Autonomous capillary systems for life science research and medical diagnostics

In autonomous capillary systems (CS) minute amounts of liquid are transported owing to capillary forces. Such CSs are appealing due to their portability, flexibility, and the exceptional physical behavior of liquids in micrometer sized microchannels, in particular, capillarity and short diffusion times. CSs have shown to be a promising technology for miniaturized immunoassays in life science research and diagnostics. Building on existing experimental demonstrations of immunoassays in CSs, a theoretical model of such immunoassays is implemented, tools and CSs for performing immunoassays are developed, key functional elements of CSs such as capillary pumps and valves are explored experimentally, and a proof-of-concept of the ultimate goal of one-step immunoassays are given in this work. For the theoretical modeling of immunoassays in CSs a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance. A method using evaporation for controlling the flow rate in CSs was developed for maximum flexibility for developing assays. The method allows to use small CSs that initially are filled by capillary forces and then provide a well defined area of the liquid-air interface from which liquid can evaporate. Temperature and humidity are continuously measured and Peltier-elements are used to adjust the temperatures in multiple areas of the CSs relative to the dew-point. Thereby flow rates in the range from ~1.2 nL s−1 to ~30 pL s−1 could be achieved in the microchannels. This method was then used for screening cells for surface receptors. CSs, that do not need any peripherals for controlling flow rates become even more appealing. We explored the filling behavior of such CSs having microchannels of various length and large capillary pumps. The capillary pumps comprise microstructures of various sizes and shapes, which are spaced to encode certain capillary pressures. The spacing and shape of the microstructures is also used to orient the filling front to obtain a reliable filling behavior and to minimize the risk of entrapping air. We show how two capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in CSs. Liquid filling CSs can hardly be stopped, but in some cases it might be beneficial to do so. In a separate chapter we explore how microstructures need to be designed to use capillary forces to stop, time, or trigger liquids. Besides well-defined flow rates in CSs accurately patterned capture antibodies (cAbs) are key for performing high-sensitive surface immunoassays in CSs. We present a method compatible with mass fabrication for patterning cAbs in dense lines of up to 8 lines per millimeter. These cAbs are used with CSs that are optimized for convenient handling, pipetting of solutions, pumping of liquids such as human serum, and visualization of signals for fluorescence immunoassays to detect c-reactive protein (CRP) with a sensitivity of 0.9 ng mL−1 (7.8 pM) from 1 uL of CRP-spiked human serum, within 11 minutes, with 4 pipetting steps, and a total volume of sample and reagents of <1.5 uL. CSs for diagnostic applications have different requirements than CSs that are used as a research tool in life sciences, where a high flexibility and performance primes over the ease of use and portability of the CSs. We give a proof-of-concept for one-step immunoassays based on CSs which we think can be the base for developing portable diagnostics for point-of-care applications. All reagents are preloaded in the CSs. A sample loaded in the CSs redissolves and reconstitutes the detection antibodies (dAbs), analyte-dAb-complexes are formed and detected downstream in the CSs. A user only needs to load a sample and measure the result using a fluorescence microscope or scanner. C-reactive protein was detected in human serum at clinical concentrations within 10 minutes and using only 2 uL of sample

Indikatoren für eine ergebnisorientierte Honorierung von Tierschutzleistungen in der Milchviehhaltung

Regulations in organic farming and in animal welfare policies are action-oriented: they refer to resources such as space allowance and management, i.e. access to pasture. With this approach, the prerequisites for the exercise of normal behaviour can be created; but direct impacts on animal health and welfare are largely left out. The aim of our project is to develop a concept for a result-oriented approach to improve animal welfare in the framework of organic farming and the EUs rural development programmes. One of the challenges is the selection of suitable indicators. To select indicators which address major welfare-problems in dairy farming, a two-phased sampling process was carried out. First, scientists from Germany, Switzerland and Austria (n = 20) were asked in a written survey to select the most appropriate from a list of 82 indicators derived from literature. The questionnaire was designed as a Delphi survey to reduce heterogeneity among researchers and resulted in a list of 23 indicators. Then a practitioner-workshop with farmers, lobby groups, administration and control took place (n = 20) to address practicability issues. With great match, a list of 10 indicators was adopted, which was subjected to a practical test on 119 dairy farms, in which the full Welfare Quality® protocol was carried out for validation and comparison. The presentation will suggest this set of indicators

Untersuchungen zum Einfluss der Wirtschaftsweise auf das Tierwohl von Milchkühen auf Basis des Welfare Quality® Protokolls

Organic farming attempts to ensure animal welfare on the basis of preventive measures. The aim of the present study was to investigate the effects of the production system on animal welfare of dairy cows in organic and conventional farms. The results of the application of the Welfare Quality® protocol for dairy cows shows significant differences between organic and conventional farms while concurrently disclosing considerable differences between farms of the same production system. To further improve the animal welfare situation in organic farming and especially to better include animal health issues, result based components should be included into the current (purely action-oriented) EU organic farming regulation

Can autonomous vehicles improve the quality of life? – A simulation study

Simulation of automated road transport: In a simulation study, the DLR Institute of Transportation Systems examined the effects of autonomous vehicles on indicators like travel time, land use and emission. The aim was to use simulations to demonstrate the potential of individual applications of ART. Several simulation scenarios with the tool SUMO (Simulation of Urban Mobility) were carried out. The focus was on two key use cases. One focussed on self-driving vehicles that drop off travellers at their destination and then park themselves in public garages. The second use case was an automated on-demand shuttle service that combined the travel requests of different passengers. Simulation des automatisierten Straßenverkehrs: In einer Simulationsstudie untersuchte das DLR-Institut für Verkehrssystemtechnik die Auswirkungen von autonomen Fahrzeugen auf Indikatoren wie Reisezeit, Flächenverbrauch und Emissionen. Ziel war es, durch Simulationen das Potenzial einzelner Anwendungen des automatisierten und vernetzten Fahrens aufzuzeigen. Es wurden mehrere Simulationsszenarien mit dem Tool SUMO (Simulation of Urban Mobility) durchgeführt. Der Fokus lag dabei auf zwei zentralen Anwendungsfällen. Im ersten Anwendungsfall wurden selbstfahrende Fahrzeuge untersucht, die Reisende am Zielort absetzen und sich dann selbstständig in öffentlichen Quartiersgaragen einparken (Quarter Valet Parking). Der zweite Anwendungsfall war ein automatisierter On-Demand-Shuttleservice, der die Reisewünsche verschiedener Fahrgäste kombiniert

Welchen Einfluss hat der Weidegang auf das Tierwohl von Milchkühen? Erste Ergebnisse des Welfare Quality® Protokolls bei ganzjähriger Stallhaltung und Sommerweidegang

Grazing provides livestock better opportunities to act out their species specific behaviour compared to the restrictive stable conditions. Studies on the effect of grazing on animal welfare in dairy farming in Germany are rare and have not been conducted under the specific conditions of organic dairy farming. The aim of the present study was to examine the effects of grazing on animal welfare of dairy cows in organic and conventional farming based on the Welfare Quality® protocol for dairy cattle. In this paper, we present the initial evaluation of a comparison between zero grazing and summer grazing. The first results indicate an improvement in most welfare principles during the summer months for dairy cows with summer grazing, except for between winter and summer in zero grazing farms. In conclusion, grazing offers a great potential for improved animal welfare, while the benefitial effects of grazing are not guaranteed in event of suboptimal management

Eiweiß- und Energieversorgung in 34 konventionellen und ökologischen Milchviehherden Ergebnisse aus einem Netzwerk von Pilotbetrieben

For reasons of animal health, milk yield and for environmental issues an optimal supply of protein and energy for dairy cows is essential. The aim of this study is to examine potential protein and energy malnutrition and surplus in dairy herds. We present a comparison of the protein and energy supply between 18 conventional and 16 organic dairy herds. Initial results indicate an undersupply on energy in the first one hundred days of lactation in both the organic and conventional feeding regimes. It is easier to provide an adequate protein supply in conventional conditions in the first one hundred days than in organic ones. A balanced supply is guaranteed for a mere 21,3 % of the organic cows. In the second one hundred days this increases to 26,5 %, compared to 51,7 % in conventional ones. In high yielding organic dairy herds there is a shortage in energy and protein in the second one hundred days as well. In low yielding organic herds a surplus on protein over the whole lactation with possible negative effects on animal health and ammonia emissions is observed. Once more these first results demonstrate the difficulty of providing a balanced nutrition in organic dairy herds

AtPTR4 and AtPTR6 are differentially expressed, tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectivel

Diversidad patogénica dentro de poblaciones de jopo de girasol (O. cumana)

Resúmes del XII Congreso Nacional de la Sociedad Española de FitopatologíaPeer reviewe

microRNA miR-142-3p Inhibits Breast Cancer Cell Invasiveness by Synchronous Targeting of WASL, Integrin Alpha V, and Additional Cytoskeletal Elements

MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR-142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of WASL (Wiskott-Aldrich syndrome-like, protein: N-WASP), Integrin-αV, RAC1, and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased Matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a change in cell morphology as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3’-untranslated region of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNA-dependent invasion phenotype. While knockdown of WASL significantly reduced the number of membrane protrusions compared to controls, knockdown of ITGAV resulted in a decreased cell volume, indicating differential contributions of these factors to the miR-142-3p-induced phenotype. Our data identify WASL, ITGAV and several additional cytoskeleton-associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer

Human Mas-related G protein-coupled receptors-X1 induce chemokine receptor 2 expression in rat dorsal root ganglia neurons and release of chemokine ligand 2 from the human LAD-2 mast cell line

Primate-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are highly enriched in dorsal root ganglia (DRG) neurons and induce acute pain. Herein, we analyzed effects of MRGPR-X1 on serum response factors (SRF) or nuclear factors of activated T cells (NFAT), which control expression of various markers of chronic pain. Using HEK293, DRG neuron-derived F11 cells and cultured rat DRG neurons recombinantly expressing human MRGPR-X1, we found activation of a SRF reporter gene construct and induction of the early growth response protein-1 via extracellular signal-regulated kinases-1/2 known to play a significant role in the development of inflammatory pain. Furthermore, we observed MRGPR-X1-induced up-regulation of the chemokine receptor 2 (CCR2) via NFAT, which is considered as a key event in the onset of neuropathic pain and, so far, has not yet been described for any endogenous neuropeptide. Up-regulation of CCR2 is often associated with increased release of its endogenous agonist chemokine ligand 2 (CCL2). We also found MRGPR-X1-promoted release of CCL2 in a human connective tissue mast cell line endogenously expressing MRGPR-X1. Thus, we provide first evidence to suggest that MRGPR-X1 induce expression of chronic pain markers in DRG neurons and propose a so far unidentified signaling circuit that enhances chemokine signaling by acting on two distinct yet functionally co-operating cell types. Given the important role of chemokine signaling in pain chronification, we propose that interruption of this signaling circuit might be a promising new strategy to alleviate chemokine-promoted pain