a confined space under the assumption humans are the only CO2source. This can be exploited by means of a low-cost infrared CO2sensor to indirectly monitor the aerosol content and to provide targeted ventilation if predefined thresholds are exceeded. The distributed CO2monitoring network presented in this paper extends that idea and provides an inexpensive, comprehensive and modular monitoring network based on readily available components and 3D printing. By using a long-range communication link (LoRa) to centrally collect the real-time CO2concentration in a multitude of rooms, this network is particularly suitable for larger building complexes such as kindergartens, schools and universities without requiring partial or even full WLAN coverage.

Trophic niche but not abundance of Collembola and Oribatida changes with drought and farming system.

Higher frequencies of summer droughts are predicted to change soil conditions in the future affecting soil fauna communities and their biotic interactions. In agroecosystems drought effects on soil biota may be modulated by different management practices that alter the availability of different food resources. Recent studies on the effect of drought on soil microarthropods focused on measures of abundance and diversity. We here additionally investigated shifts in trophic niches of Collembola and Oribatida as indicated by stable isotope analysis (13C and 15N). We simulated short-term summer drought by excluding 65% of the ambient precipitation in conventionally and organically managed winter wheat fields on the DOK trial in Switzerland. Stable isotope values suggest that plant litter and root exudates were the most important resources for Collembola (Isotoma caerulea, Isotomurus maculatus and Orchesella villosa) and older plant material and microorganisms for Oribatida (Scheloribates laevigatus and Tectocepheus sarekensis). Drought treatment and farming systems did not affect abundances of the studied species. However, isotope values of some species increased in organically managed fields indicating a higher proportion of microorganisms in their diet. Trophic niche size, a measure of both isotope values combined, decreased with drought and under organic farming in some species presumably due to favored use of plants as basal resource instead of algae and microorganisms. Overall, our results suggest that the flexible usage of resources may buffer effects of drought and management practices on the abundance of microarthropods in agricultural systems.

Conventional agriculture and not drought alters relationships between soil biota and functions.

Soil biodiversity constitutes the biological pillars of ecosystem services provided by soils worldwide. Soil life is threatened by intense agricultural management and shifts in climatic conditions as two important global change drivers which are not often jointly studied under field conditions. We addressed the effects of experimental short-term drought over the wheat growing season on soil organisms and ecosystem functions under organic and conventional farming in a Swiss long term trial. Our results suggest that activity and community metrics are suitable indicators for drought stress while microbial communities primarily responded to agricultural practices. Importantly, we found a significant loss of multiple pairwise positive and negative relationships between soil biota and process-related variables in response to conventional farming, but not in response to experimental drought. These results suggest a considerable weakening of the contribution of soil biota to ecosystem functions under long-term conventional agriculture. Independent of the farming system, experimental and seasonal (ambient) drought conditions directly affected soil biota and activity. A higher soil water content during early and intermediate stages of the growing season and a high number of significant relationships between soil biota to ecosystem functions suggest that organic farming provides a buffer against drought effects.

Soil microarthropods respond differently to simulated drought in organic and conventional farming systems.

In Central Europe, summer droughts are increasing in frequency which threatens production and biodiversity in agroecosystems. The potential of different farming systems to mitigate detrimental drought effects on soil animals is largely unknown. We investigated the effects of simulated drought on the abundance and community composition of soil microarthropods (Collembola and Oribatida and Meso-, Pro-, and Astigmata) in winter wheat fields under long-term conventional and organic farming in the DOK trial, Switzerland. We simulated drought by excluding 65% of the ambient precipitation during the wheat-growing season from March to June 2017. The abundance of Collembola and Oribatida declined more consistently in conventionally managed fields compared to organically managed fields under simulated drought. The abundance of Collembola as well as Meso-, Pro- and Astigmata, but not the abundance of Oribatida, increased in deeper soil layers due to simulated drought, suggesting vertical migration as a drought avoidance strategy. The species composition of Oribatida communities, but not of Collembola communities, differed significantly between drought treatments and between farming systems. Soil carbon content was a major factor structuring Oribatida communities. Our results suggest that organic farming buffers negative effects of drought on soil microarthropods, presumably due to higher soil carbon content and associated higher soil moisture and improved soil structure. This potential of organic farming systems to mitigate consequences of future droughts on soil biodiversity is promising and needs further exploration across larger climatic and spatial scales and should be extended to other groups of soil biota.

The Impact of Small Extracellular Vesicles on Lymphoblast Trafficking across the Blood-Cerebrospinal Fluid Barrier In Vitro.

Central nervous System (CNS) disease in pediatric acute lymphoblastic leukemia (ALL) is a major concern, but still, cellular mechanisms of CNS infiltration are elusive. The choroid plexus (CP) is a potential entry site, and, to some extent, invasion resembles CNS homing of lymphocytes during healthy state. Given exosomes may precondition target tissue, the present work aims to investigate if leukemia-derived exosomes contribute to a permissive phenotype of the blood-cerebrospinal fluid barrier (BCSFB). Leukemia-derived exosomes were isolated by ultracentrifugation from the cell lines SD-1, Nalm-6, and P12-Ichikawa (P12). Adhesion and uptake to CP epithelial cells and the significance on subsequent ALL transmigration across the barrier was studied in a human BCSFB in vitro model based on the HiBCPP cell line. The various cell lines markedly differed regarding exosome uptake to HiBCPP and biological significance. SD-1-derived exosomes associated to target cells unspecifically without detectable cellular effects. Whereas Nalm-6 and P12-derived exosomes incorporated by dynamin-dependent endocytosis, uptake in the latter could be diminished by integrin blocking. In addition, only P12-derived exosomes led to facilitated transmigration of the parental leukemia cells. In conclusion, we provide evidence that, to a varying extent, leukemia-derived exosomes may facilitate CNS invasion of ALL across the BCSFB without destruction of the barrier integrity.

Integrated strain- and process design enable production of 220 g L-1 itaconic acid with Ustilago maydis.

BACKGROUND: Itaconic acid is an unsaturated, dicarboxylic acid which finds a wide range of applications in the polymer industry and as a building block for fuels, solvents and pharmaceuticals. Currently, Aspergillus terreus is used for industrial production, with titers above 100 g L-1 depending on the conditions. Besides A. terreus, Ustilago maydis is also a promising itaconic acid production host due to its yeast-like morphology. Recent strain engineering efforts significantly increased the yield, titer and rate of production. RESULTS: In this study, itaconate production by U. maydis was further increased by integrated strain- and process engineering. Next-generation itaconate hyper-producing strains were generated using CRISPR/Cas9 and FLP/FRT genome editing tools for gene deletion, promoter replacement, and overexpression of genes. The handling and morphology of this engineered strain were improved by deletion of fuz7, which is part of a regulatory cascade that governs morphology and pathogenicity. These strain modifications enabled the development of an efficient fermentation process with in situ product crystallization with CaCO3. This integrated approach resulted in a maximum itaconate titer of 220 g L-1, with a total acid titer of 248 g L-1, which is a significant improvement compared to best published itaconate titers reached with U. maydis and with A. terreus. CONCLUSION: In this study, itaconic acid production could be enhanced significantly by morphological- and metabolic engineering in combination with process development, yielding the highest titer reported with any microorganism.

Optimized cultivation of porcine choroid plexus epithelial cells, a blood-cerebrospinal fluid barrier model, for studying granulocyte transmigration.

The blood-cerebrospinal fluid barrier (BCSFB) plays important roles during the transport of substances into the brain, the pathogenesis of central nervous system (CNS) diseases, and neuro-immunological processes. Along these lines, transmigration of granulocytes across the blood-cerebrospinal fluid (CSF) barrier (BCSFB) is a hallmark of inflammatory events in the CNS. Choroid plexus (CP) epithelial cells are an important tool to generate in vitro models of the BCSFB. A porcine CP epithelial cell line (PCP-R) has been shown to present properties of the BCSFB, including a strong barrier function, when cultivated on cell culture filter inserts containing a membrane with 0.4 µm pore size. For optimal analysis of pathogen and host immune cell interactions with the basolateral side of the CP epithelium, which presents the physiologically relevant "blood side", the CP epithelial cells need to be grown on the lower face of the filter in an inverted cell culture insert model, with the supporting membrane possessing a pore size of at least 3.0 µm. Here, we demonstrate that PCP-R cells cultivated in the inverted model on filter support membranes with a pore size of 3.0 µm following a "conventional" protocol grow through the pores and cross the membrane, forming a second layer on the upper face. Therefore, we developed a cell cultivation protocol, which strongly reduces crossing of the membrane by the cells. Under these conditions, PCP-R cells retain important properties of a BCSFB model, as was observed by the formation of continuous tight junctions and a strong barrier function demonstrated by a high transepithelial electrical resistance and a low permeability for macromolecules. Importantly, compared with the conventional cultivation conditions, our optimized model allows improved investigations of porcine granulocyte transmigration across the PCP-R cell layer.

Evolutionary freedom in the regulation of the conserved itaconate cluster by Ria1 in related Ustilaginaceae.

BACKGROUND: Itaconate is getting growing biotechnological significance, due to its use as a platform compound for the production of bio-based polymers, chemicals, and novel fuels. Currently, Aspergillus terreus is used for its industrial production. The Ustilaginaceae family of smut fungi, especially Ustilago maydis, has gained biotechnological interest, due to its ability to naturally produce this dicarboxylic acid. The unicellular, non-filamentous growth form makes these fungi promising alternative candidates for itaconate production. Itaconate production was also observed in other Ustilaginaceae species such as U. cynodontis, U. xerochloae, and U. vetiveriae. The investigated species and strains varied in a range of 0-8 g L-1 itaconate. The genes responsible for itaconate biosynthesis are not known for these strains and therefore not characterized to explain this variability. RESULTS: Itaconate production of 13 strains from 7 species known as itaconate producers among the family Ustilaginaceae were further characterized. The sequences of the gene cluster for itaconate synthesis were analyzed by a complete genome sequencing and comparison to the annotated itaconate cluster of U. maydis. Additionally, the phylogenetic relationship and inter-species transferability of the itaconate cluster transcription factor Ria1 was investigated in detail. Doing so, itaconate production could be activated or enhanced by overexpression of Ria1 originating from a related species, showing their narrow phylogenetic relatedness. CONCLUSION: Itaconate production by Ustilaginaceae species can be considerably increased by changing gene cluster regulation by overexpression of the Ria1 protein, thus contributing to the industrial application of these fungi for the biotechnological production of this valuable biomass derived chemical.

Pediatric acute lymphoblastic leukemia-Conquering the CNS across the choroid plexus.

Despite the high prevalence of central nervous system (CNS) involvement in relapsing pediatric acute lymphoblastic leukemia (ALL), our understanding of CNS invasion is still vague. As lymphoblasts have to overcome the physiological blood-CNS barriers to enter the CNS, we investigated the cellular interactions of lymphoblasts with the choroid plexus (CP) epithelium of the blood-cerebrospinal fluid barrier (BCSFB). Both a precurser B cell ALL (pB-ALL) cell line (SD-1) and a T cell ALL (T-ALL) cell line (P12-Ishikawa) were able to actively cross the CP epithelium in a human in vitro model. We could illustrate a transcellular and (supposedly) paracellular transmigration by 3-dimensional immunofluorescence microscopy as well as electron microscopy. Chemotactic stimulation with CXCL12 during this process led to a significantly increased transmigration and blocking CXCL12/CXCR4-signaling by the CXCR4-inhibitor AMD3100 inhibited this effect. However, CXCR4 expression in primary ALL samples did not correlate to CNS disease, indicating that CXCR4-driven CNS invasion across the BCSFB might be a general property of pediatric ALL. Notably, we present a unique in vitro BCSFB model suitable to study CNS invasion of lymphoblasts in a human setting, providing the opportunity to investigate experimental variables, which may determine CNS disease childhood ALL.

Conversion of MapMan to allow the analysis of transcript data from Solanaceous species: effects of genetic and environmental alterations in energy metabolism in the leaf.

The tomato microarray TOM1 offers the possibility to monitor the levels of several thousand transcripts in parallel. The microelements represented on this tomato microarray have been putatively assigned to unigenes, and organised in functional classes using the MapMan ontology (Thimm et al., 2004. Plant J. 37: 914-939). This ontology was initially developed for use with the Arabidopsis ATH1 array, has a low level of redundancy, and can be combined with the MapMan software to provide a biologically structured overview of changes of transcripts, metabolites and enzyme activities. Use of this application is illustrated using three case studies with published or novel TOM1 array data sets for Solanaceous species. Comparison of previously reported data on transcript levels in potato leaves in the middle of the day and the middle of the night identified coordinated changes in the levels of transcripts of genes involved in various metabolic pathways and cellular events. Comparison with diurnal changes of gene expression in Arabidopsis revealed common features, illustrating how MapMan can be used to compare responses in different organisms. Comparison of transcript levels in new experiments performed on the leaves of the cultivated tomato S. lycopersicum and the wild relative S. pennellii revealed a general decrease of levels of transcripts of genes involved in terpene and, phenylpropanoid metabolism as well as chorismate biosynthesis in the crop compared to the wild relative. This matches the recently reported decrease of the levels of secondary metabolites in the latter. In the third case study, new expression array data for two genotypes deficient in TCA cycle enzymes is analysed to show that these genotypes have elevated levels of transcripts associated with photosynthesis. This in part explains the previously documented enhanced rates of photosynthesis in these genotypes. Since the Solanaceous MapMan is intended to be a community resource it will be regularly updated on improvements in tomato gene annotation and transcript profiling resources.

High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes, and comparison with known responses.

MapMan is a user-driven tool that displays large genomics datasets onto diagrams of metabolic pathways or other processes. Here, we present new developments, including improvements of the gene assignments and the user interface, a strategy to visualize multilayered datasets, the incorporation of statistics packages, and extensions of the software to incorporate more biological information including visualization of corresponding genes and horizontal searches for similar global responses across large numbers of arrays.

In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system.

In the postgenomic era many experiments rely on the availability of transcript sequence for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. If a good library is generated it is desirable to use a versatile cloning system suitable for many different kinds of applications. The cloning systems based on in vitro recombination proves fitting for this task. However, the use of this method for shuttling entire cDNA libraries between different vectors has not yet been studied in great detail. Here we describe the construction of four cDNA libraries from different tissues of Arabidopsis thaliana, the shuttling of the libraries into expression vectors, and evaluation of this method as well as its suitability for downstream applications. Libraries were constructed from seedlings, hormone treated seedlings, flowers, developing seeds and primary leaves in the "entry vector" of the Gateway cloning system. After initial characterization of the libraries, they were shuttled into an expression vector (a yeast two-hybrid prey vector). To monitor for a size bias generally assumed to be inherent to in vitro recombination methods, the libraries were characterized before and after the transfer into the expression vector. However no significant difference could be detected. The functionality of the in vitro recombination system for the shuttling of entire libraries was then further tested by protein-protein interaction screens. The results of the library characterization and of the yeast two-hybrid screens and their implications for large-scale proteomic approaches are discussed.

PoMaMo--a comprehensive database for potato genome data.

A database for potato genome data (PoMaMo, Potato Maps and More) was established. The database contains molecular maps of all twelve potato chromosomes with about 1000 mapped elements, sequence data, putative gene functions, results from BLAST analysis, SNP and InDel information from different diploid and tetraploid potato genotypes, publication references, links to other public databases like GenBank (http://www.ncbi.nlm.nih.gov/) or SGN (Solanaceae Genomics Network, http://www.sgn.cornell.edu/), etc. Flexible search and data visualization interfaces enable easy access to the data via internet (https://gabi.rzpd.de/PoMaMo.html). The Java servlet tool YAMB (Yet Another Map Browser) was designed to interactively display chromosomal maps. Maps can be zoomed in and out, and detailed information about mapped elements can be obtained by clicking on an element of interest. The GreenCards interface allows a text-based data search by marker-, sequence- or genotype name, by sequence accession number, gene function, BLAST Hit or publication reference. The PoMaMo database is a comprehensive database for different potato genome data, and to date the only database containing SNP and InDel data from diploid and tetraploid potato genotypes.

MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes.

MAPMAN is a user-driven tool that displays large data sets onto diagrams of metabolic pathways or other processes. SCAVENGER modules assign the measured parameters to hierarchical categories (formed 'BINs', 'subBINs'). A first build of TRANSCRIPTSCAVENGER groups genes on the Arabidopsis Affymetrix 22K array into >200 hierarchical categories, providing a breakdown of central metabolism (for several pathways, down to the single enzyme level), and an overview of secondary metabolism and cellular processes. METABOLITESCAVENGER groups hundreds of metabolites into pathways or groups of structurally related compounds. An IMAGEANNOTATOR module uses these groupings to organise and display experimental data sets onto diagrams of the users' choice. A modular structure allows users to edit existing categories, add new categories and develop SCAVENGER modules for other sorts of data. MAPMAN is used to analyse two sets of 22K Affymetrix arrays that investigate the response of Arabidopsis rosettes to low sugar: one investigates the response to a 6-h extension of the night, and the other compares wild-type Columbia-0 (Col-0) and the starchless pgm mutant (plastid phosphoglucomutase) at the end of the night. There were qualitatively similar responses in both treatments. Many genes involved in photosynthesis, nutrient acquisition, amino acid, nucleotide, lipid and cell wall synthesis, cell wall modification, and RNA and protein synthesis were repressed. Many genes assigned to amino acid, nucleotide, lipid and cell wall breakdown were induced. Changed expression of genes for trehalose metabolism point to a role for trehalose-6-phosphate (Tre6P) as a starvation signal. Widespread changes in the expression of genes encoding receptor kinases, transcription factors, components of signalling pathways, proteins involved in post-translational modification and turnover, and proteins involved in the synthesis and sensing of cytokinins, abscisic acid (ABA) and ethylene revealing large-scale rewiring of the regulatory network is an early response to sugar depletion.

First-generation SNP/InDel markers tagging loci for pathogen resistance in the potato genome.

A panel of 17 tetraploid and 11 diploid potato genotypes was screened by comparative sequence analysis of polymerase chain reaction (PCR) products for single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), in regions of the potato genome where genes for qualitative and/or quantitative resistance to different pathogens have been localized. Most SNP and InDel markers were derived from bacterial artificial chromosome (BAC) insertions that contain sequences similar to the family of plant genes for pathogen resistance having nucleotide-binding-site and leucine-rich-repeat domains (NBS-LRR-type genes). Forty-four such NBS-LRR-type genes containing BAC-insertions were mapped to 14 loci, which tag most known resistance quantitative trait loci (QTL) in potato. Resistance QTL not linked to known resistance-gene-like (RGL) sequences were tagged with other markers. In total, 78 genomic DNA fragments with an overall length of 31 kb were comparatively sequenced in the panel of 28 genotypes. 1498 SNPs and 127 InDels were identified, which corresponded, on average, to one SNP every 21 base pairs and one InDel every 243 base pairs. The nucleotide diversity of the tetraploid genotypes (pi = 0.72 x 10(-3)) was lower when compared with diploid genotypes (pi = 2.31 x 10(-3)). RGL sequences showed higher nucleotide diversity when compared with other sequences, suggesting evolution by divergent selection. Information on sequences, sequence similarities, SNPs and InDels is provided in a database that can be queried via the Internet.

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes.

Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G + C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol 2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0.1 to 650 mU (mg protein)-1. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains.