Metabolite compositions on skins of eastern hellbenders Cryptobranchus alleganiensis alleganiensis differ with location and captivity.

Eastern hellbenders Cryptobranchus alleganiensis alleganiensis, large aquatic salamanders, are declining over most of their range. The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has contributed to global amphibian declines and has been detected on eastern hellbenders, but infection intensities were lower than those of species that are more susceptible to Bd. The factors limiting Bd on hellbenders may include antifungal metabolites produced by their skin microbiota. We used a metabolite fingerprinting technique to noninvasively identify the presence, but not identity, of metabolites associated with eastern hellbenders. We surveyed the skin of wild eastern hellbenders to test whether the composition and richness (i.e. number of metabolites) of their metabolites are explained by Bd status or location. Furthermore, we surveyed for metabolites on captive eastern hellbenders to test whether metabolite compositions were different between captive and wild eastern hellbenders. Bd detection was not associated with either metabolite richness or composition. Both metabolite composition and richness differed significantly on hellbenders from different locations (i.e. states). For metabolite composition, there was a statistical interaction between location and Bd status. Metabolite richness was greater on captive eastern hellbenders compared to wild hellbenders, and metabolite compositions differed between wild and captive eastern hellbenders. The methods we employed to detect metabolite profiles effectively grouped individuals by location even though metabolite composition and richness have high levels of intraspecific variation. Understanding the drivers and functional consequences of assemblages of skin metabolites on amphibian health will be an important step toward understanding the mechanisms that result in disease vulnerability.

Catalytic activities of mammalian epoxide hydrolases with cis and trans fatty acid epoxides relevant to skin barrier function.

Lipoxygenase (LOX)-catalyzed oxidation of the essential fatty acid, linoleate, represents a vital step in construction of the mammalian epidermal permeability barrier. Analysis of epidermal lipids indicates that linoleate is converted to a trihydroxy derivative by hydrolysis of an epoxy-hydroxy precursor. We evaluated different epoxide hydrolase (EH) enzymes in the hydrolysis of skin-relevant fatty acid epoxides and compared the products to those of acid-catalyzed hydrolysis. In the absence of enzyme, exposure to pH 5 or pH 6 at 37°C for 30 min hydrolyzed fatty acid allylic epoxyalcohols to four trihydroxy products. By contrast, human soluble EH [sEH (EPHX2)] and human or murine epoxide hydrolase-3 [EH3 (EPHX3)] hydrolyzed cis or trans allylic epoxides to single diastereomers, identical to the major isomers detected in epidermis. Microsomal EH [mEH (EPHX1)] was inactive with these substrates. At low substrate concentrations (<10 µM), EPHX2 hydrolyzed 14,15-epoxyeicosatrienoic acid (EET) at twice the rate of the epidermal epoxyalcohol, 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoic acid, whereas human or murine EPHX3 hydrolyzed the allylic epoxyalcohol at 31-fold and 39-fold higher rates, respectively. These data implicate the activities of EPHX2 and EPHX3 in production of the linoleate triols detected as end products of the 12R-LOX pathway in the epidermis and implicate their functioning in formation of the mammalian water permeability barrier.

Stereocontrolled synthesis of four isomeric linoleate triols of relevance to skin barrier formation and function.

Linoleate triol esters are intermediates along the pathway of formation of the mammalian skin permeability barrier. In connection with the study of their involvement in barrier formation we required access to isomerically pure and defined samples of four linoleate triol esters. A common synthetic strategy was developed starting from isomeric alkynols derived from d-tartaric acid and 2-deoxy-d-ribose.

The antibacterial activity of 4,4'-bipyridinium amphiphiles with conventional, bicephalic and gemini architectures.

Dialkyl 4,4'-bipyridinium compounds are widely employed for their useful redox properties, and are commonly known as viologens due to their intense coloration upon reduction. Despite their prevalence and amphiphilic nature, the antibacterial activity of these compounds remains largely unreported. We have thus prepared a series of mono- and bis-alkylated analogs of 4,4'-bipyridine to investigate structure-activity relationships in their inhibition of a battery of Gram positive and Gram negative bacteria. The prepared cationic compounds were conventional (one cationic head, one non-polar tail), bicephalic (two heads, one tail), or gemini (two heads, two tails) in their amphiphilic structure. Additionally, an isomeric series of six bis-alkylated compounds ranging from symmetric (PQ-11,11) to highly asymmetric (PQ-20,2) were prepared. Four themes of bioactivity emerged: (1) the most bioactive compounds were gemini in structure; (2) 22 carbons in the alkyl chains, with little to modest asymmetry, led to optimal activity; (3) bicephalic compounds were generally comparable to conventional amphiphiles, though only about 12 carbons in the alkyl chains were solubilized in water by each cationic nitrogen; (4) the effects of counterion identity were not evident between chlorides and bromides; however, the presence of the iodide counterion inhibited dissolution in all compounds tested. Three isomeric compounds with little to no asymmetry in tail length, PQ-11,11, PQ-12,10, and PQ-14,8, prepared as the bromide salts, showed comparable and highly potent activity, with MIC levels around 2 µM against 3 of 4 bacteria tested. The simple (one- to two-step) syntheses of potent antimicrobials portend well for future optimization.

Toxicity and horizontal transfer of 0.5% fipronil dust against Formosan subterranean termites.

The toxicity and horizontal transfer of a new formulation of fipronil, 0.5% fipronil dust, was tested against Coptotermes formosanus Shiraki in the laboratory. The formulation was applied in three different ways: 1) Directly applied to termites (donors) and mixed with untreated termites (recipients) at three ratios, viz., 50 donors: 50 recipients, 20 donors: 80 recipients and 10 donors: 90 recipients. 2) Applied onto the surface of 3 mm thick sand or soil substrate in a petri dish and then topped with another 3 mm thick sand or soil layer whereupon termites were released. 3) Applied to the inner surface of a tube (either 5 cm or 15 cm long) that connected two foraging dishes, one containing dry sand and the other moist sand plus a wood block and termites were released into the dry sand dish. All donors and >93% of the recipients were dead by 42 h after treatment in the direct treatment experiment. Significant mortalities of both donors and recipients were observed at 5 h after treatment at all donor: recipient ratios. During this period, the mortality of the recipients (but not donors) at 10:90 was significantly lower than those at the other two ratios. All termites were dead at 65 h after exposure (HAE) on the sand treatment and at 190 HAE on soil treatment. More than 96% mortality was observed at 40 HAE on the sand treatment as compared with only 6% mortality onsoil treatment during the same time period. In the tube treatment experiment, > 97% mortality was observed at 90 h after release for both tube lengths as compared with < 3% mortality in controls. About half of the termites were dead by 15 h after release regardless of the tube length. Our results showed that 0.5% fipronil dust is nonrepellent and readily transferred from treated to nontreated termites.

Area-Wide Elimination of Subterranean Termite Colonies Using a Novaluron Bait.

We investigated the use of termite baiting, a proven system of targeted colony elimination, in an overall area-wide control strategy against subterranean termites. At two field sites, we used microsatellite markers to estimate the total number of Reticulitermes colonies, their spatial partitioning, and breeding structure. Termite pressure was recorded for two years before and after the introduction of Trelona® (active ingredient novaluron) to a large area of one of the sites. Roughly 70% of the colonies in the treatment site that were present at the time of baiting were not found in the site within two months after the introduction of novaluron. Feeding activity of the remaining colonies subsequently ceased over time and new invading colonies were unable to establish within this site. Our study provides novel field data on the efficacy of novaluron in colony elimination of Reticulitermes flavipes, as well as evidence that an area-wide baiting program is feasible to maintain a termite-free area within its native range.

Influence of temperature on rate of uptake and subsequent horizontal transfer of [14C]fipronil by eastern subterranean termites (Isoptera: Rhinotermitidae).

The effect of temperature on [14C]fipronil uptake and transfer from donor (D) to recipient (R) Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) workers was evaluated. Test chambers used in the fipronil uptake study were constructed from petri dishes containing autoclaved soil treated with 1 ppm [14C]fipronil (1.14 microCi of total radioactivity per petri dish), distilled water, and R. flavipes workers. Test chambers were held in environmental growth chambers preset at 12, 17, 22, 27, and 32 degrees C. For the fipronil transfer study, donor termites stained with Nile blue-A were exposed to soil treated with 1 ppm [14C]fipronil for 2 h. Donors were then combined with unexposed recipient termite workers at either 1D:5R, 1D:10R, or 1D:20R ratios. Test chambers consisted of a nest and feeding chamber connected by a piece of polyethylene tube and held in growth chambers at 12, 17, 22, 27, and 32 degrees C. Worker termites were sampled over time and the amount of [14C]fipronil present was measured by scintillation counting. Some degree of uptake and transfer occurred at all temperatures and ratios in this study. The highest level of uptake occurred by termites held at 22-32 degrees C, followed decreasingly by 17 and 12 degrees C. Maximum transfer of [14C]fipronil occurred at the higher ratios (1:5 > 1:10 > 1:20) of donors to recipients. Data presented in this study suggest that temperature is one of the key factors affecting the rate of uptake and subsequent horizontal transfer of [14C]fipronil in subterranean termites.

Refractive surgery policy and its effect on visual acuity qualification rates for the 2004 and 2011 Army ROTC flight applicant pools.

OBJECTIVES: The goal of this study was to ascertain the effect of recent U.S. Army Aviation corneal refractive surgery (CRS) policy changes on the size of flight school applicant pools by comparing statistics from the 2004 and 2011 Warrior Forge Army Reserve Officers' Training Corps training camps. METHODS: A retrospective chart review was performed using the United States Army Aeromedical Activity's Aeromedical Electronic Resource Online database. RESULTS: Of the 607 applicants from 2004, 100 (16%) were disqualified for decreased visual acuity and had refractive errors correctable by CRS. Thirty-four cadets (6%) had prior CRS, and 21 qualified for flight school in 2004. Of the 625 applicants from 2011, 105 (17%) were disqualified for decreased visual acuity and had correctable refractive errors. Sixty-four applicants (10%) had prior CRS, and 54 subsequently qualified in 2011. CONCLUSIONS: Changes to Army Aviation CRS policy over the last decade have been associated with an increased number of initial flight applicants who now meet visual acuity standards. However, only a small percentage is using this resource. Army Aviation has the potential to significantly widen its applicant pool if more candidates are made aware of accepted CRS techniques that can help them to meet rigorous vision standards.

Mobility, longevity and activity of chlorfenapyr in soils treated at a termiticidal rate.

BACKGROUND: The mobility, longevity and termiticidal activity of chlorfenapyr applied to soils at the termiticidal labeled rate was evaluated for 30 months after treatment (MAT) in a greenhouse study. RESULTS: There was little dissipation of chlorfenapyr in soil treated at the labeled rate for perimeter treatments for the prevention and control of termite infestations. Chlorfenapyr was detected in soil immediately below the initially treated soil in the packed soil columns. This was likely due to settling of soil. The treated soil remained toxic to subterranean termites in 3 and 7 day bioassays over the duration of the study. The treated soil displayed slow-acting properties regarding toxicity to termites. Trace amounts of chlorfenapyr were detected in the eluates of packed soil cones. CONCLUSION: The commercial formulation of chlorfenapyr used in this study (21.45% concentrate diluted to 0.125% prior to application) killed 100% of the tested subterranean termites for at least 30 months.

Bicephalic amphiphile architecture affects antibacterial activity.

A series of cationic amphiphiles, each with an aromatic core, was prepared and investigated for antimicrobial properties. The synthesized amphiphiles in this study are bicephalic (double-headed) in that they each possess two trimethylammonium head groups and a single linear alkoxy tail. Minimum inhibitory and minimum bactericidal concentrations of these amphiphiles were in the low micromolar range. Antimicrobial activities are highly sensitive to the chain length of the hydrophobic region, and modestly reliant on the relative positioning of the head groups on the aromatic core. These trends were more pronounced in time kill assays, wherein longer chain compounds required significantly shorter times to completely kill bacteria. Microscopy suggested that the mode of cell death was lysis. Strong inhibition was observed with both biscationic compounds and monocationic comparisons against Gram-positive bacteria; only biscationic amphiphiles maintained good activity versus the Gram-negative bacteria tested. These observations provide direction for future antimicrobial structural investigations.