Oriented protein adsorption to gold nanoparticles through a genetically encodable binding motif.

Simple, stable, and specific methods for immobilizing proteins on gold surfaces are needed for the development of applications that rely on the oriented attachment of proteins to gold surfaces. We report a direct, stable, genetically encodable method for the oriented chemisorption of proteins to gold nanoparticles (Au NPs) through the tetracysteine motif (C-C-P-G-C-C) while simultaneously suppressing protein physisorption. Mutants of ubiquitin (Ub) and enhanced green fluorescent protein (eGFP) containing the tetracysteine motif were produced and displayed stronger adsorption to the NPs than did native proteins. An eGFP mutant with a dicysteine motif (G-C-C) did not show a significant improvement in binding to Au NPs compared to that of the wild-type protein. The binding of the proteins to Au NPs of various sizes (14, 18, 28, and 39 nm) was explored. The small Ub tetracysteine mutant stabilized several sizes of Au NPs, and the eGFP tetracysteine mutant clearly had the strongest chemisorption to the 18 nm NPs. The control of binding orientation for proteins bearing a tetracysteine motif was demonstrated through the enhanced specific binding of protein-NP conjugates to immobilized targets.

Effects of mid-season avermectin treatments on pyrethroid resistance in horn fly (Diptera: Muscidae) populations at three locations in Louisiana.

Between 1999 and 2002, the effect of mid-season doramectin treatments on the level of resistance in pyrethroid-resistant horn fly populations was examined at three separate Louisiana State University Agricultural Center research stations. The cattle were treated with pyrethroid ear tags in all years at all farms, and each farm received a mid-season doramectin treatment in 1 year. The number of weeks of control at Red River was 11 weeks higher in the year following the mid-season treatment of doramectin. At Macon Ridge, the number of weeks of control was 2 weeks higher in the year following the doramectin treatment. No change was observed at St. Joseph. The LC50s for fly populations tested at Macon Ridge and St. Joseph were found to increase for pyrethroids from the spring populations to the fall populations between 2000 and 2002. The LC50s for fly populations at Red River followed the same trends except in 2000, the year when the doramectin treatment was administered. Flies collected pre and post-treatment each year from St. Joseph and Red River were assayed for two alleles (kdr and skdr) associated with target site resistance to pyrethroids. Flies collected pretreatment at Macon Ridge in 1999 also were assayed for the kdr and skdr, and this population of flies had a frequency of 85.6% R-kdr alleles. At St. Joseph and Red River there was a general decline in the frequency of homozygous susceptible skdr (SS-skdr) and homozygous susceptible kdr (SS-kdr) individuals, as well as a general increase in homozygous resistant skdr (RR-skdr) and homozygous resistant kdr (RR-kdr) individuals, during the 4-year study. At both sites, the frequency of R-kdr alleles increased significantly in flies collected in the fall compared to flies collected in the spring with the exception of Red River in 2000, when dormacetin was applied. The frequency of the R-kdr alleles was significantly higher in flies collected in the fall compared to flies collected in the spring in the following year at both sites in two out of three comparisons. The frequency of R-skdr alleles was significantly lower in fly populations tested in the spring compared to fly populations tested in the fall at both farms in years when doramectin was not applied but there were no differences in the years when doramectin was applied. The frequency of R-skdr alleles was significantly higher in fly populations tested in the fall compared to in the spring the following year during all three comparisons at Red River and in one of three comparisons at St. Joseph.

Association of the kdr and superkdr sodium channel mutations with resistance to pyrethroids in Louisiana populations of the horn fly, Haematobia irritans irritans (L.).

Pyrethroid resistance in three horn fly populations in Louisiana was monitored by weekly fly counts, filter paper bioassays, and diagnostic PCR assays for the presence of pyrethroid resistance-associated mutations in the sodium channel gene coding region. The PCR assay for the knockdown resistance (kdr) and superkdr sodium channel mutations was used to determine the frequency of the target site insensitivity mechanism in the populations of horn flies, which possessed varying degrees of insecticide resistance. The bioassays and frequency of homozygous-resistant (RR) kdr genotypes were relative predictors of the fly control subsequently observed. Flies exposed to filter paper impregnated with a discriminating concentration of one of four different insecticides were collected when 50% mortality was estimated. Genotypes for the dead flies and the survivors were determined by the PCR assay. The results of the PCR assays indicated that the genotype at the kdr locus of the flies exposed to the two pyrethroids had an effect upon whether the flies were considered to be alive or dead at the time of collection. The kdr genotype of flies exposed to either diazinon or doramectin was unrelated to whether the flies were considered to be alive or dead, except for a single comparison of flies exposed to diazinon. When possible interactions of the kdr and superkdr mutations were compared, we found that there were no associations with the response to diazinon and doramectin. For one location, there were no survivors of the 75 flies with the SS-SS (superkdr-kdr) homozygous susceptible wild type genotype exposed to pyrethroids, while there were survivors in all of the other five genotypes. The SS-RR genotype flies were more susceptible to the pyrethroids than the SR-RR flies, but that was not the case for exposure to diazinon or doramectin. In the St. Joseph population, there were an adequate number of flies to demonstrate that the SS-SR genotype was more susceptible to pyrethroids than the SS-RR and that flies with the SR-SR genotype were more susceptible to pyrethroids than the flies with the SR-RR genotype.

Use of the polymerase chain reaction to investigate the dynamics of pyrethroid resistance in Haematobia irritans irritans (Diptera: Muscidae).

A field study was conducted from 1991 through 1997 to evaluate the use of pyrethroid and organophosphate (OP) ear tags, alternated yearly, for the control of a pyrethroid resistant horn fly, Haematobia irritans (L.), population in Louisiana. Fly resistance was monitored by weekly fly counts, filter paper bioassays and diagnostic polymerase chain reaction (PCR) assays for the presence of pyrethroid resistance-associated mutations in the sodium channel gene coding region. Fly control in the first study year was poor, as pyrethroid ear tags were effective for only 7 wk. The following year, OP ear tags provided 15 wk of fly control. However, in all subsequent years, fly control was poor with both types of ear tags. The PCR assays showed that there were very few female flies homozygous for the pyrethroid susceptible sodium channel allele, never rising above 10% of the total females in the population. A fitness cost appeared to be associated with the pyrethroid resistant allele, as the resistant form was selected against in the absence of the pyrethroid ear tags. Despite this selection in favor of the susceptible allele and the annual alternation of pyrethroid and OP ear tags, the percentage of homozygous susceptible flies never reached over 19% of the population, resistant alleles of the sodium channel remained at high levels in the population, and horn fly control on cattle with either type of tag quickly became minimal.

Evaluation of a yearly insecticidal ear tag rotation for control of pyrethroid-resistant horn flies (Diptera: Muscidae).

From 1991 to 1997, the yearly alternated use of synergized pyrethroid (lambda-cyhalothrin + piperonyl butoxide) and organophosphate (pirimiphos-methyl) ear tags was evaluated for the control of two pyrethroid-resistant horn fly populations in Louisiana. At each site, weekly fly counts were used to assess product efficacy. Control achieved by synergized pyrethroid ear tag treatments was reduced from 7 to 2 weeks and from 4 to 0 weeks at St. Joseph and Winnsboro, respectively. Control by organophosphate ear tags decreased from 15 to 3 weeks and from 10 to 7 weeks at St. Joseph and Winnsboro, respectively. The rotation of synergized lambda-cyhalothrin and pirimiphos-methyl ear tags did not improve pyrethroid ear tag efficacy or prevent further development of resistance to the pyrethroid or OP compound.