THE DYNAMICS OF RECIPROCAL SELECTIVE SWEEPS OF HOST RESISTANCE AND A PARASITE COUNTER‐ADAPTATION IN DROSOPHILA

Host–parasite coevolution can result in consecutive selective sweeps of host resistance alleles and parasite counter‐adaptations. To illustrate the dynamics of this important but little studied form of coevolution, we have modeled an ongoing arms race between Drosophila melanogaster and the vertically transmitted sigma virus, using parameters we estimated in the field. We integrate these results with previous work showing that the spread of a resistance allele of the ref(2)P gene in the host was followed by the spread of a virus genotype, which overcomes this resistance. In line with these observations, our model predicts that there can be rapid selective sweeps in both the host and parasite, which can drive large changes in the prevalence of infection. The virus will tend to be ahead in the arms race, as incomplete dominance slows down host adaptation and selection for host resistance is weaker than selection for parasites to overcome resistance—the “life‐dinner” principle. This asymmetry in the adaptation rates results in a partial sweep of the host resistance allele, as it loses its advantage part way through the selective sweep. This well‐understood natural system illustrates how the outcome of host–parasite coevolution is determined by different population genetic parameters in the field.     

A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway and is a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40-μl assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)–thiosemicarbazide (TSC) or DAMO–antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives, including dimethyl sulfoxide, reducing agents, detergents, and bovine serum albumin. DAMO–TSC (Z′-factors 0.7–0.8) was determined to be superior to DAMO–antipyrine (Z′-factors 0.5–0.6) with significantly less variability within replicates. An HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO–TSC. This robust method has no heating requirement, which was the main obstacle to applying previous assays to HTS. More important, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large-scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.     

Very rapid phosphorylation kinetics suggest a unique role for Lhcb2 during state transitions in Arabidopsis

Light‐harvesting complex II (LHCII) contains three highly homologous chlorophyll‐a/b‐binding proteins (Lhcb1, Lhcb2 and Lhcb3), which can be assembled into both homo‐ and heterotrimers. Lhcb1 and Lhcb2 are reversibly phosphorylated by the action of STN7 kinase and PPH1/TAP38 phosphatase in the so‐called state‐transition process. We have developed antibodies that are specific for the phosphorylated forms of Lhcb1 and Lhcb2. We found that Lhcb2 is more rapidly phosphorylated than Lhcb1: 10 sec of ‘state 2 light’ results in Lhcb2 phosphorylation to 30% of the maximum level. Phosphorylated and non‐phosphorylated forms of the proteins showed no difference in electrophoretic mobility and dephosphorylation kinetics did not differ between the two proteins. In state 2, most of the phosphorylated forms of Lhcb1 and Lhcb2 were present in super‐ and mega‐complexes that comprised both photosystem (PS)I and PSII, and the state 2‐specific PSI–LHCII complex was highly enriched in the phosphorylated forms of Lhcb2. Our results imply distinct and specific roles for Lhcb1 and Lhcb2 in the regulation of photosynthetic light harvesting.     

Design,synthesis and structure–activity relationships of zwitterionic spirocyclic compounds as potent CCR1 antagonists

A series of zwitterionic spirocyclic compounds were synthesised. In vitro data revealed that these compounds were potent CCR1 antagonists. In particular, 2, 4, 11 and 20 inhibited CCR1 mediated chemotaxis of THP-1 cells in a functional assay.     

Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.     

Genetic biodiversity in the Baltic Sea: species-specific patterns challenge management

Information on spatial and temporal patterns of genetic diversity is a prerequisite to understanding the demography of populations, and is fundamental to successful management and conservation of species. In the sea, it has been observed that oceanographic and other physical forces can constitute barriers to gene flow that may result in similar population genetic structures in different species. Such similarities among species would greatly simplify management of genetic biodiversity. Here, we tested for shared genetic patterns in a complex marine area, the Baltic Sea. We assessed spatial patterns of intraspecific genetic diversity and differentiation in seven ecologically important species of the Baltic ecosystem—Atlantic herring (Clupea harengus), northern pike (Esox lucius), European whitefish (Coregonus lavaretus), three-spined stickleback (Gasterosteus aculeatus), nine-spined stickleback (Pungitius pungitius), blue mussel (Mytilus spp.), and bladderwrack (Fucus vesiculosus). We used nuclear genetic data of putatively neutral microsatellite and SNP loci from samples collected from seven regions throughout the Baltic Sea, and reference samples from North Atlantic areas. Overall, patterns of genetic diversity and differentiation among sampling regions were unique for each species, although all six species with Atlantic samples indicated strong resistence to Atlantic-Baltic gene-flow. Major genetic barriers were not shared among species within the Baltic Sea; most species show genetic heterogeneity, but significant isolation by distance was only detected in pike and whitefish. These species-specific patterns of genetic structure preclude generalizations and emphasize the need to undertake genetic surveys for species separately, and to design management plans taking into consideration the specific structures of each species.