The Cytokinin-Activating LOG-Family Proteins Are Not Lysine Decarboxylases

© 2018 Elsevier Ltd A conserved PGGxGTxxE motif misleads the cytokinin (CK) converting LONELY GUY enzymes to be wrongly annotated as lysine decarboxylases (LDCs). However, so far PGGxGTxxE motif-containing LDCs do not show any LDC activity. Instead, they show phosphoribohydrolase activity by converting inactive CK nucleotides into active free-base forms to invoke CK responses

Protein phosphorylation prediction: limitations, merits and pitfalls

Protein phosphorylation is a major protein post-translational modification process that plays a pivotal role in numerous cellular processes, such as recognition, signaling or degradation. It can be studied experimentally by various methodologies, including western blot analysis, site-directed mutagenesis, 2D gel electrophoresis, mass spectrometry etc. A number of in silico tools have also been developed in order to predict plausible phosphorylation sites in a given protein. In this review, we conducted a benchmark study including the leading protein phosphorylation prediction software, in an effort to determine which performs best. The first place was taken by GPS 2.2, having predicted all phosphorylation sites with a 83% fidelity while in second place came NetPhos 2.0 with 69%.  

Aspergillus fumigatus challenged by human dendritic cells: metabolic and regulatory pathway responses testify a tight battle

Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other

Complement sensitivity and factor H binding of European Francisella tularensis ssp. holarctica strains in selected animal species

Francisella tularensis is a Gram-negative bacterium, the causative agent of the zoonotic disease tularaemia. The bacterium has developed several extracellular and intracellular strategies to evade the hosts’ innate and adaptive immune responses. The aims of the study were to examine complement sensitivity of wild and attenuated F. tularensis ssp. holarctica strains in animal hosts of distinct sensitivity to the bacterium, to compare the complement-evading ability of wild strains of different phylogeographic background, and to examine the role of factor H in the host–pathogen interactions. Complement sensitivity assays were carried out on various F. tularensis ssp. holarctica wild strains and on the attenuated live vaccine strain (LVS) with sera of the highly sensitive house mouse (Mus musculus), the moderately sensitive European brown hare (Lepus europaeus) and the relatively resistant cattle (Bos taurus). Specific binding of complement regulator factor H to bacterial membrane proteins was examined by Western blot assays. All wild strains interacted with the hosts’ complement system and showed no significant differences in their survivability. The attenuated LVS was resistant to serum killing in mouse, but was lysed in the sera of hare and cattle. Direct binding of factor H to F. tularensis membrane proteins was not detected

Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems

© 2020, The Author(s). Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions

Ovulatory shift, hormonal changes, and no effects on incentivized decision-making

Employing an incentivized controlled lab experiment, we investigate the effects of ovulatory shift on salient behavioral outcomes related to (i) risk preferences, (ii) rule violation, and (iii) exploratory attitude. As evolutionary psychology suggests, these outcomes may play an important role in economic decision-making and represent behavioral aspects that may systematically vary over the menstrual cycle to increase the reproductive success. Exploiting a within-subjects design, 124 naturally cycling females participated in experimental sessions during their ovulation and menstruation, the phases between which the difference in the investigated behavior should be the largest. In each session, hormonal samples for cortisol, estradiol, and testosterone were collected. The group of women was also contrasted against an auxiliary reference group composed of 47 males, who are not subject to hormonal variations of this nature. Our results reveal no systematic behavioral differences between the ovulation and menstruation phases

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants

Delivery of secretory immunoglobulin A (sIgA) to mucosal surfaces as a passive immunotherapy agent is a promising strategy to prevent infectious diseases. Recombinant sIgA production in plants requires the co-expression of four transcriptional units encoding the light chain (LC), heavy chain (HC), joining chain (JC) and secretory component (SC). As a way to optimize sIgA production in plants, we tested the combinatorial expression of 16 versions of a human sIgA against the VP8* rotavirus antigen in Nicotiana benthamiana, using the recently developed GoldenBraid multigene assembly system. Each sIgA version was obtained by combining one of the two types of HC (alpha 1 and alpha 2) with one of the two LC types (k and lambda) and linking or not a KDEL peptide to the HC and/or SC. From the analysis of the anti-VP8* activity, it was concluded that those sIgA versions carrying HC alpha 1 and LC lambda provided the highest yields. Moreover, ER retention significantly increased antibody production, particularly when the KDEL signal was linked to the SC. Maximum expression levels of 32.5 mu g IgA/g fresh weight (FW) were obtained in the best performing combination, with an estimated 33% of it in the form of a secretory complex.This work has been funded by Grant BIO2010-15384 from Plan Nacional I + D of the Spanish Ministry of Science. Juarez P. is a recipient of a FPU fellowship, and Sarrion-Perdigones A. and Huet-Trujillo E. are recipients of a FPI fellowship. We want to thank Monedero for kindly providing scFv and VP8* clones.Juárez Ortega, P.; Huet Trujillo, E.; Sarrion-Perdigones, A.; Falconi, E.; Granell Richart, A.; Orzáez Calatayud, DV. (2013). Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants. International Journal of Molecular Sciences. 14(3):6205-6222. https://doi.org/10.3390/ijms14036205S62056222143Reichert, J. M., & Valge-Archer, V. E. (2007). 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Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnology Journal, 8(5), 564-587. doi:10.1111/j.1467-7652.2009.00497.xSarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE, 6(7), e21622. doi:10.1371/journal.pone.0021622Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE, 6(2), e16765. doi:10.1371/journal.pone.0016765Monedero, V., Rodriguez-Diaz, J., Viana, R., Buesa, J., & Perez-Martinez, G. (2004). Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei. 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The Journal of Immunology, 178(3), 1589-1597. doi:10.4049/jimmunol.178.3.1589Murthy, A. K., Chaganty, B. K. R., Troutman, T., Guentzel, M. N., Yu, J.-J., Ali, S. K., … Arulanandam, B. P. (2011). Mannose-Containing Oligosaccharides of Non-Specific Human Secretory Immunoglobulin A Mediate Inhibition of Vibrio cholerae Biofilm Formation. PLoS ONE, 6(2), e16847. doi:10.1371/journal.pone.0016847Mathias, A., & Corthésy, B. (2011). N-Glycans on secretory component. Gut Microbes, 2(5), 287-293. doi:10.4161/gmic.2.5.18269Hu, L., Crawford, S. E., Czako, R., Cortes-Penfield, N. W., Smith, D. F., Le Pendu, J., … Prasad, B. V. V. (2012). Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen. Nature, 485(7397), 256-259. doi:10.1038/nature10996Yu, X., Dang, V. T., Fleming, F. E., von Itzstein, M., Coulson, B. S., & Blanchard, H. (2012). Structural Basis of Rotavirus Strain Preference toward N-Acetyl- or N-Glycolylneuraminic Acid-Containing Receptors. Journal of Virology, 86(24), 13456-13466. doi:10.1128/jvi.06975-11Haselhorst, T., Fleming, F. E., Dyason, J. C., Hartnell, R. D., Yu, X., Holloway, G., … von Itzstein, M. (2008). Sialic acid dependence in rotavirus host cell invasion. Nature Chemical Biology, 5(2), 91-93. doi:10.1038/nchembio.134Bonner, A., Almogren, A., Furtado, P. B., Kerr, M. A., & Perkins, S. J. (2008). The Nonplanar Secretory IgA2 and Near Planar Secretory IgA1 Solution Structures Rationalize Their Different Mucosal Immune Responses. Journal of Biological Chemistry, 284(8), 5077-5087. doi:10.1074/jbc.m807529200Yoo, E. M., & Morrison, S. L. (2005). IgA: An immune glycoprotein. Clinical Immunology, 116(1), 3-10. doi:10.1016/j.clim.2005.03.010Karnoup, A. S., Turkelson, V., & Anderson, W. H. K. (2005). O-Linked glycosylation in maize-expressed human IgA1. 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Klimapflanzen und biologische Wege zu negativen Kohlendioxidemissionen

Climate plants are critical to prevent global warming as all efforts to save carbon dioxide are too slow and climate disasters on the rise. For best carbon dioxide harvesting we compare algae, trees and crop plants and use metagenomic analysis of environmental samples. We compare different pathways, carbon harvesting potentials of different plants as well as synthetic modifications including carbon dioxide flux balance analysis. For implementation, agriculture and modern forestry are important