Exome-wide association of deltamethrin resistance in Aedes aegypti from Mexico.

Aedes aegypti is the major vector of a number of arboviruses that cause disease in humans. Without vaccines or pharmaceuticals, pyrethroid insecticides remain the major tool for public health protection. Pyrethroid resistance is now widespread. Replacement substitutions in the voltage-gated sodium channel (vgsc) that reduce the stability of pyrethroid binding account for most of the resistance, but metabolic mechanisms also inactivate pyrethroids. High-throughput sequencing and the A. aegypti L5 annotated physical map has allowed interrogation of the exome for genes and single-nucleotide polymorphisms associated with pyrethroid resistance. We exposed females of A. aegypti from Mexico to a deltamethrin discriminating dose to designate them as resistant (active after 1 h) or susceptible (knocked down with no recovery after 4 h). The vgsc on chromosome 3 had the highest association, followed by genes proximal to vgsc. We identified potential detoxification genes located singly (eg HPX8C) or within clusters in chromosome 2 [three esterase clusters, two of cytochrome P450 monooxygenases (CYP)] and chromosome 3 (one cluster of 16 CYP325 and seven CYP9 genes). Deltamethrin resistance in A. aegypti is associated with mutations in the vgsc gene and a large assortment of genes.

Effects of different wavelengths of light on the biology, behavior, and production of grow-out Pekin ducks.

Previous research has shown that red light conditions may improve growth and decrease aggressive behaviors in chickens and turkeys; however, more recent studies suggest that blue-green light may improve production of broilers over red light. To date, no research has been conducted to examine whether different wavelengths of light have an impact on production in the Pekin duck. To determine this, we raised Pekin ducks under aviary conditions that were similar to standard commercial barns. The ducks were kept in 3 different pens: red light (approximately 625 nm), blue light (approximately 425 nm), and white light. Light sources in each pen were standardized to produce a peak energy at 1.6 × 10³ µM photons/m²/s at the level of the ducks' heads. Ducks were given ad libitum access to water and commercial duck diet, and were housed on pine shavings at a density of 0.43 m²/duck. Ducks were evaluated weekly for BW and condition and a subjective measure of the duck's anxiety levels was determined. We found that ducks housed under blue light had significantly (P < 0.01) reduced BW at every age until the end of the study (processing age; 35 d). Unlike ducks housed under red or white light, ducks housed in the blue pen showed a higher level of anxiety; while evaluators were in the pen a majority of them began panting, they were much less inquisitive than other ducks, they took longer to exhibit normal social behavior once evaluation was completed, and they frequently "swarmed" when no people were present. There were no differences in any measurements between the red and white-lighted pens. These data suggest that unlike the chicken, blue lights may be inappropriate for raising Pekin ducks in a commercial setting.

MicroRNA levels are modulated in Aedes aegypti after exposure to Dengue-2.

To define microRNA (miRNA) involvement during arbovirus infection of Aedes aegypti, we mined deep sequencing libraries of Dengue type 2 (DENV2)-exposed mosquitoes. Three biological replicates for each timepoint [2, 4 and 9 days post-exposure (dpe)] and treatment group allowed us to remove the outliers associated with sample-to-sample variability. Using edgeR (R Bioconductor), designed for use with replicate deep sequencing data, we determined the log fold-change (logFC) of miRNA levels [18-23 nucleotides (nt)]. The number of significantly modulated miRNAs increased from ≤ 5 at 2 and 4 dpe to 23 unique miRNAs by 9 dpe. Putative miRNA targets were predicted by aligning miRNAs to the transcriptome, and the list was reduced to include the intersection of hits found using the Miranda, PITA, and TargetScan algorithms. To further reduce false-positives, putative targets were validated by cross-checking them with mRNAs reported in recent DENV2 host response transcriptome reports; 4076 targets were identified. Of these, 464 gene targets have predicted miRNA-binding sites in 3' untranslated regions. Context-specific target functional groups include proteins involved in transport, transcriptional regulation, mitochondrial function, chromatin modification and signal transduction processes known to be required for viral replication and dissemination. The miRNA response is placed in context with other vector host response studies by comparing the predicted targets with those of transcriptome studies. Together, these data are consistent with the hypothesis that profound and persistent changes to gene expression occur in DENV2-exposed mosquitoes.

A role for endosomal proteins in alphavirus dissemination in mosquitoes.

Little is known about endosomal pathway proteins involved in arthropod-borne virus (arbovirus) assembly and cell-to-cell spread in vector mosquitoes. UNC93A and synaptic vesicle-2 (SV2) proteins are involved in intracellular transport in mammals. They show amino acid sequence conservation from mosquitoes to humans, and their transcripts are highly enriched in Aedes aegypti during arbovirus infection. Transient gene silencing of SV2 or UNC93A in mosquitoes infected with the recombinant alphavirus Sindbis MRE16-enhanced green fluorescent protein (SINV; family Togaviridae) resulted in the accumulation of viral positive- and negative-strand RNA, congregation of virus envelope antigen in intracellular networks, and reduced virus dissemination outside of the midgut. Further, UNC93A silencing, but not SV2 silencing, resulted in a 10-fold reduction in viral titres at 4 days post-infection. Together, these data support a role for UNC93A and SV2 in virus assembly or budding. Cis-regulatory elements (CREs) were identified at the 5'-ends of genes from the original data set in which SV2 and UNC93A were identified. Common CREs at the 5'-end genomic regions of a subset of enriched transcripts support the hypothesis that UNC93A transcription may be co-regulated with that of other ion transport and endosomal trafficking proteins.

Kinetics of the normal folate enterohepatic cycle.

Detailed studies were undertaken to better define the role of the liver and the folate enterohepatic cycle in folate homeostasis. Three isotopes of folate were employed in a rat model to study several parameters: (a) intestinal transport; (b) variation in hepatic uptake after different routes of administration; (c) hepatic reduction, methylation, and polyglutamate formation; (d) biliary excretion; (e) transport of folate to tissue and its return to liver for re-entry into the enterohepatic cycle. Folate absorption was not affected by the type of folate administered, but subsequent liver accumulation was greater when PteGlu(1) was given rather than CH(3)H(4)PteGlu(1). After liver uptake, CH(3)H(4)PteGlu(1) is rapidly and quantitatively excreted into bile, whereas nonmethylated folates are either methylated and transported into bile or incorporated into a hepatic polyglutamate pool. Bile folate is then reabsorbed for distribution to both tissue and liver, completing the enterohepatic cycle. The importance of this cycle was demonstrated by long-term bile drainage and by transport studies with two isotopes of CH(3)H(4)PteGlu(1). With bile drainage, serum folate levels fell to 30-40% of normal within 6 h, a much more dramatic drop than that seen with folate-free diets alone. Studies with labeled CH(3)H(4)PteGlu(1) demonstrated that about one-third was taken up by tissue, demethylated, and returned to liver for remethylation and recirculation through the bile and gut. This establishes the enterohepatic cycle as a major factor in folate homeostasis and, for the first time, demonstrates a transport pathway between tissue and liver for nonmethylated folate.

Challenges in quantifying biosphere-atmosphere exchange of nitrogen species.

Recent research in nitrogen exchange with the atmosphere has separated research communities according to N form. The integrated perspective needed to quantify the net effect of N on greenhouse-gas balance is being addressed by the NitroEurope Integrated Project (NEU). Recent advances have depended on improved methodologies, while ongoing challenges include gas-aerosol interactions, organic nitrogen and N(2) fluxes. The NEU strategy applies a 3-tier Flux Network together with a Manipulation Network of global-change experiments, linked by common protocols to facilitate model application. Substantial progress has been made in modelling N fluxes, especially for N(2)O, NO and bi-directional NH(3) exchange. Landscape analysis represents an emerging challenge to address the spatial interactions between farms, fields, ecosystems, catchments and air dispersion/deposition. European up-scaling of N fluxes is highly uncertain and a key priority is for better data on agricultural practices. Finally, attention is needed to develop N flux verification procedures to assess compliance with international protocols.

Mitochondrial morphological and functional defects in yeast caused by yme1 are suppressed by mutation of a 26S protease subunit homologue.

The absence of functional Yme1p, a putative ATP and zinc-dependent protease localized to mitochondria of yeast, results in abnormal mitochondrial function and morphology. Yeast lacking Yme1p lose DNA from mitochondria at an accelerated rate, fail to grow on nonfermentable carbon sources at 37 degrees C, and have severely deficient growth if mitochondrial DNA suffers large deletions or is completely lost. In place of the normal reticulated mitochondrial network, strains lacking Yme1p have punctate mitochondria with some grossly swollen compartments. The growth phenotypes and morphological alterations evident in these mutant yeast can be compensated by a mutation in YNT1, an essential gene in yeast. The sequence of the YNT1 gene product indicates that it is one of a number of related regulatory subunits of the 26S protease. This proteolytic activity is necessary for progression through the cell cycle and has been implicated in the regulation of transcription. Ynt1p is more distantly related to Yme1p.

Beverly T. Galloway: visionary administrator.

With a career that began at the University of Missouri in the early 1880s and culminated at the USDA in the 1930s, Beverly Galloway devoted his life to practical botany and agriculture. He became a driving force in the movement for "New Botany" during a period that stressed an experimental approach as well as new disciplines such as plant pathology. As administrator and scientist, he was arguably the single, most influential figure involved in the early growth and development of plant pathology and the plant sciences generally in the USDA. From assistant mycologist in the Section of Mycology to Chief of the Bureau of Plant Industry to Assistant Secretary of the USDA, Galloway displayed exceptional administrative acumen. His administrative and scientific skills were instrumental in laying the foundations for the science of plant pathology during its formative period in the United States.

Enterohepatic circulation of methotrexate in rats in vivo.

The pharmacokinetics of the methotrexate enterohepatic cycle were studied in rats in vivo. For plasma levels of methotrexate between 10(-5) and 10(-8) M, biliary levels were directly proportional and concentrated 27-fold. When labeled methotrexate was administered in doses sufficient to achieve plasma levels of 10(-6) M, approximately 50% of methotrexate appeared in the bile in normal animals and up to 80% appeared in anephric animals. In spite of the high percentage of administered methotrexate which appeared in the bile, complete interruption of the enterohepatic cycle in otherwise normal animals did not affect the plasma decay curve of a bolus of methotrexate. The increased biliary excretion which occurred in animals with renal impairment was utilized with possible therapeutic implications. Bile drainage in these animals rapidly decreased plasma methotrexate levels compared to nondrained controls. This suggests that interruption of the methotrexate enterohepatic cycle may provide an alternative for the management of methotrexate toxicity associated with renal insufficiency.

Monte Carlo modelling of photodynamic therapy treatments comparing clustered three dimensional tumour structures with homogeneous tissue structures.

We explore the effects of three dimensional (3D) tumour structures on depth dependent fluence rates, photodynamic doses (PDD) and fluorescence images through Monte Carlo radiation transfer modelling of photodynamic therapy. The aim with this work was to compare the commonly used uniform tumour densities with non-uniform densities to determine the importance of including 3D models in theoretical investigations. It was found that fractal 3D models resulted in deeper penetration on average of therapeutic radiation and higher PDD. An increase in effective treatment depth of 1 mm was observed for one of the investigated fractal structures, when comparing to the equivalent smooth model. Wide field fluorescence images were simulated, revealing information about the relationship between tumour structure and the appearance of the fluorescence intensity. Our models indicate that the 3D tumour structure strongly affects the spatial distribution of therapeutic light, the PDD and the wide field appearance of surface fluorescence images.

Modelling topical photodynamic therapy treatment including the continuous production of Protoporphyrin IX.

Most existing theoretical models of photodynamic therapy (PDT) assume a uniform initial distribution of the photosensitive molecule, Protoporphyrin IX (PpIX). This is an adequate assumption when the prodrug is systematically administered; however for topical PDT this is no longer a valid assumption. Topical application and subsequent diffusion of the prodrug results in an inhomogeneous distribution of PpIX, especially after short incubation times, prior to light illumination. In this work a theoretical simulation of PDT where the PpIX distribution depends on the incubation time and the treatment modality is described. Three steps of the PpIX production are considered. The first is the distribution of the topically applied prodrug, the second in the conversion from the prodrug to PpIX and the third is the light distribution which affects the PpIX distribution through photobleaching. The light distribution is modelled using a Monte Carlo radiation transfer model and indicates treatment depths of around 2 mm during daylight PDT and approximately 3 mm during conventional PDT. The results suggest that treatment depths are not only limited by the light penetration but also by the PpIX distribution.

Variability in response to aspirin: do we understand the clinical relevance?

Aspirin, an irreversible inhibitor of platelet prostaglandin synthase activity, is the cornerstone of therapy for acute coronary syndromes. In recent years, laboratory and clinical data have accumulated that suggest there may be significant individual variability in the response to aspirin and that the effects of aspirin therapy vary significantly over time. There is, as of yet, no cohesive explanation for this variability. The term 'aspirin resistance' has been loosely applied to situations in which the clinical or ex vivo effects of aspirin are less than expected. In this review we discuss the clinical data regarding this phenomenon and the need for prospective evaluation of aspirin non-responders.

Monte Carlo modelling of daylight activated photodynamic therapy.

The treatment of superficial skin lesions via daylight activated photodynamic therapy (PDT) has been explored theoretically with three dimensional (3D) Monte Carlo radiation transfer simulations. For similar parameters and conditions, daylight activated PDT was compared to conventional PDT using a commercially available light source. Under reasonable assumptions for the optical properties of the tissue, protoporphyrin IX (PpIX) concentration and a treatment dose of 75 J cm(-2), it was found that during a clear summer day an effective treatment depth of over 2 mm can be achieved after 30 min of daylight illumination at a latitude of 56 degrees North. The same light dose would require 2.5 h of daylight illumination during an overcast summer day where a treatment depth of about 2 mm can be achieved. For conventional PDT the developed model suggests that 15 min of illumination is required to deliver a light dose of 75 J cm(-2), which would result in an effective treatment depth of about 3 mm. The model developed here allows for the determination of photo-toxicity in skin tissue as a function of depth for different weather conditions as well as for conventional light sources. Our theoretical investigation supports clinical studies and shows that daylight activated PDT has the potential for treating superficial skin lesions during different weather conditions.

Coexpression of granulocyte colony stimulating factor and its receptor in primary ovarian carcinomas.

Immunohistochemistry was used to determine the expression of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in primary ovarian carcinomas. The expression of G-CSFR was observed in the malignant cells of each of the 46 primary carcinomas examined; G-CSF was coexpressed in both the malignant epithelial cells and the stroma of 56.5% of the specimens. Thus the majority of ovarian carcinomas harbor both potential autocrine and paracrine G-CSF axes. In 37% of the samples, G-CSF was expressed only within stromal cells, suggesting that only a potential paracrine system is in place. In a preliminary, retrospective, evaluation, the survival of patients whose tumors expressed only the apparent paracrine loop was significantly worse than patients whose tumors expressed both potential autocrine and paracrine G-CSF-based regulatory loops (14.5 vs. 42.5 months, respectively). Studies on the potential function of G-CSF were performed using the G-CSFR-expressing OVCAR-3 ovarian carcinoma line. As a single agent, rhG-CSF failed to stimulate [3H]-thymidine incorporation in these cells, but enhanced the mitogenic action of epidermal growth factor (EGF) in a dose-dependent manner. Thus, potential autocrine and/or paracrine loops involving G-CSF and its receptor occur in over 90% of primary ovarian carcinomas, and may act to modulate the action of growth factors.

Oral infection of Culicoides sonorensis (Diptera: Ceratopogonidae) by vesicular stomatitis virus.

Vesicular stomatitis virus serotype New Jersey (VSNJV) was mixed with bovine blood or fetal bovine serum (FBS) and fed across silicone membranes to laboratory populations of Culicoides sonorensis Wirth & Jones. In an initial study, virus was detected after 13 d in 21% of the midges that received an FBS/VSNJV mixture. In subsequent time-course experiments, engorged females were collected and maintained at 20.0 degrees C and assayed for VSNJV immediately after feeding and at 1, 3, 7, 10 and 13 d after feeding. Virus was detected after 13 d in 3% of the midges that received a bovine blood/VSNJV mixture and in 9% of the midges that received an FBS/VSNJV mixture. The results indicate that C. sonorensis should be considered as a potential biological vector of VSNJV.

Suppression of phytophthora blight in bell pepper by a no-till wheat cover crop.

ABSTRACT Four mechanisms of dispersal of propagules of Phytophthora capsici were investigated through modifications in cultural practices and fungicide applications in field plots of bell pepper (Capsicum annuum). Dispersal of soil inoculum was suppressed, and final incidence of disease was 2.5 to 43% when stubble from a fall-sown, no-till, wheat cover crop was present. Final disease incidence was 71 to 72% and pathogen spread occurred within and across rows when all dispersal mechanisms were operative in plots of pepper planted into bare soil. Final disease incidence was 42 to 78% with black plastic mulch when a sporulating pepper fruit placed on the surface served as the source of initial inoculum. The fungicide metalaxyl applied in the irrigation system did not suppress within-row spread of surface inoculum from a sporulating fruit on plastic, but did limit across-row spread; final disease incidence in metalaxyl-treated plots was 11.5 to 14%. Pathogen dispersal mechanisms were modified most dramatically by the no-till cropping system. Thus, simple changes in cultural practices can have dramatic effects on the development of Phytophthora epidemics. Ecologically based disease management strategies have the potential to reduce our reliance on agrichemicals in this and similar pathosystems.

Influence of acidity level in simulated rain on disease progress in four plant pathosystems.

Field investigations were performed in 1984-1985 on the effects of acidity level in simulated rain on disease dynamics in four pathosystems: alfalfa leaf spot (ALS), peanut leaf spot (PLS), potato late blight (PLB), and soybean brown spot (SBS). Studies were performed in an ambient rain exclusion apparatus with simulated rain acidity levels of pH 2.8, 3.6, 4.2, 4.8, or 5.6 and four plots per pH level. In 1984 for ALS and PLB, rain was simulated three times a week at 6-8 mm per event. For PLS and SBS in 1984 and for PLB, PLS, and SBS in 1985, rain was simulated twice a week at 13-16 mm per event with a 10 min pause halfway through each event. Disease was assessed three times a week. In 1984, no significant effect of acidity level in simulated rain on disease severity was detected in any pathosystem. Severity of PLB differed among treatments in 1985 with significant quadratic and cubic components for the dose-response relationship. PLS severity decreased with increasing level of acidity in simulated rain in 1985 and the dose-response relationship was linear. No differences in severity of ALS or SBS due to acidity of simulated rain were found. Thus, disease response to acidity of simulated rain is system dependent.

Prevalence and Ecological Association of Foliar Pathogens of Cucumber in North Carolina, 1996-1998.

During the fall growing seasons of 1996-98, 5,400 leaves exhibiting leaf spots were collected from cucumber (Cucumis sativus L.) fields and microscopically examined to identify the organisms associated with these symptoms. Five fungal pathogens were associated with leaf lesions: Alternaria cucumerina, Colletotrichum orbiculare, Corynespora cassiicola, Didymella bryoniae, and Pseudoperonospora cubensis; D. bryoniae and C. orbiculare occurred most frequently. When pathogens were paired on five or more leaves, associations between pathogen pairs were tested for independence via a 2-by-2 contingency table χ2 analysis. In all, 66 two-way pathogen associations were tested. Of these, 39 associations were negative (occurred together less often than expected at random), 1 was positive (occurred together more often than expected at random), and, in 16 cases, the pathogens were not associated. An association between C. orbiculare and D. bryoniae occurred 24 times and, each time, the relationship was negative. This result, combined with different environmental requirements for infection, suggests that these pathogens either occupy different niches in the plant canopy or are antagonistic. No relationship between the cultivars grown or the fungicides applied and the pathogens isolated from specific field sites was found. Information on the dominant pathogens responsible for leaf spot epidemics in North Carolina's cucumber fields will be useful to target breeding and disease control strategies.

Famoxadone: the discovery and optimisation of a new agricultural fungicide.

Famoxadone (3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione), is a new agricultural fungicide recently commercialized by DuPont under the trade name Famoxate. Famoxadone is a member of a new class of oxazolidinone fungicides that demonstrate excellent control of plant pathogens in the Ascomycete, Basidiomycete, and Oomycete classes that infect grapes, cereals, tomatoes, potatoes and other crops. DuPont's entry into the oxazolidinone area resulted from the procurement of 5-methyl-5-phenyl-3-phenylamino-2-thioxo-4-oxazolidinone (1) from Professor Detlef Geffken, then at the University of Bonn. An extensive analog program was initiated immediately after the fungicidal activity of 1 was discovered through routine greenhouse testing. The discovery program in the oxazolidinone area eventually culminated in the advancement of famoxadone to commercial development in the early 1990s. The synthesis of various oxazolidinone ring systems and the development of the structure-activity relationships that led to the discovery of famoxadone are described.

Sampling for regional monitoring of nematode communities in agricultural soils.

Regional assessment of nematode communities to monitor the condition or ecological health of agricultural soils requires sampling programs with measures of known reliability and the ability to detect differences over time. Numbers of fields sampled in a region, samples taken per field, and subsamples assayed per sample must be balanced with cost to provide the best sampling scheme. We used components of variance from statewide surveys in North Carolina (1992) and Nebraska (1993) to estimate number of (i) fields to be sampled; (ii) 20-core, composite soil samples to be obtained for each field; and (iii) subsamples to be assayed for each composite sample to detect a specified amount of change in index values within a geographic region. Variances for these three components were used to estimate the degree of reliability for five ecologically based indices (four measures of maturity and one of diversity) of nematode communities. Total variance for maturity and diversity indices, based upon communities of free-living nematodes, was greater in North Carolina than in Nebraska; the opposite was true for indices based strictly upon maturity of communities of plant-parasitic nematodes or of all nematodes in soil. Variability within samples was greater in North Carolina than in Nebraska, especially for maturity indices based only upon free-living nematodes. We identified two possible sampling strategies for a regional survey: Option 1, with two independent samples per field and a single subsample assayed per sample, which would provide a reliability ratio value >/=0.6 for most indices; and Option 2, with three independent samples per field and two subsamples assayed per sample, which would provide a reliability ratio value >/=0.7 for several indices. When cost was considered, Option 1 was the better strategy. Number of fields to be sampled within a region or state varied with the index chosen; with specific indices, however, a 10% change in mean index value could be detected with a sample of 50 to 100 fields.