Reaction optimization of a Suzuki‐Miyaura cross‐coupling using design of experiments

The combination of lab automation and design of experiments for the execution of screening experiments increases productivity and reduces error-prone manual work. A self-developed software tool allows for creating fractional-factorial experimental design (FFED). Application of FFED on the screening of a Suzuki-Miyaura cross-coupling leads to a 93 % reduced design compared to full-factorial design. The resulting regression model qualitatively shows the positive effect of educt concentrations, time, and temperature and reveals the decrease in conversion at high base concentrations

Quantitative in vivo analysis of chromatin binding of Polycomb and Trithorax group proteins reveals retention of ASH1 on mitotic chromatin

The Polycomb (PcG) and Trithorax (TrxG) group proteins work antagonistically on several hundred developmentally important target genes, giving stable mitotic memory, but also allowing flexibility of gene expression states. How this is achieved in quantitative terms is poorly understood. Here, we present a quantitative kinetic analysis in living Drosophila of the PcG proteins Enhancer of Zeste, (E(Z)), Pleiohomeotic (PHO) and Polycomb (PC) and the TrxG protein absent, small or homeotic discs 1 (ASH1). Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy reveal highly dynamic chromatin binding behaviour for all proteins, with exchange occurring within seconds. We show that although the PcG proteins substantially dissociate from mitotic chromatin, ASH1 remains robustly associated with chromatin throughout mitosis. Finally, we show that chromatin binding by ASH1 and PC switches from an antagonistic relationship in interphase, to a cooperative one during mitosis. These results provide quantitative insights into PcG and TrxG chromatin-binding dynamics and have implications for our understanding of the molecular nature of epigenetic memor

The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila

BACKGROUND: The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. RESULTS AND DISCUSSION: Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. CONCLUSIONS: Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes

Design and characterization of a flow reaction calorimeter based on FlowPlate® Lab and Peltier elements

Continuous manufacturing and development of flow processes depend significantly on an optimized and adapted determination of thermokinetic data of chemical reactions. Reaction calorimetry represents a prominent technique to quantify the heat release of exothermic reactions. This work presents a continuous flow calorimetric measurement system based on a commercially available hastelloy C-22 microreactor. A sensor array of Peltier elements is added to the existing microreactor setup to enable the additional functionality of flow calorimetry. The calorimeter and its additional equipment are connected to open-source soft- and hardware for data acquisition and processing as well as automated reaction screening. The reaction calorimeter can be operated in both isoperibolic and isothermal operation mode. The calorimeter's performance is investigated on the basis of model reactions, where good agreement with literature was obtained for determined reaction enthalpies

Tuning heterologous glucan biosynthesis in yeast to understand and exploit plant starch diversity

Background: Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. Results: We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. Conclusions: Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process. Keywords: Amylopectin structure; Arabidopsis thaliana; Heterologous expression in yeast; Parallel reaction monitoring, Proteomics; Starch biosynthesis; YFP reporter

Quantitative proteomics identifies reduced NRF2 activity and mitochondrial dysfunction in Atopic Dermatitis

Atopic Dermatitis (AD) is the most common inflammatory skin disease and characterized by a deficient epidermal barrier and cutaneous inflammation. Genetic studies suggest a key role of keratinocytes in AD pathogenesis, but the alterations in the proteome that occur in the full epidermis have not been defined. Using a pressure-cycling technology and data-independent acquisition approach, we performed quantitative proteomics of epidermis from healthy volunteers and lesional and non-lesional patient skin. Results were validated by targeted proteomics using parallel reaction monitoring mass spectrometry and immunofluorescence staining. Proteins that were differentially abundant in the epidermis of AD vs. control patients reflect the strong inflammation in lesional skin and the defect in keratinocyte differentiation and epidermal stratification that already characterizes non-lesional skin. Most importantly, they reveal impaired activation of the NRF2-antioxidant pathway and reduced abundance of mitochondrial proteins involved in key metabolic pathways in the affected epidermis. Analysis of primary human keratinocytes with siRNA-mediated NRF2 knock-down revealed that the impaired NRF2 activation and mitochondrial abnormalities are partially interlinked. These results provide insight into the molecular alterations in the epidermis of AD patients and identify potential targets for pharmaceutical intervention

The rawrr R Package: Direct Access to Orbitrap Data and Beyond

The Bioconductor project (Nat. Methods2015, 12 (2), 115–121) has shown that the R statistical environment is a highly valuable tool for genomics data analysis, but with respect to proteomics, we are still missing low-level infrastructure to enable performant and robust analysis workflows in R. Fundamentally important are libraries that provide raw data access. Our R package rawDiag (J. Proteome Res.2018, 17 (8), 2908–2914) has provided the proof-of-principle how access to mass spectrometry raw files can be realized by wrapping a vendor-provided advanced programming interface (API) for the purpose of metadata analysis and visualization. Our novel package rawrr now provides complete, OS-independent access to all spectral data logged in Thermo Fisher Scientific raw files. In this technical note, we present implementation details and describe the main functionalities provided by the rawrr package. In addition, we report two use cases inspired by real-world research tasks that demonstrate the application of the package. The raw data used for demonstration purposes was deposited as MassIVE data set MSV000086542. Availability: https://github.com/fgcz/rawrr.ISSN:1535-3893ISSN:1535-390

Quantitative proteomics analysis of Angiostrongylus vasorum-induced alterations in dog serum sheds light on the pathogenesis of canine angiostrongylosis

Blood contains hundreds of proteins, reflecting ongoing cellular processes and immune reactions. Infections with the blood-dwelling cardiopulmonary nematode Angiostrongylus vasorum in dogs manifest with a broad spectrum of clinical signs including respiratory distress, bleeding diathesis and neurological signs, and are associated with a perturbed blood protein profile in dogs. However, current knowledge does not completely explain the observed pathologies induced by A. vasorum infections, including bleeding disorders. Using sera from experimentally infected dogs, dog serum proteome was analysed by quantitative mass spectrometry methods over several time points before and after inoculation. Following computational analysis, we identified 139 up- and downregulated proteins after infection (log2 ratio cut-off ≥ 1.0; q-value ≤ 0.05). Among upregulated proteins were chitinase 3-like 1 and pulmonary surfactant-associated protein B (log2 fold-changes ≥ 5). Pathway enrichment revealed the complement (especially the lectin pathway) and coagulation cascades as significantly affected upon analysis of downregulated proteins. Among them were mannan-binding lectin serine peptidases, ficolin, and coagulation factor XIII-B. These results bring new elements towards understanding the underlying pathomechanisms of bleeding diatheses observed in some A. vasorum-infected dogs

The Angiostrongylus vasorum Excretory/Secretory and Surface Proteome Contains Putative Modulators of the Host Coagulation

Angiostrongylus vasorum is a cardiopulmonary nematode of canids and is, among others, associated with bleeding disorders in dogs. The pathogenesis of such coagulopathies remains unclear. A deep proteomic characterization of sex specific A. vasorum excretory/secretory proteins (ESP) and of cuticular surface proteins was performed, and the effect of ESP on host coagulation and fibrinolysis was evaluated in vitro. Proteins were quantified by liquid chromatography coupled to mass spectrometry and functionally characterized through gene ontology and pathway enrichment analysis. In total, 1069 ESP (944 from female and 959 from male specimens) and 1195 surface proteins (705 and 1135, respectively) were identified. Among these were putative modulators of host coagulation, e.g., von Willebrand factor type D domain protein orthologues as well as several proteases, including serine type proteases, protease inhibitors and proteasome subunits. The effect of ESP on dog coagulation and fibrinolysis was evaluated on canine endothelial cells and by rotational thromboelastometry (ROTEM). After stimulation with ESP, tissue factor and serpin E1 transcript expression increased. ROTEM revealed minimal interaction of ESP with dog blood and ESP did not influence the onset of fibrinolysis, leading to the conclusion that Angiostrongylus vasorum ESP and surface proteins are not solely responsible for bleeding in dogs and that the interaction with the host’s vascular hemostasis is limited. It is likely that coagulopathies in A. vasorum infected dogs are the result of a multifactorial response of the host to this parasitic infection