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Electrochemical methods offer the simple characterization of the synthesis of molecularly imprinted polymers (MIPs) and the readouts of target binding. The binding of electroinactive analytes can be detected indirectly by their modulating effect on the diffusional permeability of a redox marker through thin MIP films. However, this process generates an overall signal, which may include nonspecific interactions with the nonimprinted surface and adsorption at the electrode surface in addition to (specific) binding to the cavities. Redox-active low-molecular-weight targets and metalloproteins enable a more specific direct quantification of their binding to MIPs by measuring the faradaic current. The in situ characterization of enzymes, MIP-based mimics of redox enzymes or enzyme-labeled targets, is based on the indication of an electroactive product. This approach allows the determination of both the activity of the bio(mimetic) catalyst and of the substrate concentration.
Interplay of Dietary Fatty Acids and Cholesterol Impacts Brain Mitochondria and Insulin Action
(2020)
Overconsumption of high-fat and cholesterol-containing diets is detrimental for metabolism and mitochondrial function, causes inflammatory responses and impairs insulin action in peripheral tissues. Dietary fatty acids can enter the brain to mediate the nutritional status, but also to influence neuronal homeostasis. Yet, it is unclear whether cholesterol-containing high-fat diets (HFDs) with different combinations of fatty acids exert metabolic stress and impact mitochondrial function in the brain. To investigate whether cholesterol in combination with different fatty acids impacts neuronal metabolism and mitochondrial function, C57BL/6J mice received different cholesterol-containing diets with either high concentrations of long-chain saturated fatty acids or soybean oil-derived poly-unsaturated fatty acids. In addition, CLU183 neurons were stimulated with combinations of palmitate, linoleic acid and cholesterol to assess their effects on metabolic stress, mitochondrial function and insulin action. The dietary interventions resulted in a molecular signature of metabolic stress in the hypothalamus with decreased expression of occludin and subunits of mitochondrial electron chain complexes, elevated protein carbonylation, as well as c-Jun N-terminal kinase (JNK) activation. Palmitate caused mitochondrial dysfunction, oxidative stress, insulin and insulin-like growth factor-1 (IGF-1) resistance, while cholesterol and linoleic acid did not cause functional alterations. Finally, we defined insulin receptor as a novel negative regulator of metabolically stress-induced JNK activation.
Interplay of Dietary Fatty Acids and Cholesterol Impacts Brain Mitochondria and Insulin Action
(2020)
Overconsumption of high-fat and cholesterol-containing diets is detrimental for metabolism and mitochondrial function, causes inflammatory responses and impairs insulin action in peripheral tissues. Dietary fatty acids can enter the brain to mediate the nutritional status, but also to influence neuronal homeostasis. Yet, it is unclear whether cholesterol-containing high-fat diets (HFDs) with different combinations of fatty acids exert metabolic stress and impact mitochondrial function in the brain. To investigate whether cholesterol in combination with different fatty acids impacts neuronal metabolism and mitochondrial function, C57BL/6J mice received different cholesterol-containing diets with either high concentrations of long-chain saturated fatty acids or soybean oil-derived poly-unsaturated fatty acids. In addition, CLU183 neurons were stimulated with combinations of palmitate, linoleic acid and cholesterol to assess their effects on metabolic stress, mitochondrial function and insulin action. The dietary interventions resulted in a molecular signature of metabolic stress in the hypothalamus with decreased expression of occludin and subunits of mitochondrial electron chain complexes, elevated protein carbonylation, as well as c-Jun N-terminal kinase (JNK) activation. Palmitate caused mitochondrial dysfunction, oxidative stress, insulin and insulin-like growth factor-1 (IGF-1) resistance, while cholesterol and linoleic acid did not cause functional alterations. Finally, we defined insulin receptor as a novel negative regulator of metabolically stress-induced JNK activation.
Similar Yet Different
(2020)
The importance of intrinsically disordered late embryogenesis abundant (LEA) proteins in the tolerance to abiotic stresses involving cellular dehydration is undisputed. While structural transitions of LEA proteins in response to changes in water availability are commonly observed and several molecular functions have been suggested, a systematic, comprehensive and comparative study of possible underlying sequence-structure-function relationships is still lacking. We performed molecular dynamics (MD) simulations as well as spectroscopic and light scattering experiments to characterize six members of two distinct, lowly homologous clades of LEA_4 family proteins from Arabidopsis thaliana. We compared structural and functional characteristics to elucidate to what degree structure and function are encoded in LEA protein sequences and complemented these findings with physicochemical properties identified in a systematic bioinformatics study of the entire Arabidopsis thaliana LEA_4 family. Our results demonstrate that although the six experimentally characterized LEA_4 proteins have similar structural and functional characteristics, differences concerning their folding propensity and membrane stabilization capacity during a freeze/thaw cycle are obvious. These differences cannot be easily attributed to sequence conservation, simple physicochemical characteristics or the abundance of sequence motifs. Moreover, the folding propensity does not appear to be correlated with membrane stabilization capacity. Therefore, the refinement of LEA_4 structural and functional properties is likely encoded in specific patterns of their physicochemical characteristics.
Similar Yet Different
(2020)
The importance of intrinsically disordered late embryogenesis abundant (LEA) proteins in the tolerance to abiotic stresses involving cellular dehydration is undisputed. While structural transitions of LEA proteins in response to changes in water availability are commonly observed and several molecular functions have been suggested, a systematic, comprehensive and comparative study of possible underlying sequence-structure-function relationships is still lacking. We performed molecular dynamics (MD) simulations as well as spectroscopic and light scattering experiments to characterize six members of two distinct, lowly homologous clades of LEA_4 family proteins from Arabidopsis thaliana. We compared structural and functional characteristics to elucidate to what degree structure and function are encoded in LEA protein sequences and complemented these findings with physicochemical properties identified in a systematic bioinformatics study of the entire Arabidopsis thaliana LEA_4 family. Our results demonstrate that although the six experimentally characterized LEA_4 proteins have similar structural and functional characteristics, differences concerning their folding propensity and membrane stabilization capacity during a freeze/thaw cycle are obvious. These differences cannot be easily attributed to sequence conservation, simple physicochemical characteristics or the abundance of sequence motifs. Moreover, the folding propensity does not appear to be correlated with membrane stabilization capacity. Therefore, the refinement of LEA_4 structural and functional properties is likely encoded in specific patterns of their physicochemical characteristics.
Optical excitation of spin-ordered rare earth metals triggers a complex response of the crystal lattice since expansive stresses from electron and phonon excitations compete with a contractive stress induced by spin disorder. Using ultrafast x-ray diffraction experiments, we study the layer specific strain response of a dysprosium film within a metallic heterostructure upon femtosecond laser-excitation. The elastic and diffusive transport of energy to an adjacent, non-excited detection layer clearly separates the contributions of strain pulses and thermal excitations in the time domain. We find that energy transfer processes to magnetic excitations significantly modify the observed conventional bipolar strain wave into a unipolar pulse. By modeling the spin system as a saturable energy reservoir that generates substantial contractive stress on ultrafast timescales, we can reproduce the observed strain response and estimate the time- and space dependent magnetic stress. The saturation of the magnetic stress contribution yields a non-monotonous total stress within the nanolayer, which leads to unconventional picosecond strain pulses.
Optical excitation of spin-ordered rare earth metals triggers a complex response of the crystal lattice since expansive stresses from electron and phonon excitations compete with a contractive stress induced by spin disorder. Using ultrafast x-ray diffraction experiments, we study the layer specific strain response of a dysprosium film within a metallic heterostructure upon femtosecond laser-excitation. The elastic and diffusive transport of energy to an adjacent, non-excited detection layer clearly separates the contributions of strain pulses and thermal excitations in the time domain. We find that energy transfer processes to magnetic excitations significantly modify the observed conventional bipolar strain wave into a unipolar pulse. By modeling the spin system as a saturable energy reservoir that generates substantial contractive stress on ultrafast timescales, we can reproduce the observed strain response and estimate the time- and space dependent magnetic stress. The saturation of the magnetic stress contribution yields a non-monotonous total stress within the nanolayer, which leads to unconventional picosecond strain pulses.
The European rabbit (Oryctolagus cuniculus) is declining in large parts of Europe but populations in some German cities remained so far unaffected by this decline. The question arises of how urbanization affects patterns of population genetic variation and differentiation in German rabbit populations, as urban habitat fragmentation may result in altered meta-population dynamics. To address this question, we used microsatellite markers to genotype rabbit populations occurring along a rural-to-urban gradient in and around the city of Frankfurt, Germany. We found no effect of urbanization on allelic richness. However, the observed heterozygosity was significantly higher in urban than rural populations and also the inbreeding coefficients were lower, most likely reflecting the small population sizes and possibly on-going loss of genetic diversity in structurally impoverished rural areas. Global FST and G'ST-values suggest moderate but significant differentiation between populations. Multiple matrix regression with randomization ascribed this differentiation to isolation-by-environment rather than isolation-by-distance. Analyses of migration rates revealed asymmetrical gene flow, which was higher from rural into urban populations than vice versa and may again reflect intensified agricultural land-use practices in rural areas. We discuss that populations inhabiting urban areas will likely play an important role in the future distribution of European rabbits.
The European rabbit (Oryctolagus cuniculus) is declining in large parts of Europe but populations in some German cities remained so far unaffected by this decline. The question arises of how urbanization affects patterns of population genetic variation and differentiation in German rabbit populations, as urban habitat fragmentation may result in altered meta-population dynamics. To address this question, we used microsatellite markers to genotype rabbit populations occurring along a rural-to-urban gradient in and around the city of Frankfurt, Germany. We found no effect of urbanization on allelic richness. However, the observed heterozygosity was significantly higher in urban than rural populations and also the inbreeding coefficients were lower, most likely reflecting the small population sizes and possibly on-going loss of genetic diversity in structurally impoverished rural areas. Global FST and G'ST-values suggest moderate but significant differentiation between populations. Multiple matrix regression with randomization ascribed this differentiation to isolation-by-environment rather than isolation-by-distance. Analyses of migration rates revealed asymmetrical gene flow, which was higher from rural into urban populations than vice versa and may again reflect intensified agricultural land-use practices in rural areas. We discuss that populations inhabiting urban areas will likely play an important role in the future distribution of European rabbits.
The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.