Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.     

A genetic analysis of body size in pink salmon (Oncorhynchus gorbuscha)

Two small-sized and two large-sized male pink salmon (Oncorhynchus gorbuscha) were mated to each of four females, producing eight families sired by small males and eight sired by large males. The juveniles were reared for 500 d after fry emergence. Juvenile weight in the two male size classes was similar until the spring of the year of maturity, when juveniles sired by large males grew faster than those sired by small ones. Heritability estimates of weight based upon the dam component of variance increased during 500 d of rearing from 0.4 to 0.8. Heritability of weight based upon the sire component of variance generally ranged between 0.1 and 0.3. The large variation in male body size in spawning pink salmon populations may have resulted from different male breeding strategies.     

Inflorescence architecture: A developmental genetics approach

We are characterizing a suiteof Pisum sativum mutants that alter inflorescence architecture to construct a model for the genetic regulation of inflorescence development in a plant with a compound raceme. Such a model, when compared with those created forAntirrhinum majus andArabidopsis thaliana, both of which have simple racemes, should provide insight into the evolution of the development of inflorescence architecture. The highly conserved nature of cloned genes that regulate reproductive development in plants and the morphological similarities among our mutants and those identified inA. majus andA. thaliana enhance the probability that a developmental genetics approach will be fruitful. Here we describe sixP. sativum mutants that affect morphologically and architecturally distinct aspects of the inflorescence, and we analyze interactions among these genes. Both vegetative and inflorescence growth of the primary axis is affected byUNIFOLIA TA, which is necessary for the function ofDETERMINATE (DET).DET maintains indeterminacy in the first-order axis. In its absence, the meristem differentiates as a stub covered with epidermal hairs.DET interacts withVEGETATIVE1 (VEG1).VEG1 appears essential for second-order inflorescence (I₂) development.veg1 mutants fail to flower or differentiate the I₂ meristem into a rudimentary stub,det veg1 double mutants produce true terminal flowers with no stubs, indicating that two genes must be eliminated for terminal flower formation inP. sativum, whereas elimination of a single gene accomplishes this inA. thaliana andA. majus. NEPTUNE also affects I₂ development by limiting to two the number of flowers produced prior to stub formation. Its role is independent ofDET, as indicated by the additive nature of the double mutantdet nep. UNI, BROC, and PIM all play roles in assigning floral meristem identity to the third-order branch.pim mutants continue to produce inflorescence branches, resulting in a highly complex architecture and aberrant flowers.uni mutants initiate a whorl of sepals, but floral organogenesis is aberrant beyond that developmental point, and the double mutantuni pim lacks identifiable floral organs. A wild-type phenotype is observed inbroc plants, butbroc enhancesthe pim phenotype in the double mutant, producing inflorescences that resemble broccoli. Collectively these genes ensure that only the third-order meristem, not higher- or lower-order meristems, generates floral organs, thus precisely regulating the overall architecture of the plant. Gene symbols used in this article: For clarity a common symbolization is used for genes of all species discussed in this article. Genes are symbolized with italicized capital letters. Mutant alleles are represented by lowercase, italicized letters. In both cases, the number immediately following the gene symbol differentiates among genes with the same symbol. If there are multiple alleles, a hyphen followed by a number is used to distinguish alleles. Protein products are represented by capital letters without italics.     

Single and dual task gait training in people with Parkinson's Disease: A protocol for a randomised controlled trial

Background  

Difficulty performing more than one task at a time (dual tasking) is a common and disabling problem experienced by people with Parkinson disease (PD). If asked to perform another task when walking, people with PD often take shorter steps or walk more slowly. Currently there is uncertainty about whether clinicians should teach people with PD to avoid dual tasking or whether they should encourage them to practice dual tasking with the hope that practice will lead to enhanced performance. This study will address this issue by comparing single to dual task gait training.     

The application of fluorescence spectroscopy to organic matter characterisation in drinking water treatment

Key to effective disinfection byproduct (DBP) management is source water control and management, and more specifically, organic matter (OM) control and management. However, the content and character of OM in source waters is spatially and temporally variable, and the prediction of its composition is challenging. Water treatment companies require adequate analytical techniques for OM characterisation to maintain the operation of the water supply and treatment systems adjusted to constantly changing environmental conditions. There is a requirement, therefore, for an improved understanding of OM composition and character in source water, how that composition and character varies with flow conditions, and how this impacts on drinking water treatment. This paper demonstrates that fluorescence spectroscopy offers a potential alternative to other analytical methods of OM characterisation. The advantages of fluorescence include rapid, sensitive and selective characterisation of OM, no sample pre-treatment, small sample volume, and the potential for on-line monitoring incorporation. Fluorescence can provide useful information on OM reactivity and treatability together with an indication of the OM sources (allochthonous or autochthonous). The paper discusses a body of literature which has identified relationships between fluorescence spectra and OM physico-chemical properties (i.e. degree of hydrophobicity, microbial content), has applied fluorescence spectroscopy to characterise the changes in OM upon disinfection, and has related the fluorescence properties to DBP formation. Further work is required in the robust management of data arising from fluorescence spectroscopy analysis and, in particular, Excitation Emission Matrices. Consideration must be given as to how the data might best be employed to greatest effect on a routine basis at WTW.     

Design and synthesis of potent antagonists containing rigid spirocyclic privileged structures for the CGRP receptor

We report the synthesis of rigid spirocyclic systems as conformationally constrained variants of the Ala-Phe-NH(2) dipeptide amide C-terminus of the calcitonin gene-related peptide (CGRP). CGRP receptor antagonists containing these moieties displayed potent affinity, functional antagonism and excellent oxidative stability. Structure-activity relationship studies demonstrated the relative importance of hydrogen bond donor/acceptor functionalities and the preferred orientation of an aromatic ring. Antagonists showed potent and full reversal of CGRP-induced dilation of ex vivo human intracranial arteries.     

The design and synthesis of indazole and pyrazolopyridine based glucokinase activators for the treatment of Type 2 diabetes mellitus

Glucokinase activators represent a promising potential treatment for patients with Type 2 diabetes. Herein, we report the identification and optimization of a series of novel indazole and pyrazolopyridine based activators leading to the identification of 4-(6-(azetidine-1-carbonyl)-5-fluoropyridin-3-yloxy)-2-ethyl-N-(5-methylpyrazin-2-yl)-2H-indazole-6-carboxamide (42) as a potent activator with favorable preclinical pharmacokinetic properties and in vivo efficacy.     

Targeting conserved water molecules: Design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.     

Ageing increases vulnerability to aβ42 toxicity in Drosophila

Age is the major risk factor for many neurodegenerative diseases, including Alzheimer's Disease (AD), for reasons that are not clear. The association could indicate that the duration or degree of exposure to toxic proteins is important for pathology, or that age itself increases susceptibility to protein toxicity. Using an inducible Drosophila model of AD, we investigated these possibilities by varying the expression of an Aβ42 transgene in neurons at different adult ages and measuring the effects on Aβ42 levels and associated pathological phenotypes. Acute induction of Arctic Aβ42 in young adult flies resulted in rapid expression and clearance of mRNA and soluble Arctic Aβ42 protein, but in irreversible expression of insoluble Arctic Aβ42 peptide. Arctic Aβ42 peptide levels accumulated with longer durations of induction, and this led to a dose-dependent reduction in negative geotaxis and lifespan. For a standardised level of mRNA expression, older flies had higher levels of Arctic Aβ42 peptide and associated toxicity, and this correlated with an age-dependent reduction in proteasome activity. Equalising Aβ42 protein at different ages shortened lifespan in correlation with the duration of exposure to the peptide, suggesting that Aβ42 expression accumulates damage over time. However, the relative reduction in lifespan compared to controls was greater in flies first exposed to the peptide at older ages, suggesting that ageing itself also increases susceptibility to Aβ42 toxicity. Indeed older flies were more vulnerable to chronic Aβ42 toxicity even with a much lower lifetime exposure to the peptide. Finally, the persistence of insoluble Aβ42 in both young and old induced flies suggests that aggregated forms of the peptide cause toxicity in later life. Our results suggest that reduced protein turnover, increased duration of exposure and increased vulnerability to protein toxicity at later ages in combination could explain the late age-of-onset of neurodegenerative phenotypes.