Protein Bait Research Articles

Cucumber Volatile Blend Attractive to Female Melon Fly, Bactrocera cucurbitae (Coquillett)

The melon fly, Bactrocera cucurbitae (Coquillett), is a serious crop pest throughout the Asia Pacific sub-continent and Southeast Asia, causing damage to tree fruits, cucurbits, and related crops. Attractants for female melon flies are of particular interest as they could be used in control tactics to reduce pest levels. Previous work has shown that freshly sliced cucumbers are attractive to female melon flies, but the compounds responsible for this attraction were not identified. The objective of the present study was to create a synthetic lure for female B. cucurbitae based on its close association with Cucurbitaceae. Coupled gas chromatography-electroantennogram detection (GC-EAD) analysis of fresh and aged puréed cucumbers identified 31 compounds that were detected by females. Compounds that elicited EAD responses initially were screened as single components in glass McPhail traps in outdoor rotating olfactometer experiments. Four criteria were used to select compounds for testing in blends: a) strength of EAD response elicited; b) amount of compound present; c) relative attractiveness of a single compound; and d) compound novelty to Cucurbitaceae. Several synthetic blends attracted significant numbers of females in outdoor rotating olfactometer experiments; a nine-component blend (lure #7) was the most attractive. Field captures of female B. cucurbitae in traps baited with lure #7 were twice those in traps baited with Solulys protein bait. Besides having a female-biased attraction, this lure may have several advantages over protein baits: it can be used with a dry trap, is long lasting, and it captured low numbers of non-target species. Possible applications of this lure include trapping (for detection and/or monitoring/delimitation) and control/eradication (e.g., mass trapping, attract-and-kill, or as an attractant for existing protein insecticide bait sprays such as GF-120).

Establishment of a Protein Frequency Library and Its Application in the Reliable Identification of Specific Protein Interaction Partners

The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static “bead proteomes.” The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.

Effective Sampling Range of Food-Based Attractants for Female <I>Anastrepha suspensa</I> (Diptera: Tephritidae)

Release-recapture studies were conducted with both feral and sterile females of the Caribbean fruit fly, Anastrepha suspensa (Loew) (Diptera: Tephritidae), to determine sampling range for a liquid protein bait (torula yeast/borax) and for a two-component synthetic lure (ammonium acetate and putrescine). Tests were done in a guava, Psidium guajava L., grove and involved releasing flies at a central point and recording the numbers captured after 7 h and 1, 2, 3, and 6 d in an array of 25 Multilure traps located 9-46 m from the release point. In all tests, highest rate of recapture occurred within the first day of release, so estimations of sampling range were based on a 24-h period. Trap distances were grouped into four categories (<10, 10-20, 20-30, and >30 m from release point) and relative trapping efficiency (percentage of capture) was determined for each distance group. Effective sampling range was defined as the maximum distance at which relative trapping efficiency was > or = 25%. This corresponded to the area in which 90% of the recaptures occured. Contour analysis was also performed to document spatial distribution of fly dispersal. In tests with sterile flies, immature females dispersed farther and were recovered in higher numbers than mature females, regardless of attractant, and recapture of both cohorts was higher with torula yeast. For mature feral flies, range of the synthetic lure was determined to be 30 m. With sterile females, effective range of both attractants was 20 m. Contour maps indicated that wind direction had a strong influence on the active space of attractants, as reflected by distribution of captured flies.

Micropatterning of Plasma Membrane Proteins to Analyze Raft Localization in Living Cells

We have developed an assay for quantitative analysis of the interaction between a fluorescently marked protein (prey) and a membrane protein (bait) using microstructured surfaces covered with biotinylated ligands (antibodies) targeted against the bait. The proof-of-concept was demonstrated for the interaction between CD4, a major co-receptor in T-cell signalling, and Lck, a protein tyrosine kinase essential for early T cell signalling. Here we present improvements and a more precise characterization of the method as well as the applicability of the assay for the analysis of protein interactions within lipid rafts in the inner and outer leaflet of the plasma membrane. We stably expressed fluorescently labelled raft and non-raft proteins in the human T24 cell line as prey proteins and determined the degree of interaction with the antibody-targeted bait proteins CD59 (GPI-anchored protein, raft marker) and CD71 (Transferrin-receptor, non-raft marker), respectively. We found strong interaction of CD59 with putative raft markers including various GPI-GFP constructs, the inner-leaflet associated proteins Lck and Flottilin1 and a Pleckstrin-Homology domain fused to GFP. Importantly, we did not find interaction of CD59 with CD71-GFP and other potential non-raft proteins. When CD71 was used as the bait protein we did not find interaction with the putative raft markers. While the detected absence of CD71 from and the presence of CD59 in lipid rafts confirm current knowledge, it is still very unclear if a lipid-raft dependent coupling of proteins and certain especially negatively charged lipids across the plasma-membrane bilayer exists. Thus, our micropatterning assay will be of great interest to address this question.

Long-Distance Movement of Bactrocera dorsalis (Diptera: Tephritidae) in Puna, Hawaii: How far can they go?

The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is considered a major economic threat in many regions worldwide, including the island of Hawaii in the Hawaiian archipelago. The need to control large populations over large areas helped initiate the USDA-ARS (United States Department of Agriculture–Agricultural Research Service) area-wide program. There is some discussion concerning the feasibility of eradication of the oriental fruit fly in areas on the island of Hawaii. An important aspect of population suppression is concerned with the ability of a species to move long distances. Suppression of an area may be futile if source populations outside of that area are capable of moving into and re-establishing populations within the suppression area. While most movement studies focus on the short-distance dispersal of flies from a single release point, this study aimed to explore the longer tails of the dispersal distribution suspected for many tephritids. Four releases of double-marked, sterile, laboratory-reared oriental fruit flies were completed. The releases took place at four different distances from an experimental fruit fly suppression zone within the Puna District on the island of Hawaii. Flies captured in methyl eugenol traps and protein bait traps were collected and examined for the mark. Many flies were recovered at unprecedented long distances (between 2–11.39 km) from the release point. Studies of this nature are notoriously difficult, as replication is not feasible and countless variables complicate procedures. However, we feel extremely fortunate to have collected data confirming results that, to our knowledge, have not been previously observed. These long-distance recaptures aid in understanding the long tails of spatial distribution of fly movement that have been suspected of this species, and will benefit consideration of dimensions for buffer zones that would be needed for the establishment of infestation-free or low-prevalence zones.

Dynamics of pH Modification of an Acidic Protein Bait Used for Tropical Fruit Flies (Diptera: Tephritidae)

Several species of Anastrepha and Bactrocera fruit flies (Diptera: Tephritidae) are captured in traps baited with the protein bait NuLure combined with borax (sodium tetraborate decahydrate) in an aqueous solution, typically 9% NuLure (vol:vol) with 3% borax (wt:vol). NuLure is an acid hydrolysate of corn and has an acidic pH. Addition of borax makes the solution more alkaline, and increase in alkalinity results in increase of ammonia release from the bait solution. This is a very dynamic system, with resultant pH affected by factors such as the amount of borax added, the pH of the water used for preparation, the age of the bait solution, and the development of microbial growth. Problems with borax include amount needed to increase alkalinity of NuLure solutions, which creates difficulties in disposing of spent bait in fruit fly trapping programs. Therefore, research was conducted to evaluate NaOH as an alternative method to increase alkalinity of NuLure solutions. Laboratory experiments compared effect of NaOH versus borax for pH modification on changes in pH and ammonia content of NuLure solutions over time. Although NuLure/NaOH solutions could be adjusted to a more alkaline pH than NuLure/borax solutions, borax plays a critical role in pH stability over time. However, the pH of NuLure/NaOH is stabilized when propylene glycol (10% vol:vol) was used to prepare the bait solution. The use of NaOH can provide an alternative to the use of borax to increase bait solution alkalinity.

Temperature and a dominant dolichoderine ant species affect ant diversity in Indonesian cacao plantations

Agricultural land conversion and climate change play a major role in shaping tropical landscapes, but the direct and indirect links to biodiversity and species community composition remain little understood. We tested how landscape and environmental factors and management techniques, affect the diversity of ground and tree living ants in cacao plantations in Sulawesi (Indonesia). In addition, we investigated the occurrence of an aggressive, numerically dominant dolichoderine ant species (genus Philidris). Half of the 43 study plots, which differed in canopy cover, shade tree diversity, cacao tree age and their distance to the nearest rainforest, were weeded manually every 3 month, the others biannually. Each plot was divided into two subplots, one was fertilized twice a year whereas the other remained unfertilized. Using protein and sugar-solution baits, we examined species richness, abundances and interspecific interactions of ants on the ground and in cacao trees. In total we collected 160 ant morphospecies. Reduced ant species richness on the ground and in the trees was significantly correlated with higher mean temperatures while the other factors, including number of shade trees did not have any significant influence. The abundant and aggressive Philidris species, reduced arboreal ant species richness. It occurred more frequently in warmer, less shaded plots and on older cacao trees, which offer more nesting sites. In our study we show, that micro-climatic conditions and the occurrence of single ecologically dominant species are the major factors predicting species diversity in tropical agricultural ecosystems.

Bimolecular affinity purification (BAP): tandem affinity purification using two protein baits.

The tandem affinity purification (TAP) procedure was pioneered in yeast for the purpose of purifying and characterizing protein complexes. While affinity purification is relatively easy to perform, nonspecific protein interactions can plague the identification of true interacting partners of the given bait utilized in the purification. To alleviate this problem, two sequential affinity purification steps are employed in the TAP procedure. Since its inception in yeast, TAP has gone through many adaptations and has been employed multiple times in diverse organisms, including mammalian systems. In all these approaches, two out of many possible affinity moieties are employed and are usually expressed as a fusion polypeptide in the amino or carboxyl-terminal region of the protein bait. In this protocol, we describe a variation on the TAP procedure in which the affinity moieties are placed on two different proteins of a molecular complex to isolate or detect components present in the complex. This variation, which we refer to as bimolecular affinity purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known components.

For ant‐protected plants, the best defense is a hungry offense

Animal foraging has been characterized as an attempt to maximize the intake of carbon and nitrogen at appropriate ratios. Plant species in over 90 families produce carbohydrate-rich extrafloral nectar (EFN), a resource attractive to ants and other omnivorous insects. This attraction can benefit the plant if those arthropods subsequently attack herbivores. This protective response has been attributed to the increased visitation and "ownership" of plants that provide a predictable source of fuel. Here, we propose and test an alternative (but non-mutually exclusive) hypothesis, that access to C-rich carbohydrates increases the ants' desire for N-rich protein and hence the likelihood that they will attack herbivorous insects on the host plant. This "deficit hypothesis" would be rejected if (1) EFN were itself a sufficiently balanced food source in terms of C and N, (2) ant dietary preferences were similar in the presence vs. absence of EFN, (3) protein-hungry ants were not more predaceous, or (4) ants provided access to protein were more aggressive toward potential prey items than were ants provided access to carbohydrates. We test these predictions in a protective mutualism between a guild of desert ants and the barrel cactus Ferocactus wislizeni. C:N ratios of EFN exceeded that of ants or potential prey items by an order of magnitude (i.e., EFN is an N-poor food for ants). Baiting studies demonstrated that plant-tending ant species recruited more workers to N-rich protein baits than to C-rich sugar baits; this difference was more pronounced when the ants had access to F. wislizeni EFN. From these data, we infer that protein is a valuable resource and that its relative value increases when carbohydrates are readily available. Moreover, ant colonies provided access to supplemental carbohydrates responded more aggressively to surrogate herbivores than did control colonies (to which no additional resources were provided) or colonies provided protein. These results support the predictions of the "deficit" hypothesis, wherein plant protection is elicited by plant-mediated dietary imbalances.

Oral and Topical Toxicity of Fipronil to Melon Fly and Oriental Fruit Fly (Diptera:Tephritidae)

The objective of this study was to develop oral and topical toxicity data for fipronil in Solulys protein bait to wild melon fly, Bactrocera cucurbitae (Coquillett), and the oriental fruit fly, Bactrocera dorsalis (Hendel). For the oral study, both females and males were evaluated, whereas in the contact study only females were evaluated. The 24 h oral LC50 estimates for female B. cucurbitae and B. dorsalis were 113 and 108 mg ai/l, respectively. Female B. cucurbitae were more susceptible than males, but female and male B. dorsalis were equally susceptible to fipronil after the oral route of exposure. Female B. cucurbitae were significantly less susceptible to the fipronil-bait mixture after topical exposure compared with feeding exposure. However, female B. dorsalis were equally susceptible to either route of exposure. At the LC50, B. dorsalis was significantly more susceptible than B. cucurbitae by the topical route of exposure. At the LC90, B. dorsalis was significantly more susceptible than B. cucurbitae by both oral and topical routes of exposure. Results of this study indicate that there are differences In susceptibility between B. cucurbitae and B. dorsalis to fipronil, especially at the LC90. Bactrocera dorsalis was more susceptible to fipronil than B. cucurbitae by oral and topical routes of exposure. LC90 estimates were significantly lower than the 5,333 mg ai/l applied to Amulet Attract and Kill Stations for control of B. cucurbitae and B. dorsalis.

Purification and characterisation of proteins secreted by the entomopathogenic fungus Metarhizium anisopliae with insecticidal activity against adults of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae)

The control of the Mediterranean fruit fly (medfly) Ceratitis capitata (Wied) is usually performed with protein bait sprays incorporating chemical insecticides that may have adverse effects on humans, non-target organisms and the environment. In recent years, scientists have sought more environmentally friendly insecticides for medfly control, such as plant- and microorganism-derived compounds. Among these compounds, entomopathogenic fungi are an unexplored source of natural insecticides. The crude soluble protein extract (CSPE) of the entomopathogenic fungus Metarhizium anisopliae (Mestch.) (strain EAMa 01/58-Su) shows chronic insecticidal activity when administered per os. Mortality in flies exhibits a dose response. The CSPE produces an antifeedant effect in adult flies, a result probably due to a progressive deterioration of the fly midgut after ingestion of the extract. Protease and temperature treatments show that insecticidal activity against C. capitata is due to proteinaceous compounds that are highly thermostable. Four monomeric proteins from this crude extract have been purified by liquid chromatography and gel electroelution. Although all four monomers seem to be involved in the insecticidal activity of the CSPE, the 15 kDa and the 11 kDa proteins appear to be mainly responsible for the observed insecticidal effect. Four new fungal proteins with insecticidal activity have been purified and identified. These proteins might be combined with insect baits for C. capitata biocontrol.

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed "yeast surface two-hybrid (YS2H)," to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two alpha-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pM to 10 microM. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pM to 100 microM. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

Managing Oriental Fruit Fly (Diptera: Tephritidae), With Spinosad-Based Protein Bait Sprays and Sanitation in Papaya Orchards in Hawaii

The efficacy of GF-120 NF Naturalyte Fruit Fly Bait in combination with field sanitation was assessed as a control for female oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), in papaya (Carica papaya L.) orchards in Hawaii. Three different bait spray regimes were evaluated: every row (high use of the bait), every fifth row (moderate use), and every 10th row (low use). Orchard plots in which no bait was applied served as controls. For five of the seven biweekly periods that followed the first bait spray, trapping data revealed significantly fewer female B. dorsalis captured in plots subject to high and moderate bait use than in control plots. Differences in incidence of infestation among treatments were detected only by the third (12 wk after first spray) fruit sampling with significantly fewer infested one-fourth to one-half ripe papaya fruit in plots subject to high and moderate bait use than in control plots. Parasitism rates by Fopius arisanus (Sonan) (Hymenoptera: Braconidae) were not negatively affected by bait application. Results indicate that foliar applications of GF-120 NF Naturalyte Fruit Fly Bait either to all rows (every other tree), or to every fifth row (every tree) in combination with good sanitation can effectively reduce infestation by B. dorsalis in papaya orchards in Hawaii.

Competition for food between the exotic wasp Vespula germanica and the native ant assemblage of NW Patagonia: evidence of biotic resistance?

The success of a biological invasion may depend on the interactions between the invader and the native biota. However, little experimental evidence demonstrates whether local species can successfully compete with exotics. We experimentally determined the existence of competition for food between the exotic wasp Vespula germanica, one of the most recent Patagonian invaders, and the native ant assemblage. Both wasps and ants are generalist predators and scavengers, sharing habitat and food resources. We selected 30 sites within scrubland habitats where both ants and wasps were present. At each site, we placed containers with protein baits under three treatments: wasp exclusion, ant exclusion, and control (i.e., free access for wasps and ants). Ant exclusion increased the number of wasps (with regard to a control), but wasp exclusion did not affect ant abundance. This result suggests that native ants affect the foraging activity of exotic wasps but not vice versa. Aggressive behaviors and worker aggregation may explain the competitive advantage of ants. Ants bite wasp legs and massively aggregate on food sources, physically limiting the landing of wasps on baits. If the outcome of interactions at baits reported here influence wasp population-level parameters, this competitive interaction could be one of the factors explaining the low abundance of this exotic wasp in NW Patagonia in comparison with other invaded regions.

Puzzle of protein complexes in vivo: a present and future challenge for functional proteomics

Complete description of the complex network of cellular mechanisms and use of the network to predict the full range of cellular behaviors are major goals of systems biology. A key role in contemporary biology can be played by functional proteomics, which focuses on the elucidation of protein functions and the definition of cellular mechanisms at the molecular level. The attainment of these targets is strictly dependent on the identification of individual proteins within functional complexes in vivo. Isolation of interacting proteins relies on either affinity-based or immunoprecipitation procedures in which the protein bait and its specific partners can be fished out by their specific binding to ligand molecules immobilized on insoluble supports. These approaches led to the final identification of several proteins belonging to distinct complexes endowed with different biological functions. Assignment of each protein to a specific complex constitutes a tremendous problem that can only be partially solved using protein–protein interaction databases and literature information. The development of prefractionation methodologies to separate individual protein complexes while preserving their native interactions might then represent an essential tool for the future of functional proteomics. Prepurification of single complexes can only be pursued under native conditions on the basis of their physicochemical features, such as size, dimension (gel filtration chromatography) and density (gradient ultracentrifugation). Following prefractionation, the complex associated to a specific biological function can be isolated using affinity purification techniques. Functional proteomics approaches able to describe individual proteins belonging to complexes involved in specific cellular functions will have a terrific impact on future systems biology studies.

Chemical Alternatives to Malathion for Controlling &lt;I&gt;Ceratitis capitata&lt;/I&gt; (Diptera: Tephritidae), and Their Side Effects on Natural Enemies in Spanish Citrus Orchards

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is one of the most important fruit pests worldwide. Mediterranean fruit fly control in Spain has been based on organophosphate sprays, especially malathion, mixed with protein baits. However, this insecticide has recently been excluded from annex 1 of the Directive 91/414 CEE, which lists authorized active ingredients for pest control in the European Union. This article reports on the efficacy of four alternative baited insecticides on Mediterranean fruit fly and their side effects on three natural enemies [Cryptolaemus montrouzieri (Mulsant), Neoseiulus californicus (McGregor), and Aphidius colemani (Viereck)] relevant for pest control in citrus agroecosystems. A high Mediterranean fruit fly mortality was obtained for all baited insecticides (phosmet and spinosad) except lambdacyhalothrin, which caused the lowest mortality and showed a novel disabling effect on surviving Mediterranean fruit fly adults. Spinosad proved to be the most selective bait treatment for C. montrouzieri and N. californicus, whereas for A. colemani the most selective bait was phosmet and lambda-cyhalothrin. These findings would contribute to a sustainable chemical control of C. capitata populations under an integrated pest management system in Spanish citrus orchards.

Proteome-Wide Identification of Novel Binding Partners to the Oncogenic Fusion Gene Protein, NPM-ALK, using Tandem Affinity Purification and Mass Spectrometry

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

The use of Gene Ontology terms for predicting highly-connected 'hub' nodes in protein-protein interaction networks

BackgroundProtein-protein interactions mediate a wide range of cellular functions and responses and have been studied rigorously through recent large-scale proteomics experiments and bioinformatics analyses. One of the most important findings of those endeavours was the observation that 'hub' proteins participate in significant numbers of protein interactions and play critical roles in the organization and function of cellular protein interaction networks (PINs) [1,2]. It has also been demonstrated that such hub proteins may constitute an important pool of attractive drug targets.Thus, it is crucial to be able to identify hub proteins based not only on experimental data but also by means of bioinformatics predictions.ResultsA hub protein classifier has been developed based on the available interaction data and Gene Ontology (GO) annotations for proteins in the Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster and Homo sapiens genomes. In particular, by utilizing the machine learning method of boosting trees we were able to create a predictive bioinformatics tool for the identification of proteins that are likely to play the role of a hub in protein interaction networks. Testing the developed hub classifier on external sets of experimental protein interaction data in Methicillin-resistant Staphylococcus aureus (MRSA) 252 and Caenorhabditis elegans demonstrated that our approach can predict hub proteins with a high degree of accuracy.A practical application of the developed bioinformatics method has been illustrated by the effective protein bait selection for large-scale pull-down experiments that aim to map complete protein-protein interaction networks for several species.ConclusionThe successful development of an accurate hub classifier demonstrated that highly-connected proteins tend to share certain relevant functional properties reflected in their Gene Ontology annotations. It is anticipated that the developed bioinformatics hub classifier will represent a useful tool for the theoretical prediction of highly-interacting proteins, the study of cellular network organizations, and the identification of prospective drug targets – even in those organisms that currently lack large-scale protein interaction data.

Mortality of Rhagoletis indifferens exposed to hydrolyzed protein baits and spinosad in the absence and presence of yeast extract

AbstractWestern cherry fruit fly, Rhagoletis indifferens Curran (Diptera: Tephritidae), is the major quarantine pest of sweet cherry, Prunus avium (L.) L. (Rosaceae), in the Pacific northwest of the USA and in British Columbia in Canada. Although spinosad bait (GF‐120 NF Naturalyte® Fruit Fly Bait) is used for the control of R. indifferens in this region, the effects of alternate food sources on fly responses to this bait have not been studied. In this study, experiments were conducted to determine mortalities of flies exposed to hydrolyzed protein baits in the presence of sugar only and sugar + yeast extract food. All baits contained Entrust® (termed ‘spinosad alone’). When flies were exposed to GF‐120 with or without added ammonia compounds (uric acid, ammonium acetate, and ammonium carbonate) for 48 h, mortalities were higher in the presence of sugar only than in the presence of sugar + yeast extract, but when flies were exposed to spinosad alone, mortalities were similar in presence of either of the two foods. In another experiment comparing GF‐120, Nu‐Lure, Mazoferm, Baker's yeast extract, and spinosad alone, mortalities in the GF‐120, Mazoferm, and Baker's yeast extract treatments were higher in the presence of sugar only than in the presence of sugar + yeast extract, but in the Nu‐Lure and spinosad alone treatments, mortalities were similar in the presence of either of the two foods. Overall results suggest that the indirect effects of yeast extract food on mortality are dependent on bait type and that mortalities caused by spinosad alone and baits are similar. Nu‐Lure and spinosad alone may have an advantage over other treatments for fly control, because their effects do not appear to be affected by the presence of nitrogenous food.

Yeast surface 2-hybrid to detect protein-protein interactions via the secretory pathway as a platform for antibody discovery

AbstractHigh throughput methods to measure protein-protein interactions will facilitate uncovering pairs of unknown interactions as well as designing new interactions. We have developed a platform to detect protein interactions on the surface of yeast, where one protein (bait) is covalently anchored to the cell wall and the other (prey) is expressed in secretory form. The prey is released either outside of the cells or remains on the cell surface by its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag fused to the prey or direct readout of split fluorescence protein complementation. Our novel 'yeast surface 2-hybrid' system was found to differentiate 6-log difference in binding affinities between coiled coil associations and to measure specific interactions of antibodies and antigens. We demonstrate the use of YS2H in exploring activation allostery in integrins and isolating heavy chain only antibodies against botulinum neurotoxin.