Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase

This research was supported by the German Academic Exchange Service (DAAD), the Wellcome Trust (TKS and WNH as Trust Senior Research fellows), the Biotechnology and Biological Science Research Council (Structural Proteomics of Rational Targets) and the European Synchrotron Radiation Facility.Background: Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components. Results: Putative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)-over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of ( R)mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix alpha 2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding. Conclusion: Our new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.Publisher PDFPeer reviewe

Lipidomic analysis of fats and oils – a lot more than just omega-3

TKS research is supported in part by the Wellcome Trust, SUSLA, BBSRC and the European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDReD)Edible oils and fats are among the most abundant cooking ingredients in the world, and are an important part of a healthy balanced diet, especially if they are high in omega-6 and omega-3 polyunsaturated fatty acids. Rather than just the total fatty acid compositions, the analysis of individual lipid species within these oils and fats has become increasingly important. Within the past decade several mass spectrometric lipidomics methods have been adapted and applied to the analysis of edible oils and fats. These methodologies are vital for the analysis of a plethora of lipid species that will be important for numerous health and sustainability issues in the future.PostprintPeer reviewe

Cultivating 21st century competencies in a virtual worlds learning environment

Education reforms in recent years have pressured schools to show achievement results through testing and conformity to standards. Problems of low student engagement in the current test-heavy environment have been a serious barrier to learning in schools across the United States, especially in low socioeconomic areas. After years of unsuccessful testing programs, educators and researchers are calling for approaches that enhance student engagement and foster the 21st century competencies that students need to succeed. Researchers have found that engagement, 21st century competencies, and learning can be enhanced using virtual worlds approaches (Arici, 2008; Barab, Dodge, & Ingram-Goble, 2006; Dede, Nelson, Ketelhut, Clarke, & Bowman, 2004; Klopfer, Osterweil, &Salen, 2009; Ludgate, 2008). Research in learning supports socialization and situated experiences in which content is learned in a meaningful, active context such as is provided by virtual worlds (Brown, Collins, & Duguid, 1989; Gee, 2003; Lave & Wenger, 1991). This mixed-methods study used existing quantitative student data from the Quest Atlantis Project at Indiana University, and qualitative survey data from trained teachers experienced with the Quest Atlantis virtual worlds learning environment. Research questions addressed teacher observations of 21st century competencies, the degree that students were engaged with Quest Atlantis, and looked for other benefits seen by teachers. Findings showed (a) Quest Atlantis fosters 21st century competencies as reported by teachers; (b) Quest Atlantis is highly engaging for students; and (c) Academic content learned in Quest Atlantis transfers to traditional testing formats. Future research is recommended to examine why teachers in this study reported relatively lower levels of student creativity. Additionally, because students of low socioeconomic status showed equal or better results in 21st century competencies, further study of socioeconomic variables relating to learning in virtual worlds is recommended. The National Education Technology Plan (2010) recommends fostering 21st century competencies and new learning approaches such as virtual worlds, games, and other interactive technologies. Continued study of virtual worlds holds potential for innovative solutions for improving student engagement and learning in America\u27s classrooms

Damping of Electron Density Structures and Implications for Interstellar Scintillation

The forms of electron density structures in kinetic Alfven wave turbulence are studied in connection with scintillation. The focus is on small scales $L \sim 10^8-10^{10}$ cm where the Kinetic Alfv\'en wave (KAW) regime is active in the interstellar medium. MHD turbulence converts to a KAW cascade, starting at 10 times the ion gyroradius and continuing to smaller scales. These scales are inferred to dominate scintillation in the theory of Boldyrev et al. From numerical solutions of a decaying kinetic Alfv\'en wave turbulence model, structure morphology reveals two types of localized structures, filaments and sheets, and shows that they arise in different regimes of resistive and diffusive damping. Minimal resistive damping yields localized current filaments that form out of Gaussian-distributed initial conditions. When resistive damping is large relative to diffusive damping, sheet-like structures form. In the filamentary regime, each filament is associated with a non-localized magnetic and density structure, circularly symmetric in cross section. Density and magnetic fields have Gaussian statistics (as inferred from Gaussian-valued kurtosis) while density gradients are strongly non-Gaussian, more so than current. This enhancement of non-Gaussian statistics in a derivative field is expected since gradient operations enhance small-scale fluctuations. The enhancement of density gradient kurtosis over current kurtosis is not obvious, yet it suggests that modest fluctuation levels in electron density may yield large scintillation events during pulsar signal propagation in the interstellar medium. In the sheet regime the same statistical observations hold, despite the absence of localized filamentary structures. Probability density functions are constructed from statistical ensembles in both regimes, showing clear formation of long, highly non-Gaussian tails

A brief journey into the history of, and future sources and uses of fatty acids

The authors would like to thank the Engineering and Physical Sciences Research Council, University of St. Andrews, and the EPSRC Centre for Doctoral Training in Critical Resource Catalysis (CRITICAT) for financial support (Ph.D. studentship to MC; Grant code: EP/L016419/1).Fats and lipids have always had a primarily role in the history of humankind, from the ancient civilisations to the modern and contemporary time, going from domestic and cosmetic uses, to the first medical applications and later to the large scale industrial uses for food, pharmaceutical, cosmetics and biofuel production. Sources and uses of those have changed during time following the development of chemical sciences and industrial technological advances. Plants, fish and animal fats have represented the primary source of lipids and fats for century. Nowadays the use of fatty acid sources has taken a turn: industries are mainly interested in polyunsaturated fatty acids (PUFAs), which have beneficial properties in human health; and also, for high-value fatty acids product for innovative and green production of biofuel and feedstocks. Thus, the constant increase in demand of fatty acids, the fact that marine and vegetable sources are not adequate to meet the high level of fatty acids required worldwide and climate change, have determined the necessity of the search for renewable and sustainable sources for fatty acids. Biotechnological advances and bioengineering have started looking at the genetic modification of algae, bacteria, yeasts, seeds and plants to develop cell-factory able to produce high value fatty acid products in renewable and sustainable manner. This innovative approach applied to FAs industry is a peculiar example of how biotechnology can serve as powerful mean to drive the production of high value fatty acid derivatives on the concept of circular bioeconomy, based on the reutilisation of organic resources for alternative and sustainable productive patterns that are environmentally friendly.Publisher PDFPeer reviewe

Coupled Enzyme Activity and Thermal Shift Screening of the Maybridge Rule of 3 Fragment Library Against Trypanosoma brucei Choline Kinase; A Genetically Validated Drug Target

In this study we interrogate 630 compounds of the Maybridge Rule of 3 Fragment Library for compounds that interact with, and inhibit TbCK. The Maybridge Rule of 3 Fragment Library is a small collection of quantifiable diverse, pharmacophoric rich, chemical entities that comply with the following criteria; MW ≤ 300, cLogP ≤ 3, H-Bond Acceptors ≤ 3, H-Bond Donors ≤ 3, Rotatable bonds (Flexibility Index) ≤ 3, Polar Surface Area ≤ 60 Å2 and aqueous solubility ≥ 1 mM using LogS and high purity (≥ 95%). Comparisons between two different screening methods, a coupled enzyme activity assay and differential scanning fluorimetry, has allowed identification of compounds that interact and inhibit the T. brucei choline kinase, several of which possess selective trypanocidal activity. Screening of a comparatively small fragment library by two different screening methods has allowed identification of several compounds that interact with and inhibit TbCK, a genetically validated drug target against African sleeping sickness. Some of the inhibitory fragments were also selectively trypanocidal, considering these are relatively simple molecules with no optimization, finding low μΜ inhibitors is very encouraging. Moreover some of the morphological phenotypes of these trypanocidal compounds include cell-cycle arrests similar to those observed for the TbCK conditional knockout grown under permissive conditions

Lipids and Liposomes in the Enhancement of Health and Treatment of Disease

TKS research is supported in part by the Wellcome Trust, SUSLA, BBSRC and the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDReD).Publisher PD

The lipidome of Crithidia fasiculata and its plasticity

We would like to thank the Engineering and Physical Sciences Research Council, University of St. Andrews, and the EPSRC Centre for Doctoral Training in Critical Resource Catalysis (CRITICAT) for financial support [Ph.D. studentship to MC; Grant code: EP/L016419/1].Crithidia fasiculata belongs to the trypanosomatidae order of protozoan parasites, bearing close relation to other kinetoplastid parasites such as Trypanosoma brucei and Leishmania spp. As an early diverging lineage of eukaryotes, the study of kinetoplastid parasites has provided unique insights into alternative mechanisms to traditional eukaryotic metabolic pathways. Crithidia are a monogenetic parasite for mosquito species and have two distinct lifecycle stages both taking place in the mosquito gut. These consist of a motile choanomastigote form and an immotile amastigote form morphologically similar to amastigotes in Leishmania. Owing to their close relation to Leishmania, Crithidia are a growing research tool, with continuing interest in its use as a model organism for kinetoplastid research with the added benefit that they are non-pathogenic to humans and can be grown with no special equipment or requirements for biological containment. Although comparatively little research has taken place on Crithidia, similarities to other kinetoplast species has been shown in terms of energy metabolism and genetics. Crithidia also show similarities to kinetoplastids in their production of the monosaccharide D-arabinopyranose similar to Leishmania, which is incorporated into a lipoarabinogalactan a major cell surface GPI-anchored molecule. Additionally, Crithidia have been used as a eukaryotic expression system to express proteins from other kinetoplastids and potentially other eukaryotes including human proteins allowing various co- and post-translational protein modifications to the recombinant proteins. Despite the obvious usefulness and potential of this organism very little is known about its lipid metabolism. Here we describe a detailed lipidomic analyses and demonstrate the possible placidity of Crithidia’s lipid metabolis. This could have important implications for biotechnology approaches and how other kinetoplastids interact with, and scavenge nutrients from their hosts.Publisher PDFPeer reviewe