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NMR conformational analysis of a hydroxyethylamine peptide isostere developed as an aspartic protease inhibitor shows that it is a flexible architecture. Cyclization to form pyrrolidines, piperidines, or morpholines results in a preorganization of the whole system in solution. The resulting conformation is similar to the conformation of the inhibitor in the active site of BACE-1. This entropic gain results in increased affinity for the enzyme when compared with the acyclic system. For morpholines 27 and 29, the combination of steric and electronic factors is exploited to orient substituents toward S1, S1', and S2' pockets both in the solution and in the bound states. These highly preorganized molecules proved to be the most potent compounds of the series. Additionally, the morpholines, unlike the pyrrolidine and piperidine analogues, have been found to be brain penetrant BACE-1 inhibitors.

The genus Ecdytolopha Zeller, 1875 includes 14 described species whose geographic distributions encompass most of temperate North America and a large part of the northern Neotropics. Two new species are described: Ecdytolopha nigrita Brown, new species, from southeastern Arizona, U.S.A.; and Ecdytolopha meridana Brown, new species, from Merida, Venezuela. A neighbor-joining tree based on COI sequences of Ecdytolopha insiticiana Zeller, E. mana (Kearfott), E. occidentana Adamski and Brown, E. nigrita, and E. fabivora (Meyrick) is presented. Although percent divergence within species is typical for Lepidoptera, divergences between species of Ecdytolopha are fairly large, with the most similar pair (i.e., E. insiticiana and E. mana) 9.1% different.

The wheat curl mite, Aceria tosichella Keifer, one of the most destructive arthropod pests of bread wheat worldwide, inflicts significant annual reductions in grain yields. Moreover, A. tosichella is the only vector for several economically important wheat viruses in the Americas, Australia and Europe. To date, mite-resistant wheat genotypes have proven to be one of the most effective methods of controlling the A. tosichella-virus complex. Thus, it is important to elucidate A. tosichella population genetic structure, in order to better predict improved mite and virus management. Two genetically distinct A. tosichella lineages occur as pests of wheat in Australia, Europe, North America, South America and the Middle East. These lineages are known as type 1 and type 2 in Australia and North America and in Europe and South America as MT-8 and MT-1, respectively. Type 1 and type 2 mites in Australia and North America are delineated by internal transcribed spacer 1 region (ITS1) and cytochrome oxidase I region (COI) sequence differences. In North America, two A. tosichella genotypes known as biotypes are recognized by their response to the Cmc3 mite resistance gene in wheat. Aceria tosichella biotype 1 is susceptible to Cmc3 and biotype 2 is virulent to Cmc3. In this study, ITS1 and COI sequence differences in 25 different populations of A. tosichella of known biotype 1 or biotype 2 composition were characterized for ITS1 and COI sequence differences and used to model spatio-temporal dynamics based on biotype prevalence. Results showed that the proportion of biotype 1 and 2 varies both spatially and temporally. Greater ranges of cropland and grassland within 5000m of the sample site, as well as higher mean monthly precipitation during the month prior to sampling appear to reduce the probability of occurrence of biotype 1 and increase the probability of occurrence of biotype 2. The results suggest that spatio-temporal modeling can effectively improve A. tosichella management. Continual integration of additional current and future precipitation and ground cover data into the existing model will further improve the accuracy of predicting the occurrence of A. tosichella in annual wheat crops, allowing producers to make informed decisions about the selection of varieties with different A. tosichella resistance genes.

The molecular bases of aphid virulence to aphid crop plant resistance genes are poorly understood. The Russian wheat aphid, Diuraphis noxia, (Kurdjumov), and the greenbug, Schizaphis graminum (Rondani), are global pest of cereal crops. Each species damages barley, oat, rye and wheat, but S. graminum includes fescue, maize, rice and sorghum in its host range. This study was conducted to compare and contrast the transcriptomes of S. graminum biotype I and D. noxia biotype 1 when each ingested phloem from leaves of varieties of bread wheat, Triticum aestivum L., containing no aphid resistance (Dn0), resistance to D. noxia biotype 1 (Dn4), or resistance to both D. noxia biotype 1 and S. graminum biotype I (Dn7, wheat genotype 94M370). Gene ontology enrichments, k-means analysis and KEGG pathway analysis indicated that 94M370 plants containing the Dn7 D. noxia resistance gene from rye had stronger effects on the global transcriptional profiles of S. graminum and D. noxia relative to those fed Dn4 plants. S. graminum responds to ingestion of phloem sap from 94M370 plants by expression of unigenes coding for proteins involved in DNA and RNA repair, and delayed tissue and structural development. In contrast, D. noxia displays a completely different transcriptome after ingesting phloem sap from Dn4 or 94M370 plants, consisting of unigenes involved primarily in detoxification, nutrient acquisition and structural development. These variations in transcriptional responses of D. noxia and S. graminum suggest that the underlying evolutionary mechanism(s) of virulence in these aphids are likely species specific, even in cases of cross resistance.

Data pipeline platforms hosting big data analytics can span multiple clouds. Backup and restore service is typically applied to deal with data corruptions in such platforms. This paper proposes a novel approach to providing consistency to the restored state of a multi-cloud data pipeline platform from its backups, and also presents the performance of the approach demonstrated in a dry run test.

Species of Farfantepenaeus support economically important shrimp fisheries throughout the Western Hemisphere, necessitating proper fisheries management. To be effective, species management should be informed of the potential presence of cryptic species and of the evolutionary forces driving biodiversity. This is best accomplished through a robust phylogenetic framework and evidence-based species delimitation. This study represents the first comprehensive molecular phylogeny and species delimitation analyses of shrimps belonging to the genus Farfantepenaeus. Targeting three mitochondrial genes (12S, 16S, and COI), gene trees and a phylogeny for the genus were inferred using maximum likelihood and Bayesian approaches. In general, the phylogenetic relationships inferred here largely agree with those recovered from morphological data, including the most recent designation of F. isabelae as sister to F. subtilis. Molecular divergence was found between northern and southern populations of F. brasiliensis, suggesting the existence of unrecognised subspecies. However, previous recognition of F. duorarum and F. notialis as two distinct species was not supported by this study. The phylogeny inferred here also uncovers a phylogeographic signal of latitudinal speciation in the genus. The study presented here provides valuable insight into the evolutionary history of Farfantepenaeus, improving our ability to effectively manage these economically important species.

Despite the ecological importance of deep-sea cephalopods, little is known about their genetic diversity or population dynamics. The cephalopod species Cranchia scabra, Pyroteuthis margaritifera, and Vampyroteuthis infernalis are commonly collected in midwater samples from both the Gulf of Mexico and northwestern Atlantic Ocean but, despite their common appearance in trawls and important roles in marine food webs, no genetic studies of population connectivity exist for these species. Here, Sanger sequencing of three conserved genetic loci and ddRADseq techniques were used to examine population genetic dynamics in these deep-sea species. Genetic diversity is lowest in C. scabra, which appears to be in a population growth stage, and highest in V. infernalis. Population structure was unique to V. infernalis but does not appear to be the result of ocean-basin vicariance, thus possible alternative explanations are explored, specifically environmental variation in dissolved oxygen. The genetic connectivity between these geographically disparate sites suggests these three cephalopod species could be resilient to localized environmental disturbances in the Gulf of Mexico.

Inhibition of BACE1 has become an important strategy in the quest for disease modifying agents to slow the progression of Alzheimer's disease. We previously reported the fragment-based discovery of LY2811376, the first BACE1 inhibitor reported to demonstrate robust reduction of human CSF Aβ in a Phase I clinical trial. We also reported on the discovery of LY2886721, a potent BACE1 inhibitor that reached phase 2 clinical trials. Herein we describe the preparation and structure activity relationships (SAR) of a series of BACE1 inhibitors utilizing trans-cyclopropyl moieties as conformational constraints. The design, details of the stereochemically complex organic synthesis, and biological activity of these BACE1 inhibitors is described.

Heliothine larvae, especially early instars, are difficult to identify, and determinations sometimes rely on indirect information such as origin and host data. The introduction of Helicoverpa armigera (Hubner) into the New World has undermined the reliability of host and origin data to identify intercepted Helicoverpa larvae, and suspect Heliothinae/Helicoverpa larvae intercepted at U.S. ports of entry are now screened for H. armigera and Helicoverpa zea (Boddie) using molecular methods. Here, we analyze heliothine larvae intercepted during 2014-2106 to identify nontargets and evaluate morphological characters traditionally used to separate taxa. In total, nine species were identified, with Chloridea virescens (Fabricius) making up the bulk of interception records. The majority of heliothine suspects originate from Mexico and Peru on pigeon pea, chickpea, tomatillo, pea, and corn. Helicoverpa armigera is commonly intercepted from Peru on pea. Chloridea virescens is recorded from every country where interceptions were identified for this study except Guatemala and is found on multiple hosts. Identification issues and specific host/origin associations are discussed in detail.

As an early visitor to the injured loci, neutrophil-derived human Myeloperoxidase (hMPO) offers an attractive protein target to modulate the inflammation of the host tissue through suitable inhibitors. We describe a novel methodology of using low temperature ESR spectroscopy (6 K) and FAST™ technology to screen a diverse series of small molecules that inhibit the peroxidase function through reversible binding to the native state of MPO. Our initial efforts to profile molecules on the inhibition of MPO-initiated nitration of the Apo-A1 peptide (AEYHAKATEHL) assay showed several potent (with sub-micro molar IC50s) but spurious inhibitors that either do not bind to the heme pocket in the enzyme or retain high (>50 %) anti oxidant potential. Such molecules when taken forward for X-ray did not yield inhibitor-bound co-crystals. We then used ESR to confirm direct binding to the native state enzyme, by measuring the binding-induced shift in the electronic parameter g to rank order the molecules. Molecules with a higher rank order—those with g-shift R relative ≥15—yielded well-formed protein-bound crystals (n = 33 structures). The co-crystal structure with the LSN217331 inhibitor reveals that the chlorophenyl group projects away from the heme along the edges of the Phe366 and Phe407 side chain phenyl rings thereby sterically restricting the access to the heme by the substrates like H2O2. Both ESR and antioxidant screens were used to derive the mechanism of action (reversibility, competitive substrate inhibition, and percent antioxidant potential). In conclusion, our results point to a viable path forward to target the native state of MPO to tame local inflammation.

Moths in the genus Helicoverpa are some of the most important agricultural pests in the world. Two species, H. armigera (Hübner) and H. zea (Boddie), cause the majority of damage to crops and millions of dollars are spent annually on control of these pests. The recent introduction of H. armigera into the New World has prompted extensive survey efforts for this species in the United States. Surveys are conducted using bucket traps baited with H. armigera pheromone, and, because the same pheromone compounds attract both species, these traps often capture large numbers of the native H. zea. Adult H. armigera and H. zea are very similar and can only be separated morphologically by minor differences in the genitalia. Thus, a time consuming genitalic dissection by a trained specialist is necessary to reliably identify either species, and every specimen must be dissected. Several molecular methods are available for differentiating and identifying H. armigera and H. zea, including two recently developed rapid protocols using real-time PCR. However, none of the published methods are capable of screening specimens in large batches. Here we detail a droplet digital PCR (ddPCR) assay that is capable of detecting a single H. armigera in a background of up to 999 H. zea. The assay has been tested using bulk extractions of 1,000 legs from actual trap samples and is effective even when using poor quality samples. This study provides an efficient, rapid, reproducible, and scalable method for processing H. armigera survey trap samples in the U.S. and demonstrates the potential for applying ddPCR technology to screen and diagnose invasive species.

Dynamics of three MET antibody constructs (IgG1, IgG2, and IgG4) and the IgG4-MET antigen complex was investigated by creating their atomic models with an integrative experimental and computational approach. In particular, we used two-dimensional (2D) Electron Microscopy (EM) images, image class averaging, homology modeling, Rapidly exploring Random Tree (RRT) structure sampling, and fitting of models to images, to find the relative orientations of antibody domains that are consistent with the EM images. We revealed that the conformational preferences of the constructs depend on the extent of the hinge flexibility. We also quantified how the MET antigen impacts on the conformational dynamics of IgG4. These observations allow to create testable hypothesis to investigate MET biology. Our protocol may also help describe structural diversity of other antigen systems at approximately 5 A precision, as quantified by Root-Mean-Square Deviation (RMSD) among good-scoring models.

Thaumatovalva gen. n. is described and illustrated from the Afrotropical region. As currently defined the genus includes four species: T. deprinsorum sp .n. from the Democratic Republic of Congo; T. albolineana sp .n. (type species) from the Democratic Republic of Congo; T. spinai (Razowski & Trematerra), comb. n., from Ethiopia and Nigeria; and T. limbata (Diakonoff), comb. n., from the Seychelles and Kenya. Thaumatovalva limbata has been reared from the fruit of Cordia somaliensis Baker and C. monoica Roxb. (Boraginaceae) in Kenya. Although structures of the male and female genitalia are extremely similar among three of the four species, male secondary scales on the under surface of the hindwing easily distinguish them.

Objectives To evaluate variability in health literacy outcomes due to home visiting (HV) program components including PHN, Intervention, and Client. Design and Sample A comparative, correlational study evaluated PHN home visiting program data that included PHNs (N = 16); Interventions (N = 21,634); and Clients (N = 141). Client age ranged from 14 to 46 (median = 21, mean = 22.8, SD = 6.65). Clients were predominately White (75.9%), not married (84.4%), and female (99.3%). PHNS documented care using electronic health records (EHR) and the Omaha System. Measures The outcome of interest was health literacy benchmark attainment (adequate knowledge) operationalized by Omaha System Problem Rating Scale for Outcomes Knowledge scores averaged across problems. Intervention Program of individually tailored, evidence-based HV interventions provided by PHNs. Results There were 233 different interventions for 22 problems. Knowledge benchmark was attained by 16.3% of clients. Four factors explained variance in reaching the knowledge benchmark: Client (51%), Problem (17%), Intervention (16%), and PHN (16%). Conclusions The PHN and intervention tailoring are actionable components of HV programs that explain variability in health literacy outcomes. Further research should examine effects of training on PHN relationship skills and intervention tailoring to optimize outcomes of evidence-based PHN HV programs, and to evaluate whether improving health literacy may subsequently improve client problems.

Comprising over 15,000 living species, decapods (crabs, shrimp, and lobsters) are the most instantly recognizable crustaceans, representing a considerable global food source. Although decapod systematics have received much study, limitations of morphological and Sanger sequence data have yet to produce a consensus for higher-level relationships. Here we introduce a new anchored hybrid enrichment kit for decapod phylogenetics designed from genomic and transcriptomic sequences that we used to capture new high-throughput sequence data from 94 species, including 58 of 179 extant decapod families, and 11 of 12 major lineages. The enrichment kit yields 410 loci (>86,000 bp) conserved across all lineages of Decapoda, eight times more molecular data than any prior study. Phylogenomic analyses recover a robust decapod tree of life strongly supporting the monophyly of all infraorders, and monophyly of each of the reptant, ‘lobster’, and ‘crab’ groups, with some results supporting pleocyemate monophyly. We show that crown decapods diverged in the Late Ordovician and most crown lineages diverged in the Triassic-Jurassic, highlighting a cryptic Paleozoic history, and post-extinction diversification. New insights into decapod relationships provide a phylogenomic window into morphology and behavior, and a basis to rapidly and cheaply expand sampling in this economically and ecologically significant invertebrate clade.

Despite being taxonomically and phylogenetically informative, the morphology of the immature stages of cicadas has received comparatively superficial attention. One reason for this is the difficulty of positively identifying immature stages, particularly as these stages are fossorial. We present a method for identifying cicada exuviae using DNA sequence data and describe a set of characters and character states for the final instar nymph of Quintilia carinata (Thunberg). The identification of immature stages using molecular methods will increase our knowledge of African cicadas, allowing for the initiation of future phylogenetic and ecological comparisons.

Mobile healthcare applications enjoy increasing popularity and provide significant benefits to users who can take advantage of the general availability and the increasing set of sensors in mobile devices. Healthcare analytics services developed for a variety of conditions to provide insights and guidance to patients can augment the mobile applications with computationally intensive analysis at the server-side environment. Creating applications that are supported by analytics is challenging for different reasons: We must deal with the limited information to get relevant recommendations, or not enough to provide appropriate predictions (patients trajectory to reach goal such as weight loss). Moreover building an analytical model in the health domain brings its own challenges such as data ingestion, data curation, and service levels, etc.This paper proposes a software architecture framework that eases the support of analytics in the mobile health applications. The paper also discusses the various components that make up the framework by taking one analytical model as a use case.

The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion.

Synopsis Sipuncula is a relatively small taxon with roughly 150 recognized species. Many species are geographically widespread or ‘‘cosmopolitan.’’ The pelagosphera larvae of some species are estimated to spend several months in the plankton. However, recent molecular evidence suggests that many of the ‘‘cosmopolitan’’ species actually represent species-complexes, some not even monophyletic. Herein, we present data on three sipunculan species with different developmental modes that occur both in the Sea of Japan and in the Northeast Pacific. The development of the three species—Phascolosoma agassizii, Thysanocardia nigra, and Themiste pyroides—is exceptionally well studied in both regions of the Pacific. Significant differences have been observed between the two regions with respect to egg size, developmental mode, and developmental timing. In general, eggs are larger and development slower in the Northeast Pacific when compared with the Sea of Japan. These differences have been explained as a result of phenotypic plasticity exhibited under different environmental conditions, in particular temperature, but we show that the populations of all three species are also remarkably distinct genetically and that gene flow between the two regions is extremely unlikely. In Thysanocardia nigra, we even found two very distinct genetic lineages within the same location in the Northeast Pacific. The amount of genetic divergence between populations from the Sea of Japan and those from the Northeast Pacific is not correlated with developmental mode. Themiste pyroides, the species with the most abbreviated development, actually has the least degree of genetic divergence between the regions. Analyses of molecular variance show that the majority of the observed variation in all three species is between the regions. We conclude that all three ‘‘cosmopolitan’’ species actually represent complexes of cryptic or pseudo-cryptic species. These examples demonstrate that a solid taxonomic framework based on molecular and morphological evidence is a prerequisite for evaluating relationships between dispersal capabilities, species’ ranges, and the connectivity of populations.

The annual swarming of epitokes of the eunicid polychaete Palola viridis has great cultural significance in Samoa and other Indo-Pacific locations. Palola spp. occur worldwide in warm shallow-water environments, but not all populations appear to swarm. Morphological distinctions among Palola species are subtle but, based on phylogenetic analysis of mitochondrial markers, two species groups can be distinguished, each containing numerous subclades. The objectives of this study were to determine the position of P. viridis in the phylogeny of the genus, to assess the species’ genetic distinctness and to describe P. viridis epitokes morphologically. We sequenced epitokes from Samoa (locally known as palolo) and Vanuatu (un) for two mitochondrial markers and performed phylogenetic analyses in combination with previously published sequences. The samples from Samoa and Vanuatu formed a common clade in species group B. We suggest that swarming is restricted to this species group, although probably not to a single species.

Aim The use of host-specific biological control agents is widely considered an effective option for the management of invasive alien plant species. However, the formation of novel associations between released biological control agents and indigenous species poses risks. Here, we investigate whether native food webs associated with two galling biological control agents on Acacia longifolia and A. saligna are similar to those found in their introduced range. Location Gall inhabitants recorded from South Africa and Australia. Methods Non-targeted insects were collected from galls in introduced ranges for comparisons to that of the agents’ native ranges. Results We find that two host plant-specific galling biological control agents accumulate food web links with higher trophic levels in their introduced range that are similar in number, taxonomic/phylogenetic pattern and guild composition to those in their native range. Bray-Curtis percentage similarity between native (Australia) and novel (South Africa) food webs was 30–50% and 50–75% at the family and superfamily taxonomic level, respectively, and 45–50% if considering shared phylogenetic diversity. Main conclusions Trichilogaster acaciaelongifoliae and Uromycladium tepperianum accumulated food webs in South Africa that are strikingly similar in complexity and structure to those that occur in their native ranges. This indicates that the structure of food webs in the introduced range could be predicted by studying food webs in the native range of a biological control agent, potentially paving the way for more effective risk assessment of weed biological control.

At the dimer interface of the extracellular ligand-binding domain of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors a hydrophilic pocket is formed that is known to interact with two classes of positive allosteric modulators, represented by cyclothiazide and the ampakine 2H,3H,6aH-pyrrolidino(2,1–3′,2′)1,3-oxazino(6′,5′-5,4)benzo(e)1,4-dioxan-10-one (CX614). Here, we present structural and functional data on two new positive allosteric modulators of AMPA receptors, phenyl-1,4-bis-alkylsulfonamide (CMPDA) and phenyl-1,4-bis-carboxythiophene (CMPDB). Crystallographic data show that these compounds bind within the modulator-binding pocket and that substituents of each compound overlap with distinct moieties of cyclothiazide and CX614. The goals of the present study were to determine 1) the degree of modulation by CMPDA and CMPDB of AMPA receptor deactivation and desensitization; 2) whether these compounds are splice isoform-selective; and 3) whether predictions of mechanism of action could be inferred by comparing molecular interactions between the ligand-binding domain and each compound with those of cyclothiazide and CX614. CMPDB was found to be more isoform-selective than would be predicted from initial binding assays. It is noteworthy that these new compounds are both more potent and more effective and may be more clinically relevant than the AMPA receptor modulators described previously.

SummaryInterface terminologies used in electronic health records must be re-evaluated and revised to reflect current health care practice and knowledge. To enable future revisions of the Omaha System Intervention Scheme, investigators evaluated formal semantic structure of target terms and concept duplication of problem and target terms. Using linguistic principles and qualitative analysis, five themes were found. A multidimensional formal semantic structure for the intervention target term was proposed. Concept duplication was examined for 16 problem-target matches. Clinical data enabled assessment of the validity of a proposed formal semantic structure and concept duplication. Recommendations are suggested for future development of the Omaha System Intervention Scheme.

International audience; Analysis of population genetic structure is a key aspect to understand insect pest population dynamics in agricultural scenarios. Here the role of geography, hosts and time on the population genetic structure of codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae) populations is described. Temporal variation was examined in two French orchards among each of three adult flights during two successive years. Analyses were conducted using two insecticide resistance markers (variation at the sodium channel gene and enzymatic activity of cytochrome P450 oxidases) and three microsatellite loci. Levels of genetic variation among temporal populations were not significant based on variation in the sodium channel gene and microsatellite loci. However, P450 oxidase activity differed significantly during both flights and years, decreasing during the three flights of the first year and increasing during the second. These results suggest that phytosanitary measures are among the factors shaping the genetic structure of C. pomonella populations over temporal and geographical scales. We discuss the relative importance of natural and passive dispersal related to anthropogenic activities affecting C. pomonella population genetics and highlight population genetic research needs in order to design more efficient pest management practices.

Four tortricid species are of major economic importance on deciduous fruit tree crops in South Africa. They are codling moth, Cydia pomonella, oriental fruit moth, Grapholita molesta, false codling moth, Thaumatotibia leucotreta, and carnation worm, Epichoristodes acerbella. Pest management and phytosanitary practices are often hampered by the inability to distinguish between the immature stages of these four species. To address this need, the final instar larvae and pupae of E. acerbella are described and illustrated. The morphology of these immature stages of E. acerbella is compared with those of the aforementioned tortricid species. Keys are included to distinguish between the four species. In addition, as an alternative method of identification of any life stage, a means of discriminating between the four species, based on analysis of nucleotide sequences of the mitochondrial cytochrome oxidase I gene, is described.

Numerous thrips species cause economic damage to a range of agricultural crops in southern Africa. Much of this economic damage may be prevented by an accurate system for identifying pest thrips species. At present the inability to identify thrips species leads both to poor control in the field and a decrease in exports due to the presence of species of quarantine importance. The objective of this study was therefore to provide a thrips identification system for a selection of economically important species present in southern Africa. For this purpose, a molecular identification tool, based on nucleotide sequencing analysis of the mitochondrial cytochrome oxidase I (COI) gene, was developed. Nine thrips species were included in analyses: Frankliniella occidentalis, F. schultzei, Fulmekiola serrata, Haplothrips nigricornis, H. clarisetis, Heliothrips sylvanus, Scirtothrips aurantii, Synaptorthrips psoraleae and Thrips tabaci. Analysis of 163 individuals showed that each of the nine thrips species could be distinguished on the basis of their COI sequences. Information generated was integrated with that produced previously for thrips species present in Europe and allowed a rapid, accurate and simple means of identifying the species of major economic importance present in southern Africa. The molecular key will provide much-needed information on thrips identification for South African pest management officers and quarantine purposes.

Amplified fragment length polymorphism (AFLP) fingerprints were used to characterize the population genetic structure and gene flow of the oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), for the first time in the major stone-fruit growing regions in South Africa. Populations were collected from six different regions and compared with each other. Furthermore, intraregion gene flow was determined by sampling more extensively from farms and orchards within two of these six regions. Five selective AFLP primer pairs generated 250 fragments. Phylogeny analysis clustered populations from the six regions into two main groups, although those situated close together geographically were not necessarily closely related genetically. Over local scales, populations collected from closely situated orchards (

Three tortricid species are of major economic importance to the South African tropical and subtropical fruit industry. They are the false codling moth, Thaumatotibia leucotreta, the macadamia nut borer, T. batrachopa, and the litchi moth, Cryptophlebia peltastica. Identification of these species is essential for phytosanitary purposes and to aid the management of population levels in the field. For this purpose, species identification was investigated using morphological and molecular genetic techniques. For each species, final instar larvae and pupae are described and illustrated. Diagnostic characters are given for each species and keys included to facilitate identification. In addition, as an alternative to morphological identification, a diagnostic 367-bp region of the mitochondrial cytochrome oxidase I (COI) gene was sequenced from individuals of the three species. The inclusion of both morphological and molecular keys provides a reliable means of identifying the tortricids of economic importance on tropical and subtropical fruit in South Africa.

FAH (fumarylacetoacetate hydrolase) catalyses the final step of tyrosine catabolism to produce fumarate and acetoacetate. HT1 (hereditary tyrosinaemia type 1) results from deficiency of this enzyme. Previously, we prepared a partial mimic of the putative tetrahedral intermediate in the reaction catalysed by FAH co-crystallized with the enzyme to reveal details of the mechanism [Bateman, Bhanumoorthy, Witte, McClard, Grompe and Timm (2001) J. Biol. Chem. 276, 15284–15291]. We have now successfully synthesized complete mimics CEHPOBA {4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate} and COPHPAA {3-[(3-carboxy-2-oxopropyl)hydroxyphosphinyl]acrylate}, which inhibit FAH in slow-onset tight-binding mode with Ki values of 41 and 12 nM respectively. A high-resolution (1.35 Å; 1 Å=0.1 nm) crystal structure of the FAH·CEHPOBA complex was solved to reveal the affinity determinants for these compounds and to provide further insight into the mechanism of FAH catalysis. These compounds are active in vivo, and CEHPOBA demonstrated a notable dose-dependent increase in SA (succinylacetone; a metabolite seen in patients with HT1) in mouse serum after repeated injections, and, following a single injection (1 μmol/g; intraperitoneal), only a modest regain of FAH enzyme activity was detected in liver protein isolates after 24 h. These potent inhibitors provide a means to chemically phenocopy the metabolic defects of either HT1 or FAH knockout mice and promise future pharmacological utility for hepatocyte transplantation.

The BACE1 enzyme is a key target for Alzheimer’s disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core. Pursuit of S3-binding groups produced low micromolar inhibitor 6, which informed the S3-design for constrained analogs 7 and 8, themselves prepared via independent, multi-step synthetic routes. Biological characterization of BACE inhibitors 6–8 is described.

BACE1 is a key protease controlling the formation of amyloid β, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimer's disease (AD). Therefore, the development of potent and selective inhibitors of BACE1 has been a focus of many drug discovery efforts in academia and industry. Herein, we report the nonclinical and early clinical development of LY2886721, a BACE1 active site inhibitor that reached phase 2 clinical trials in AD. LY2886721 has high selectivity against key off-target proteases, which efficiently translatesin vitroactivity into robustin vivoamyloid β lowering in nonclinical animal models. Similar potent and persistent amyloid β lowering was observed in plasma and lumbar CSF when single and multiple doses of LY2886721 were administered to healthy human subjects. Collectively, these data add support for BACE1 inhibition as an effective means of amyloid lowering and as an attractive target for potential disease modification therapy in AD.

Despite the agricultural importance of species in the Grapholitini (Lepidoptera: Tortricidae), and the value of gene expression analysis for improved population management, few gene expression studies based on quantitative real-time PCR (qPCR) have been conducted for this tribe. Part of the reason for this lack of information is that suitable reference genes, which are fundamental for accurate normalization of qPCR studies, have not been identified for the tribe. Thus, the expression stability of six potential reference genes (ACT, AK, COI, EF1, ENO and TUB) was assessed in three different tissues (whole body, midgut and cuticle) of Cryptophlebia peltastica (Meyrick), Cydia pomonella (L.) and Thaumatotibia leucotreta (Meyrick). Additionally, these reference genes were tested using T. leucotreta at different temperatures (15°C, 25°C and 35°C) with and without baculovirus infection. Suitable reference genes were identified for the whole body and midgut tissue of all three species, and for cuticle tissue of Cy. pomonella and T. leucotreta. When T. leucotreta was infected with the virus at all temperature conditions ACT, AK and EF1 were found to be the most suitable reference genes for experimental normalization. In general, for all tissue types, species and stress conditions, AK and EF1 were the best-performing reference genes. However, even though the three species analysed were closely related and within the same tribe, each species required varying gene combinations for suitable normalization. This study provides the first reference gene evaluation for the Tortricidae, and paves the way for future qPCR analysis in Tortricidae.

1 The macadamia nut borer Thaumatotibia (Cryptophlebia) batrachopa and the litchi moth Cryptophlebia peltastica are Afrotropical species causing extensive damage to cultivated macadamias and litchis in the northernmost provinces in South Africa. Cryptophlebia peltastica also occurs as natural populations throughout the country. 2 To analyse the population genetic structure of the two species, amplified fragment length polymorphism analysis was conducted. Patterns of genetic diversity in C. peltastica populations in the Mpumalanga province, where the species is controlled, were compared with those in the Western Cape, where populations occur under natural conditions. 3 Gene diversity was high within T. batrachopa populations (H = 0.2219) with significant genetic differentiation among populations (Gst = 0.358). Cluster analysis showed that geographical populations were closely related and extensive divergence was found over local scales. 4 Similar to that of T. batrachopa, genetic diversity and population differentiation was high within both C. peltastica Mpumalanga and Western Cape populations (H = 0.1906 and 0.1687 and Gst = 0.4124 and 0.3799, respectively). 5 It is suggested that the population genetic structure of both species is influenced by their limited ability to disperse. In addition, chemical control of C. peltastica in the Mpumalanga province has not succeeded in reducing the population size, but the C. peltastica population subdivision in the Western Cape may be influenced by the limited distribution of host plants in this region.

Thiamine pyrophosphokinase transfers a pyrophosphate group from a nucleoside triphosphate, such as ATP, to the hydroxyl group of thiamine to produce thiamine pyrophosphate. Deficiencies in thiamine can result in the development of the neurological disorder Wernicke-Korsakoff Syndrome as well as the potentially fatal cardiovascular disease wet beriberi. Pyrithiamine is an inhibitor of thiamine metabolism that induces neurological symptoms similar to that of Wernicke-Korsakoff Syndrome in animals. However, the mechanism by which pyrithiamine interferes with cellular thiamine phosphoester homeostasis is not entirely clear. We used kinetic assays coupled with mass spectrometry of the reaction products and x-ray crystallography of an equilibrium reaction mixture of thiamine pyrophosphokinase, pyrithiamine, and Mg2+/ATP to elucidate the mechanism by which pyrithiamine inhibits the enzymatic production of thiamine pyrophosphate. Three lines of evidence support the ability of thiamine pyrophosphokinase to form pyrithiamine pyrophosphate. First, a coupled enzyme assay clearly demonstrated the ability of thiamine pyrophosphokinase to produce AMP when pyrithiamine was used as substrate. Second, an analysis of the reaction mixture by mass spectrometry directly identified pyrithiamine pyrophosphate in the reaction mixture. Last, the structure of thiamine pyrophosphokinase crystallized from an equilibrium substrate/product mixture shows clear electron density for pyrithiamine pyrophosphate bound in the enzyme active site. This structure also provides the first clear picture of the binding pocket for the nucleoside triphosphate and permits the first detailed understanding of the catalytic requirements for catalysis in this enzyme.

Conformational analysis in solution of β-secretase inhibitors 1 and 2 by NMR spectroscopy reveals that the hydroxyethylene isostere, an apparently flexible fragment widely used as a scissile bond replacement in aspartic protease inhibitors, exists in one predominant conformation in solution. This preferred conformation is similar to that adopted by the hydroxyethylene core of 1 in complex with β-secretase and that adopted by hydroxyethylene cores of related compounds when bound to aspartic proteases, indicating that this structural unit is preorganized in solution.

The protein kinase family represents an enormous opportunity for drug development. However, the current limitation in structural diversity of kinase inhibitors has complicated efforts to identify effective treatments of diseases that involve protein kinase signaling pathways. We have identified a new structural class of protein serine/threonine kinase inhibitors comprising an aminoimidazo[1,2-a]pyridine nucleus. In this report, we describe the first successful use of this class of aza-heterocycles to generate potent inhibitors of cyclin-dependent kinases that compete with ATP for binding to a catalytic subunit of the protein. Co-crystal structures of CDK2 in complex with lead compounds reveal a unique mode of binding. Using this knowledge, a structure-based design approach directed this chemical scaffold toward generating potent and selective CDK2 inhibitors, which selectively inhibited the CDK2-dependent phosphorylation of Rb and induced caspase-3-dependent apoptosis in HCT 116 tumor cells. The discovery of this new class of ATP-site-directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis for a new medicinal chemistry tool to be used in the search for effective treatments of cancer and other diseases that involve protein kinase signaling pathways.

Plant response to insect feeding appears to be highly specific with regard to the organisms in the system. Here, we report on the interaction between grapevine Vitis vinifera L. plants and a phloem-feeding insect pest, the vine mealybug Planococcus ficus (Homoptera: Pseudococcidae). Plants were exposed to P. ficus for periods of 6 and 96 h, respectively, after which they were analysed for differences in gene expression levels using microarrays. Grapevine displayed a fairly low response to mealybug feeding, with only 107 and 149 transcripts being differentially expressed compared to uninfested control plants after 6 and 96 h of mealybug feeding, respectively. Most of these genes are known to be expressed by grapevine or other plants as a response to microbial pathogen attack. In addition, intermediate exposure times (24, 48 and 72 h) of grapevine plants to P. ficus feeding were investigated using qPCR analysis of ten genes associated with known plant defence responses. Results showed that only a single gene, pathogenesis-related protein 1, was differentially expressed after 48 h of mealybug feeding compared to control plants. Therefore, our study has shown that on a transcriptional level grapevine plants respond weakly to attack by vine mealybugs, which is analogous to a range of other studies on plant responses to piercing-sucking insects.

Eighty thousand years of ice-sheet build-up came to a rapid end ~20–10 thousand years before present (ka BP), when ice sheets receded quickly, and global mean surface temperatures rose by about 4 °C. It still remains unresolved whether insolation changes due to variations of earth's tilt and orbit were sufficient to terminate glacial conditions. Using a coupled three-dimensional climate–ice-sheet model, we simulate the climate and Northern Hemisphere ice-sheet evolution from 78 to 0 ka BP in good agreement with sea level and ice topography reconstructions. Based on this simulation and a series of deglacial sensitivity experiments with individually varying orbital parameters and CO2, we find that enhanced calving led to a slow-down of ice-sheet growth already 5 to 8 ka prior to the Last Glacial Maximum (LGM), as evidenced by the change in curvature of the simulated and reconstructed ice volume time series. Increasing obliquity and precession then led to accelerated ice loss due to ablation and calving, thereby initiating the glacial termination. The deglacial sensitivity experiments further reveal that the ~100 ppmv rise of atmospheric CO2 after ~18 ka BP was a key contributor to the deglaciation. Without it, the present-day ice volume would be comparable to that of the LGM and global mean temperatures would be about 3 °C lower than today. We further demonstrate that neither orbital forcing nor CO2 forcing alone were sufficient to complete the deglaciation.

Isolating RNA from insects is becoming increasingly important in molecular entomology. Four methods including three commercial kits RNeasy Mini Kit (Qiagen), SV Total RNA isolation system (Promega), TRIzol reagent (Invitrogen), and a cetyl trimethylammonium bromide (CTAB)-based method were compared regarding their ability to isolate RNA from whole-body larvae of Thaumatotibia leucotreta (Meyrick), Thanatophilus micans (F.), Plutella xylostella (L.), and Tenebrio molitor (L.). A difference was observed among the four methods regarding RNA quality but not quantity. However, RNA quality and quantity obtained was not dependent on the insect species. The CTAB-based method produced low-quality RNA and the Trizol reagent produced partially degraded RNA, whereas the RNeasy Mini Kit and SV Total RNA isolation system produced RNA of consistently high quality. However, after reverse transcription to cDNA, RNA produced using all four extraction methods could be used to successfully amplify a 708 bp fragment of the cytochrome oxidase I gene. Of the four methods, the SV Total RNA isolation system showed the least amount of DNA contamination with the highest RNA integrity number and is thus recommended for stringent applications where high-quality RNA is required. This is the first comparison of RNA isolation methods among different insect species and the first to compare RNA isolation methods in insects in the last 20 years.

We have previously reported on the use of an open flow helium cryostat in macromolecular crystallographic data collection. This device, developed at the University of Toledo, was used at the APS IMCA beamline to test He as a cryogen and to compare data collected at He temperature to that collected at N 2 temperature. Data were collected on matched crystals of the nucleosome core particle (NCP), fumarylacetoacetate hydrolase (FAH), and the murine monoclonal antibody BV0401 (BV), and other crystals using an Oxford Cryosystems Cryostream and the Toledo Helium Cryostat. In these experiments, crystal lifetime was enhanced by using He as a cryogen. At the end of data collection with NCP, crystals cooled by N 2 were blackened; crystals cooled by He remained clear. A crystal of Mcg light chain dimer (Mcg) had undiminished diffraction in He after 90 min of exposure in the same position. Data improvement is best illustrated in the diffraction data from BV. These data showed an improvement in resolution (1.75–1.48 A) with a significant enhancement of the intensity for weak data. Data from the NCP showed a similar enhancement in intensity. However, some diffraction data exhibited split reflections at He temperature rendering processing of the data more problematic. One possible explanation for these results is a phase change occurring between 100 K and the 30 K of the helium cold stream. Planned work will characterize this phenomenon and extend the experiments to other crystal systems.

Thiamin pyrophosphate (TPP) is a coenzyme derived from vitamin B1 (thiamin). TPP synthesis in eukaryotes requires thiamin pyrophosphokinase (TPK), which catalyzes the transfer of a pyrophosphate group from ATP to thiamin. TPP is essential for central metabolic processes, including the formation of acetyl CoA from glucose and the Krebs cycle. Deficiencies in human thiamin metabolism result in beriberi and Wernicke encephalopathy. The crystal structure of mouse TPK was determined by multiwavelength anomalous diffraction at 2.4 A resolution, and the structure of TPK complexed with thiamin has been refined at 1.9 A resolution. The TPK polypeptide folds as an alpha/beta-domain and a beta-sandwich domain, which share a central ten-stranded mixed beta-sheet. TPK subunits associate as a dimer, and thiamin is bound in the dimer interface. Despite lacking apparent sequence homology with other proteins, the alpha/beta-domain resembles the Rossman fold and is similar to other kinase structures, including another pyrophosphokinase and a thiamin biosynthetic enzyme. Comparison of mouse and yeast TPK structures reveals differences that could be exploited in developing species-specific inhibitors of potential use as antimicrobial agents.

Background: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyrophosphate group from ATP to vitamin B 1 (thiamin) to form the coenzyme thiamin pyrophosphate (TPP). Thus, TPK is important for the formation of a coenzyme required for central metabolic functions. TPK has no sequence homologs in the PDB and functions by an unknown mechanism. The TPK structure has been determined as a significant step toward elucidating its catalytic action. Results: The crystal structure of Saccharomyces cerevisiae TPK complexed with thiamin has been determined at 1.8 A resolution. TPK is a homodimer, and each subunit consists of two domains. One domain resembles a Rossman fold with four α helices on each side of a 6 strand parallel β sheet. The other domain has one 4 strand and one 6 strand antiparallel β sheet, which form a flattened sandwich structure containing a jelly-roll topology. The active site is located in a cleft at the dimer interface and is formed from residues from domains of both subunits. The TPK dimer contains two compound active sites at the subunit interface. Conclusions: The structure of TPK with one substrate bound identifies the location of the thiamin binding site and probable catalytic residues. The structure also suggests a likely binding site for ATP. These findings are further supported by TPK sequence homologies. Although possessing no significant sequence homology with other pyrophospokinases, thiamin pyrophosphokinase may operate by a mechanism of pyrophosphoryl transfer similar to those described for pyrophosphokinases functioning in nucleotide biosynthesis.

Sacroiliac joint dysfunction in elite rowers [Journal of Orthopaedic & Sports Physical Therapy 1999;29(5):288-93]

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.

Homogentisate dioxygenase (HGO) cleaves the aromatic ring during the metabolic degradation of Phe and Tyr. HGO deficiency causes alkaptonuria (AKU), the first human disease shown to be inherited as a recessive Mendelian trait. Crystal structures of apo-HGO and HGO containing an iron ion have been determined at 1.9 and 2.3 A resolution, respectively. The HGO protomer, which contains a 280-residue N-terminal domain and a 140-residue C-terminal domain, associates as a hexamer arranged as a dimer of trimers. The active site iron ion is coordinated near the interface between subunits in the HGO trimer by a Glu and two His side chains. HGO represents a new structural class of dioxygenases. The largest group of AKU associated missense mutations affect residues located in regions of contact between subunits.

Additional examples of successful application of macromolecular crystal annealing are presented. A qualitative evaluation of variables related to the annealing process was conducted using a variety of macromolecular crystals to determine in which cases parameters may be varied and in which cases the original macromolecular crystal annealing protocol is preferred. A hypothesis is presented relating the solvent content of the crystal to the specific protocol necessary for the successful application of annealing.