Flavonol Glucosylation: A Structural Investigation of the Flavonol Specific 3-O Glucosyltransferase Cp3GT

Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin cleavage site was incorporated proximal to the tag, followed by cloning in Pichia pastoris, methanol-induced expression, and cobalt-affinity chromatography for initial purification stages. Notably, the recombinant tags did not exhibit a discernible influence on Cp3GT kinetics, substrate preference, pH optima, or metal interactions, maintaining its specificity towards flavonols at the 3-OH position and favoring glucosylation of quercetin and kaempferol. Subsequent purification steps, including MonoQ anion exchange and size-exclusion chromatography, yielded Cp3GT with ≥95% homogeneity. In silico molecular models of Cp3GT and its truncated variants, Cp3GTΔ80 and Cp3GTΔ10, were constructed using D-I-TASSER and COFACTOR to assess binding interactions with quercetin and kaempferol. Results indicated minimal interference of c-myc/6x-his tags with the native Cp3GT structure. This study not only lays a foundation for impending crystallographic studies, aiming to solidify the understanding of Cp3GT\u27s stringent 3-O flavonol specificity, but also accentuates the potential of microbial expression platforms and plant metabolic engineering in producing beneficial compounds. To this end, a thorough review of four pivotal classes of plant secondary metabolites, flavonoids, alkaloids, betalains, and glucosinolates, was conducted. This will open avenues for further research and applications in biotechnological, medical, and agricultural domains

The Effect of Recombinant Tags on Citrus Paradisi Flavonol-Specific 3-O Glucosyltransferase Activity

Recombinant tags are used extensively in protein expression systems to allow purification through IMAC (Immobilized Metal Affinity Chromatography), identification through Western blot, and to facilitate crystal formation for structural analysis. While widely used, their role in enzyme characterization has raised concerns with respect to potential impact on activity. In this study, a flavonol-specific 3-O glucosyltransferase (Cp3GT) from grapefruit (Citrus paradisi) was expressed in Pichia pastoris, and was assayed in its untagged form and with a C-terminal c-myc/6x His tag under various conditions to determine the effect of tags. Prior characterization of pH optima for Cp3GT obtained through expression in Escherichia coli, containing an N-terminal thioredoxin/6x His tag, indicated an optimal pH of 7–7.5, which is indicative of a normal physiological pH and agrees with other glucosyltransferase (GT) pH optima. However, characterization of Cp3GT expressed using P. pastoris with a C-terminal c-myc-6x His tag showed a higher optimal pH of 8.5–9. This suggests a possible tag effect or an effect related to physiological differences between the cell expression systems. Results testing recombinant Cp3GT expressed in Pichia with and without C-terminal tags showed a possible tag effect with regard to substrate preference and interactions with metals, but no apparent effect on enzymatic kinetics or pH optima

Metabolic Engineering and Synthetic Biology of Plant Natural Products – a Minireview

Plant natural products include a diverse array of compounds that play important roles in plant metabolism and physiology. After elucidation of biosynthetic pathways and regulatory factors, it has become possible to metabolically engineer new capabilities in planta as well as successfully engineer whole pathways into microbial systems. Microbial expression systems for producing valuable plant compounds have evolved to incorporate polyculture and co-culture consortiums for carrying out robust biosynthesis strategies. This review focuses on four classes of plant secondary metabolites and the recent advances in generating useful compounds in microbial expression platforms and in plant metabolic engineering. They are the flavonoids, alkaloids, betalains, and glucosinolates

Application of Large-Scale Synthetic Power System Models for Energy Economic Studies

Due to information confidentiality issues, there is limited access to actual power system models that represent features of actual power grids for teaching, training, and research purposes. The authors\u27 previous work describes the process of creating synthetic transmission networks, with statistics similar to those of actual power grids. Thus, this paper outlines a systematic methodology to augment the synthetic network base case for energy economic studies. The key step is to determine generator cost models by fuel type and capacity. Based on statistics summarized from the actual grids, two approaches are proposed to assign coefficients to generator cost models. To illustrate the proposed creation procedure, we describe the construction of a synthetic model for Electric Reliability Council of Texas footprint. Simulation results are presented to verify that the created test system is able to represent the behavior of actual power systems

Meaningful engagement: computer-based interactive media art in public space.

Interactive technologies, including electronic devices are increasingly being utilized as a medium for artistic expression and have been placed in freely accessible public environments with mixed results. When audiences encounter computer-based interactive media arts in a public space they are drawn by various interactivities, to play and experiment with them. However, whether the audience is able to gain a meaningful experience through those physical interactivities has remained an issue of both theoretical and practical debate. This paper will focus on these aspects, most specifically through the study of interactive art in freely accessible public space. The author proposes four new conceptual/analytical tools for examining the subject. It is anticipated that this paper will provide possible alternative strategies for both artists and art researchers in this field with a purpose to enhance intellectual engagement with their audiences, so as to succeed in leading interactors to obtain meaningful experience and rewards

Fuzzy Free Path Detection based on Dense Disparity Maps obtained from Stereo Cameras

In this paper we propose a fuzzy method to detect free paths in real-time using digital stereo images. It is based on looking for linear variations of depth in disparity maps, which are obtained by processing a pair of rectified images from two stereo cameras. By applying least-squares fitting over groups of disparity maps columns to a linear model, free paths are detected by giving a certainty using a fuzzy rule. Experimental results on real outdoor images are also presented.Nuria Ortigosa acknowledges the support of Universidad Polit'ecnica de Valencia under grant FPI-UPV 2008. Samuel Morillas acknowledges the support of Spanish Ministry of Education and Science under grant MTM 2009-12872-C02-01.Ortigosa Araque, N.; Morillas Gómez, S.; Peris Fajarnes, G.; Dunai Dunai, L. (2012). Fuzzy Free Path Detection based on Dense Disparity Maps obtained from Stereo Cameras. 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Rethinking globalised resistance : feminist activism and critical theorising in international relations

This article argues that a feminist approach to the 'politics of resistance' offers a number of important empirical insights which, in turn, open up lines of theoretical inquiry which critical theorists in IR would do well to explore. Concretely, we draw on our ongoing research into feminist 'anti-globalisation' activism to rethink the nature of the subject of the politics of resistance, the conditions under which resistance emerges and how resistance is enacted and expressed. We begin by discussing the relationship of feminism to critical IR theory as a way of situating and explaining the focus and approach of our research project. We then summarise our key empirical arguments regarding the emergence, structure, beliefs, identities and practices of feminist 'anti-globalisation' activism before exploring the implications of these for a renewed critical theoretical agenda in IR