Identification of a mutation associated with permethrin resistance in the para-type sodium channel of the stable fly (Diptera: Muscidae)

The insect sodium channel is of particular interest for evaluating resistance to pyrethroids because it is the target molecule for this major class of neurotoxic insecticides. The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), sodium channel coding sequence representing domains IS6 through IVS6 was isolated, and the sequence encoding domain II was compared among individuals of a laboratory strain selected for resistance to permethrin and the unselected, parental generation. A point mutation resulting in a leucine-to-histidine amino acid change was identified (Leul014His), and its location corresponded with that observed for knockdown resistance (kdr) mutations in other insects. As a result, the allele was designated kdr-his. A molecular assay was developed to assess the frequency of this mutation in genomic DNA of individual stable flies from the laboratory selections, which provided further evidence that the kdr-his allele accounts for the observed level ofpermethrin resistance in the selected strain. The assay was then used to evaluate the frequency of the mutation from five field-collected populations originating from three horse farms near Ocala, FL; one horse farm near Gainesville, FL; and one dairy farm near Hague, FL. Frequency of the kdr-his allele ranged from 0.46 to 0.78, supporting further investigation of allele prevalence throughout the stable fly season and in response to field insecticide application.     

Lack of interaction of rhodopsin chromophore with membrane lipids. An electron-electron double resonance study using 14N:15N pairs.

Electron-electron double resonance (ELDOR) has been applied to the study of specific interactions of 15N-spin-labeled stearic acid with the retinal chromophore of a rhodopsin analogue containing a 14N spin-labeled retinal. Both the 5 and 16 spin-labeled stearic acids were incorporated into the lipid bilayer of rod outer segment membranes containing the spin-labeled pigment. No interaction between the 15N and 14N spin-labels was observed in rhodopsin or the metarhodopsin II state with either of these labeled stearic acids. Therefore in this system the ring portion of the chromophore must be highly sequestered from the phospholipid bilayer in both the rhodopsin and metarhodopsin II forms.     

In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine-pyrimidine junctions

Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine–pyrimidine (pyr–pyr) junctions, which are cleaved 1–3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr–pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited k_obs values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited ~1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.     

The HIV-1 accessory protein Nef increases surface expression of the checkpoint receptor Tim-3 in infected CD4+ T cells

Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef''s dileucine motif LL_164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif–dependent manner. Treating HIV-1-infected CD4⁺ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4⁺ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4⁺ T cells.     

Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles

Background  

The simultaneous production of various recombinant proteins in every cell of a culture is often needed for the production of virus-like particles (VLP) or vectors for gene therapy. A common approach for such a purpose is the coinfection of insect cell cultures with different recombinant baculoviruses, each containing one or more recombinant genes. However, scarce information exists regarding kinetics during multiple infections, and to our knowledge, no studies are available on the behavior of the different populations that arise during coinfections. Such information is useful for designing infection strategies that maximize VLP or vector yield. In this work, kinetics of cell populations expressing rotavirus GFPVP2 (infected with bacGFPVP2), VP6 (infected with bacVP6), or both proteins simultaneously (coinfected with both baculoviruses) were followed by flow cytometry.     

Seasonal abundance of stable flies and filth fly pupal parasitoids (Hymenoptera: Pteromalidae) at Florida equine facilities

Beginning in November 2007 and continuing until December 2009, weekly stable fly, Stomoxys calcitrans (L.), surveillance was conducted at four equine facilities near Ocala, FL, by using alsynite sticky traps for adults and by searching immature developmental sites for pupae. Adult stable fly trap captures were highly variable throughout the year, ranging from 0 to 1,400 flies per trap per farm. The greatest adult stable fly activity was observed during the spring months of March and April, with weekly three-trap means of 121 and 136 flies per farm, respectively. The importance of cultural control measures was most apparent on the only farm with no reported insecticide use and the lowest stable fly trap captures, where an intense daily sanitation and composting program was conducted. A survey of on-site filth fly pupae revealed that 99.9% of all parasitoids recovered were Spalangia spp., consisting of Spalangia cameroni Perkins (56.5%), Spalangia nigroaenea Curtis (34.0%), Spalangia endius Walker (5.8%), and Spalangia nigra Latreille (3.7%). The implications of these findings are discussed.