Impact of phagostimulants on effectiveness of OMRI‐listed insecticides used for control of spotted‐wing drosophila (Drosophila suzukii Matsumura)

Spotted‐wing drosophila, Drosophila suzukii Matsumura, is an invasive pest in the United States that causes considerable damage to fruit crops. It is responsible for many millions of dollars of revenue loss. The female D. suzukii has a heavily sclerotized ovipositor and can lay eggs in ripening or ripe fruit. The arrival of this invasive species has disrupted existing integrated pest management programmes, and growers rely on repeated insecticide applications to protect fruit. Organic growers have few chemical control options, and their reliance on spinosad increases the risk of developing insecticide resistance. We hypothesized that combining phagostimulants with insecticides would increase insecticide efficacy by prompting flies to spend more time in contact with residues. Therefore, the objective of this study was to evaluate the effectiveness of sucrose and the yeast Saccharomyces cerevisiae as phagostimulants in combination with organic biopesticides against D. suzukii in blueberries. Adding sucrose with or without yeast did not improve insecticide efficacy in terms of adult fly mortality or fruit infestation. Spinosad was very effective in all experiments, and for this product, there is little room for improvement. The phagostimulants had no effect on residual activity of any insecticide. The addition of sucrose with or without yeast did not improve the effectiveness of organic insecticides for D. suzukii. Concentrations of these phagostimulants in our experiments (0.36%) may have been too low to elicit a response. Further research is recommended to test different types and concentrations of phagostimulants.     

Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli

Bacteria show asymmetric subcellular distribution of many proteins involved in diverse cellular processes such as chemotaxis, motility, actin polymerization, chromosome partitioning and cell division. In many cases, the specific subcellular localization of these proteins is critical for proper regulation and function. Although cellular organization of the bacterial cell clearly plays an important role in cell physiology, systematic studies to uncover asymmetrically distributed proteins have not been reported previously. In this study, we undertook a proteomics approach to uncover polar membrane proteins in Escherichia coli. We identified membrane proteins enriched in E. coli minicells using a combination of two-dimensional electrophoresis and mass spectrometry. Among a total of 173 membrane protein spots that were consistently detected, 36 spots were enriched in minicell membranes, whereas 15 spots were more abundant in rod cell membranes. The minicell-enriched proteins included the inner membrane proteins MCPs, AtpA, AtpB, YiaF and AcrA, the membrane-associated FtsZ protein and the outer membrane proteins YbhC, OmpW, Tsx, Pal, FadL, OmpT and BtuB. We immunolocalized two of the minicell-enriched proteins, OmpW and YiaF, and showed that OmpW is a bona fide polar protein whereas YiaF displays a patchy membrane distribution with a polar and septal bias.     

Quantitative and qualitative comparison of density-based purification methods for detection of Cryptosporidium oocysts in turbid environmental matrices

Purification methods for Cryptosporidium oocysts are usually selected on the basis of recovery yield, but the amount of particulate debris in environmental matrices could limit efficiency of oocyst detection by microscopic examination or PCR detection. Previous studies have shown that the standard immunomagnetic separation (IMS) procedure would not be the most suitable method for oocyst purification from turbid matrices. We compared the capacity of Percoll-sucrose flotation and six other density-based purification methods to achieve selective separation of Cryptosporidium oocysts from particulate debris. Rate of oocyst recovery and particulate loading in the purified suspensions were chosen as comparison criteria for the different purification methods. In most earlier studies, the chemical treatments employed to obtain a purified oocyst suspension modify the surface properties of oocysts in spiked samples. Assuming this produces unrealistic conditions affecting the evaluation of purification methods, we performed the present study with native oocysts. Flotation and gradient procedures were tested with and without formaldehyde ethyl acetate (FEA) separation. FEA separation was found to be unsuitable. Filtration and Percoll gradient did not allow selective oocyst separation from debris. Among the purification methods suitable for routine microscopic examination, Percoll-sucrose flotation provided the best recovery rates. For automated enumeration systems or PCR detection, potassium bromide and especially Nycodenz gradients appeared to be the most suitable purification methods. Potassium bromide and Nycodenz gradients provided the best balance between oocyst recovery and particulate load.     

Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method

The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.     

Tricyclic pyridones as functionally selective human GABAA alpha 2/3 receptor-ion channel ligands

A series of tricyclic pyridones has been evaluated as benzodiazepine site ligands with functional selectivity for the alpha(3) over the alpha(1) containing subtype of the human GABA(A) receptor ion channel. This investigation led to the identification of a high affinity, functionally selective, orally bioavailable benzodiazepine site ligand that demonstrated activity in rodent anxiolysis models and reduced sedation relative to diazepam.     

Genetic linkage of Francois-Neetens fleck (mouchetée) corneal dystrophy to chromosome 2q35

Francois-Neetens fleck (mouchetée) corneal dystrophy is an autosomal dominant corneal dystrophy characterized by scattered small white flecks occurring at all levels of the corneal stroma. We report linkage of the CFD locus to D2S2289 (Z(max)=4.46, theta=0), D2S325 (Z(max)=3.28, theta=0), D2S317 (Z(max)=3.1, theta=0), D2S143 (Z(max)=3.8, theta=0.03), and D2S2382 (Z(max)=5.0, theta=0) on chromosome 2q35. Multipoint analysis confirmed linkage to the region between D2S117 and D2S126 with a maximum multipoint lod score of 5.0 located midway between D2S2289 and D2S325. Analysis of CFD in these same families assuming a 90% penetrance increased the maximum lod score to 6.28 at D2S157.     

Normal human keratinocytes bind to the alpha3LG4/5 domain of unprocessed laminin-5 through the receptor syndecan-1

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, we recombinantly expressed the human alpha3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.     

Function of oxygen resistance proteins in the anaerobic,sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.