In plant-pathogen interactions, components of the plant ubiquitination machinery are preferred targets of pathogen-encoded effectors suppressing defense responses or co-opting host cellular functions for accommodation. Here, we employed transient and stable gene silencing-and over-expression systems in Hordeum vulgare (barley) to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15). The partial ARM1 gene was derived from an ancient gene-duplication event in a common ancestor of the Triticeae tribe of grasses comprising the major crop species H. vulgare, Triticum aestivum and Secale cereale. The barley gene HvARM1 contributed to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis, and allelic variants were found to be associated with powdery mildew-disease severity. Both HvPUB15 and HvARM1 proteins interacted in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. The results suggest a neo-functionalization HvARM1 to increase resistance against powdery mildew and provide a link to plastid function in susceptibility to biotrophic pathogen attack.

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.

Developing a robust and performant data analysis workflow that integrates all necessary components whilst still being able to scale over multiple compute nodes is a challenging task. We introduce a generic method based on the microservice architecture, where software tools are encapsulated as Docker containers that can be connected into scientific workflows and executed in parallel using the Kubernetes container orchestrator. The access point is a virtual research environment which can be launched on-demand on cloud resources and desktop computers. IT-expertise requirements on the user side are kept to a minimum, and established workflows can be re-used effortlessly by any novice user. We validate our method in the field of metabolomics on two mass spectrometry studies, one nuclear magnetic resonance spectroscopy study and one fluxomics study, showing that the method scales dynamically with increasing availability of computational resources. We achieved a complete integration of the major software suites resulting in the first turn-key workflow encompassing all steps for mass-spectrometry-based metabolomics including preprocessing, multivariate statistics, and metabolite identification. Microservices is a generic methodology that can serve any scientific discipline and opens up for new types of large-scale integrative science.

The activity and abundance of proteins within a cell are controlled precisely to ensure the regulation of cellular and physiological processes. In eukaryotes, this can be achieved by targeting specific proteins for degradation by the ubiquitinproteasome system. The N-end rule pathway, a subset of the ubiquitinproteasome system, targets proteins for degradation depending on the identity of a protein N-terminal residue or its post-translational modifications. Here, we discuss the most recent findings on the diversity of N-end rule pathways. We also focus on recently found defensive functions of the N-end rule pathway in plants. We then discuss the current understanding of N-end rule substrate formation by protease cleavage. Finally, we review state-of-the-art proteomics techniques used for N-end rule substrate identification, and discuss their usefulness and limitations for the discovery of the molecular mechanisms underlying the roles of the N-end rule pathway in plants.

The new coumarine derivate with methyl ester of 2-((Z)-1(2,4-dioxochroman-3-ylidene)ethylamino)-3-methylbutanoic acid and the corresponding palladium(II) complex are synthesized and characterized by microanalysis, infrared, 1H and 13C NMR spectroscopy. The proposed structure of the ligand was confirmed based on the X-ray structural study.

Amyloid-β (Aβ) deposition is one of the hallmarks of the amyloid hypothesis in Alzheimer’s disease (AD). Mouse models using APP-transgene overexpression to generate amyloid plaques have shown to model only certain parts of the disease. The extent to which the data from mice can be transferred to man remains controversial. Several studies have shown convincing treatment results in reducing Aβ and enhancing cognition in mice but failed totally in human. One model-dependent factor has so far been almost completely neglected: the endogenous expression of mouse APP and its effects on the transgenic models and the readout for therapeutic approaches.Here, we report that hAPP-transgenic models of amyloidosis devoid of endogenous mouse APP expression (mAPP-knockout / mAPPko) show increased amounts and higher speed of Aβ deposition than controls with mAPP. The number of senile plaques and the level of aggregated hAβ were elevated in mAPPko mice, while the deposition in cortical blood vessels was delayed, indicating an alteration in the general aggregation propensity of hAβ together with endogenous mAβ. Furthermore, the cellular response to Aβ deposition was modulated: mAPPko mice developed a pronounced and age-dependent astrogliosis, while microglial association to amyloid plaques was diminished. The expression of human and murine aggregation-prone proteins with differing amino acid sequences within the same mouse model might not only alter the extent of deposition but also modulate the route of pathogenesis, and thus, decisively influence the study outcome, especially in translational research.

Background: Type 2 diabetes mellitus (T2DM) is the most common lifestyle disease affecting all countries. Due to its asymptomatic onset, it is often diagnosed after irreversible vascular complications have been initiated. Therefore, specific markers characteristic for very early disease stages and suitable for early diagnostics are required. Glycation of plasma proteins, such as human serum albumin (HSA), has been often suggested as marker. However, the total glycation degree of HSA does not provide sufficient information about short-term fluctuations of blood glucose concentrations due to the large number of glycation sites. Analysis of individual modification sites might be more informative, but methods for reliable quantifications are still missing.Objective: The main objective of this study was to establish and qualify a method of analysis applicable to sensitive and precise quantification of glycations sites in plasma proteins.Methods: Plasma samples obtained from diabetic patients and non-diseased individuals were separated from low-molecular weight compounds, digested with trypsin, enriched for glycated peptides by boronic acid affinity chromatography (BAC), desalted by solid phase extraction (SPE), and separated by RP-HPLC coupled online to ESI-QqQ-MS. Quantification relied on multiple reaction monitoring (MRM) of multiple glycation sites identified in plasma proteins using a stable isotope dilution approach or internal standardization.Results: The data presented here suggests high selectivity and precision (relative standard deviations below 10%) of the overall approach appearing to be well suited for the identification of prospective biomarkers. Six glycated peptides corresponding to different glycation sites of HSA were present in plasma samples obtained from T2DM patients at significantly higher levels than in non-diabetic men matched for age. Additionally, each of the studied glycation site of HSA appeared to be affected at different degrees.Conclusion: The presented approach enables the sensitive and robust quantification of prospective T2D biomarkers promising for clinical diagnostics.

Type 2 diabetes mellitus (T2DM) is a complex group of disorders, characterized by hyperglycemia, insulin resistance and insulin deficiency. In human blood, hyperglycemia ultimately results in the enhancement of glycation – a posttranslational modification formed by the interaction of protein amino groups with glucose. The resulting fructosamines (Amadori compounds) readily undergo further degradation resulting in advanced glycation end products (AGEs), known to be pro-inflammatory in humans. These compounds are highly heterogeneous and characteristic of advanced stages of the disease, whereas fructosamines are recognized markers of early diabetes stages (HbA1C, glycated albumin). Recently, individual plasma protein glycation sites were proposed as promising T2DM biomarkers sensitive to short-term fluctuations of plasma glucose. However, corresponding absolute quantification strategies, applicable in regular clinical practice, are still not established. Therefore, here we propose a new analytical approach aiming at reproducible and precise quantification of multiple glycated peptides in human plasma tryptic digests. Thereby, the standard peptides comprised a 13C,15N-labeled lysyl residue, a dabsyl moiety for determination of standard amounts, and a cleavable linker. Known amounts of these peptides were spiked to plasma samples prior to tryptic digestion, quantification relying on stable isotope dilution. The method was demonstrated to be applicable for quantification of individual glycated sites in T2DM patients and non-diabetic controls.

Glycation is a non-enzymatic post-translational modification of proteins, formed by the reaction of reducing sugars and α-dicarbonyl products of their degradation with amino and guanidino groups of proteins. Resulted early glycation products are readily involved in further transformation, yielding a heterogeneous group of advanced glycation end products (AGEs). Their formation is associated with ageing, metabolic diseases, and thermal processing of foods. Therefore, individual glycation adducts are often considered as the markers of related pathologies and food quality. In this context, their quantification in biological and food matrices is required for diagnostics and establishment of food preparation technologies. For this, exhaustive protein hydrolysis with subsequent amino acid analysis is the strategy of choice. Thereby, multi-step enzymatic digestion procedures ensure good recoveries for the most of AGEs, whereas tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode with stable isotope dilution or standard addition represents “a gold standard” for their quantification. Although the spectrum of quantitatively assessed AGE structures is continuously increases, application of untargeted profiling techniques for identification of new products is desired, especially for in vivo characterization of anti-glycative systems. Thereby, due to a high glycative potential of plant metabolites, more attention needs to be paid on plant-derived AGEs.

Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs) represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus), proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress.