Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Seit Februar 2021 bietet Wolfgang Brandt, ehemaliger Leiter der Arbeitsgruppe Computerchemie am IPB, sein Citizen Science-Projekt zur Pilzbestimmung an. Dafür hat er in regelmäßigen Abständen öffentliche Vorträge zur Vielfalt…
Leonova, T.; Popova, V.; Tsarev, A.; Henning, C.; Antonova, K.; Rogovskaya, N.; Vikhnina, M.; Baldensperger, T.; Soboleva, A.; Dinastia, E.; Dorn, M.; Shiroglasova, O.; Grishina, T.; Balcke, G. U.; Ihling, C.; Smolikova, G.; Medvedev, S.; Zhukov, V. A.; Babakov, V.; Tikhonovich, I. A.; Glomb, M. A.; Bilova, T.; Frolov, A.;Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds?Int. J. Mol. Sci.21567(2020)DOI: 10.3390/ijms21020567
Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.
Publikation
Gladchuk, A.; Shumilina, J.; Kusnetsova, A.; Bureiko, K.; Billig, S.; Tsarev, A.; Alexandrova, I.; Leonova, L.; Zhukov, V. A.; Tikhonovich, I. A.; Birkemeyer, C.; Podolskaya, E.; Frolov, A.;High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass SpectrometryMethods Protoc.336(2020)DOI: 10.3390/mps3020036
Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.
Publikation
Nausch, H.; Dorn, M.; Frolov, A.; Hoedtke, S.; Wolf, P.; Broer, I.;Direct Delivery of Health Promoting β-Asp-Arg Dipeptides via Stable Co-expression of Cyanophycin and the Cyanophycinase CphE241 in Tobacco PlantsFront. Plant Sci.11842(2020)DOI: 10.3389/fpls.2020.00842
Feed supplementation with β-arginine-aspartate dipeptides (β-Asp-Arg DP) shows growth promoting effects in feeding trials with fish and might also be beneficial for pig and poultry farming. Currently, these DPs are generated from purified cyanophycin (CGP), with the help of the CGP-degrading enzyme cyanophycinase (CGPase). As alternative to an in vitro production, the DPs might be directly produced in feed crops. We already demonstrated that CGP can be produced in plastids of tobacco and potato, yielding up to 9.4% of the dry weight (DW). We also transiently co-expressed CGPases in the cytosol without degrading CGP in intact cells, while degradation occurs in the homogenized plant tissue. However, transient co-expression is not feasible for field-grown CGP plants, which is necessary for bulk production. In the present study, we proved that stable co-expression of the CGPase CphE241 in CGP-producing tobacco is sufficient to degrade 2.0% CGP/DW nearly completely within 3 h after homogenization of the leaves. In intact senescing leaves, CGP is partially released to the cytosol and degraded into DPs which limits the overall accumulation of CGP but not the level of the stable DPs. Even after 48 h, 54 μmol β-Asp-Arg DP/g DW could be detected in the extract, which correspond to 1.5% DP/DW and represents 84% of the expected amount. Thus, we developed a system for the production of β-Asp-Arg DP in field-grown plants.
Publikation
Meshalkina, D. A.; Glushchenko, A. S.; Kysil, E. V.; Mizgirev, I. V.; Frolov, A.;SpCas9- and LbCas12a-Mediated DNA Editing Produce Different Gene Knockout Outcomes in Zebrafish EmbryosGenes11740(2020)DOI: 10.3390/genes11070740
CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) genome editing is a powerful technology widely used in current genetic research. In the most simple and straightforward way it can be applied for a gene knockout resulting from repair errors, induced by dsDNA cleavage by Cas nuclease. For decades, zebrafish (Danio rerio) has been known as a convenient model object of developmental biology. Both commonly used nucleases SpCas9 (Streptococcus pyogenes Cas9) and LbCas12a (Lachnospiraceae bacterium Cas12a) are extensively used in this model. Among them, LbCas12a is featured with higher specificity and efficiency of homology-directed editing in human cells and mouse. But the editing outcomes for these two nucleases in zebrafish are still not compared quantitatively. Therefore, to reveal possible advantages of one nuclease in comparison to the other in the context of gene knockout generation, we compare here the outcomes of repair of the DNA breaks introduced by these two commonly used nucleases in zebrafish embryos. To address this question, we microinjected the ribonucleoprotein complexes of the both nucleases with the corresponding guide RNAs in zebrafish zygotes and sequenced the target gene regions after three days of development. We found that LbCas12a editing resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies (percentage of mutated copies of the target gene to all gene copies in the embryo) of both nucleases were the same. On the other hand, overlapping of protospacers resulted in similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.