Daily variation in markers of nutritional condition in wintering Black‐capped Chickadees Poecile atricapillus

The plasma metabolites triglycerides (TRIG) and β‐hydroxybutyrate (BUTY) are used as indices of nutritional condition in migrating birds during refuelling and can provide a measure of relative fattening rates in individual birds. Because non‐migratory birds wintering at northern latitudes also fatten on a daily basis to support their overnight fast, blood metabolites could provide a useful tool to measure individual performance in energy acquisition. However, daily patterns of metabolite change may differ between species and could be affected by thermoregulatory requirements. We studied daily variation in TRIG and BUTY over a complete winter in Black‐capped Chickadees to determine the pattern of daily and seasonal change in these markers. We also assessed how short‐term variation (up to 7 days) in weather parameters that influence heat exchange may affect TRIG and BUTY levels. In contrast to a linear gain of body mass, TRIG increased non‐linearly during the day, with a rapid increase in the morning that levelled off in the afternoon, whereas BUTY did not change significantly. Metabolites varied with sampling time and the seasonal change in day length, suggesting higher fat catabolism and fattening rates in mid‐winter. TRIG and BUTY also differed between capture sites, possibly due to differences in shelter quality. Weather variation did not affect TRIG levels and had a significant but marginal effect on BUTY, explaining at best 3% of the variation. Our results suggest that these markers can be used as indicators of energy turnover in resident wintering passerines.     

Biotransformation of 4‐fluoro‐N‐(1‐{2‐[(propan‐2‐yl)phenoxy]ethyl}‐8‐azabicyclo[3.2.1]octan‐3‐yl)‐benzenesulfonamide,a novel potent 5‐HT7 receptor antagonist with antidepressant‐like and anxiolytic properties: In vitro and in silico approach

The aim of the study was to investigate the metabolism of 4‐fluoro‐N‐(1‐{2‐[(propan‐2‐yl)phenoxy]ethyl}‐8‐azabicyclo[3.2.1]octan‐3‐yl)‐benzenesulfonamide (PZ‐1150), a novel 5‐HT₇ receptor antagonist with antidepressant‐like and anxiolytic properties, by the following three ways: in vitro with microsomes; in vitro employing Cunninghamella echinulata, and in silico using MetaSite. Biotransformation of PZ‐1150 with microsomes resulted in five metabolites, while transformation with C. echinulata afforded two metabolites. In both models, the predominant metabolite occurred due to hydroxylation of benzene ring. In silico data coincide with in vitro experiments, as three MetaSite metabolites matched compounds identified in microsomal samples. In human liver microsomes PZ‐1150 exhibited in vitro half‐life of 64 min, with microsomal intrinsic clearance of 54.1 μL/min/mg and intrinsic clearance of 48.7 mL/min/kg. Therefore, PZ‐1150 is predicted to be a high‐clearance agent. The study demonstrated the applicability of using microsomal model coupled with microbial model to elucidate the metabolic pathways of compounds and comparison with in silico metabolite predictions.     

Measuring ketones in the field: rapid and reliable measures of β‐hydroxybutyrate in birds

Ketone bodies such as β‐hydroxybutyrate are important indicators of metabolic condition in birds and are linked to a suite of ecologically relevant factors including migratory decision‐making, hunger level and ectoparasite load. Portable point‐of‐care (POC) devices designed to measure ketones in humans offer a cheap and easy solution to field physiologists in comparison with previous laboratory methods; however, their accuracy for use in birds has received scant attention. Here, we assessed the accuracy of a POC ketone meter (FreeStyle Precision Neo, Abbott, IL, USA) using samples from intermittently fed Red Junglefowl Gallus gallus. Although the device overestimated ketone levels in comparison with laboratory‐derived values, random error was low and laboratory vs. device values correlated well, indicating that the Precision Neo is of sufficient accuracy for use in the field and is a pragmatic choice for avian physiologists.     

Determination of (E)-5-(2-bromovinyl)-2′-deoxyuridine in plasma and urine by capillary electrophoresis

(E)-5-(2-Bromovinyl)-2′-deoxyuridine is an antiviral drug used for treatment of infections with Herpes simplex virus type 1 as well as Varicella zoster virus. Two fast methods for the determination of the drug and its metabolite in plasma and urine by capillary electrophoresis have been developed. The plasma method can be used for measurement of total as well as unbound drug and metabolite. Plasma and urine samples are prepared for measuring by liquid/liquid extraction resulting in a limit of quantification of 40 ng/ml for total and 10 ng/ml for free BVdU in plasma and 170 ng/ml in urine. Inter- as well as intra-day precision were found to be better than 10% and both methods have been used for drug monitoring of patients.     

Possible antagonism of (Z)‐2‐hexenyl (E)‐3‐hexenoate against the attractiveness of (E)‐2‐hexenyl (Z)‐3‐hexenoate to Ooencyrtus nezarae (Hymenoptera: Encyrtidae)

Ooencyrtus nezarae (Hymenoptera: Encyrtidae) is an egg parasitoid of bean bug Riptortus pedestris (Hemiptera: Alydidae) which is a major pest of beans. Females of O. nezarae are attracted to (E)‐2‐hexenyl (Z)‐3‐hexenoate (EZ), one of the components of aggregation pheromone of R. pedestris. Effects of three isomers (ZE, EE and ZZ) of EZ on the attractiveness of O. nezarae were tested using electroantennography (EAG) and field bioassays. EAG analyses revealed that the response of O. nezarae to ZE was significantly higher than those to air, hexane and two other isomers, even though the response was lower than that to EZ. ZE affected the attractiveness of EZ dose‐dependently in the field. Addition of ZE (100 mg) to EZ (10 mg) caused a significant reduction in the catches of O. nezarae females. Single or binary addition of two other isomers (EE and ZZ) to EZ could not decrease or increase significantly the number of O. nezarae catches of EZ. Even though addition of ZZ (10, 50 or 100 mg) to EZ (10 mg) caused dose‐dependent reduction in the number of O. nezarae female catches, the reductions were not significantly different from that of EZ. EZ and its three isomers were not attractive to O. nezarae males at all.     

Evaluation of the quality of bedside monitoring of the blood glucose level in a teaching hospital.

We evaluated the precision and accuracy of monitoring of the blood glucose level in the laboratory and at the bedside with one of four glucose meters by an experienced operator and by 39 nurses in a teaching hospital. For precision studies aqueous quality control materials were used. A total of 85 blood samples were tested. The precision of the glucose meters (expressed as the coefficient of variation [CV]) in the hands of the experienced operator ranged from 6.7% to 11.1%. The correlation between the values obtained by the experienced operator and the reference values obtained in the laboratory was high (0.95 to 0.98). The precision of the values obtained by the nurses using the meters ranged from 13.7% on medical wards to 45.7% in the intensive care unit (ICU). The correlation between these values and those obtained in the central laboratory ranged from 0.72 to 0.82. Twenty-four percent of the glucose values determined on medical wards and 62% of those determined in the ICU deviated from the reference value by at least 20%. Of the 85 patients 12 (14%) would have received different insulin dosages had the reference value been available at the same time as the glucose meter reading: in 3 of the patients the discrepancy was 6 units of insulin or greater. Continuous quality control of bedside monitoring of the blood glucose level is needed. In addition, personnel who use glucose meters should receive adequate training.     

Analysis of flumequine enantiomers in rat plasma by UFLC‐ESI‐MS/MS

In this study the analysis and confirmation of flumequine enantiomers in rat plasma by ultra‐fast liquid chromatography coupled with electron spray ionization mass spectrometry (using propranolol as an internal standard [IS]) was developed and validated. Plasma samples were prepared by liquid–liquid extraction using methyl tert‐butyl ether as the extraction solvent. Direct resolution of the R‐ and S‐isomers was performed on a CHIRALCEL OJ‐RH column (4.6 × 150 mm, 5 μm) using acetonitrile / 0.1% formic acid / 1 mM ammonium acetate as the mobile phase. Detection was operated by electron spray ionization in the selected ion monitoring and positive ion mode. The target ions at m/z 262.1 and m/z 260.1 were selected for the quantification of the enantiomers and IS, respectively. The linear range was 0.5–500 ng/mL. The precisions (coefficient of variation, CV%) and recoveries were 1.43–8.68 and 94.24–106.76%, respectively. The lowest quantitation limit for both enantiomers is 0.5 ng/mL, which is sensitive enough to be applied to sample analysis in other related studies.     

Determination of enantiomeric excess of nipecotic acid as 1‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl) derivatives

For the enantiopure synthesis of novel chiral GABA uptake inhibitors, nipecotic acid ( 1 ) is an important key precursor. To characterize accurately the pharmacological activity of these interesting target compounds, the determination of the correct enantiomeric purity of nipecotic acid as the starting material is indispensable. In this report, a sensitive high‐performance liquid chromatography (HPLC) based method for the separation and quantitation of both enantiomers of nipecotic acid as 1‐(7‐nitrobenzo[c ][1,2,5]oxadiazol‐4‐yl) derivatives ( 5 ) on a Chiralpak ID‐3 column (Daicel, Illkirch, France) was established. UV/Vis‐detection at 490 nm was chosen to ensure a selective determination of even highly enantioenriched samples. Reliability was demonstrated by validation of specificity, linearity, lower limit of quantification (LLOQ), accuracy, and precision. By spiking highly enantiopure samples with small amounts of racemic rac ‐ 5 , it was proven that the established HPLC method is able to detect even slight changes in enantiomeric excess (ee) values. Thus, accurate determination of ee values up to 99.87% ee for (R )‐ 5 and 99.86% ee for (S )‐ 5 over a linear concentration range of 1– 1500 μM for (R )‐ 5 and of 1– 1455 μM for (S )‐ 5 could be demonstrated.     

The Fate of Organic Sources of Carbon in Moss‐rich Tundra Soil Microbial Communities: A Laboratory Experimental Study

Effects of glucose‐carbon supplementation on soil respiration and bacterial and protist biomass were investigated in laboratory studies of three soil samples from Alaskan tundra: spring tussock sample 1 (thin surface moss), spring tussock sample 2 (thick surface moss), and a summer tundra open field sample. Addition of 1% (w/v) glucose solution produced an immediate, pronounced two to three fold increase in respiration above basal rate, which declined over 4 h to baseline levels. Less than 1% (w/w) of glucose‐C supplement was respired during the respiratory spike, relative to the 89 μg/g added. A more substantial amount of the glucose‐C became incorporated in microbial biomass. The total difference in microbial carbon (μg/g) between the experimental treatments and controls without glucose after 1 wk was as follows: spring sample 1 (8), spring sample 2 (31), and summer sample (70). The percent (w/w) of glucose‐C incorporated was: spring sample 1 (5%), spring sample 2 (17%), and summer sample (39%), most attributed to biomass of bacteria and heterotrophic nanoflagellates. Although respiratory response to pulsed glucose‐C was minimal, the overall mean basal rate after 1 wk ranged between 4 and 6 nmol/min/g soil, indicating a significant assimilation and respiration of constituent soil organic carbon.     

Evaluating the interaction of faecal pellet deposition rates and DNA degradation rates to optimize sampling design for DNA‐based mark–recapture analysis of Sonoran pronghorn

Knowledge of population demographics is important for species management but can be challenging in low‐density, wide‐ranging species. Population monitoring of the endangered Sonoran pronghorn (Antilocapra americana sonoriensis) is critical for assessing the success of recovery efforts, and noninvasive DNA sampling (NDS) could be more cost‐effective and less intrusive than traditional methods. We evaluated faecal pellet deposition rates and faecal DNA degradation rates to maximize sampling efficiency for DNA‐based mark–recapture analyses. Deposition data were collected at five watering holes using sampling intervals of 1–7 days and averaged one pellet pile per pronghorn per day. To evaluate nuclear DNA (nDNA) degradation, 20 faecal samples were exposed to local environmental conditions and sampled at eight time points from one to 124 days. Average amplification success rates for six nDNA microsatellite loci were 81% for samples on day one, 63% by day seven, 2% by day 14 and 0% by day 60. We evaluated the efficiency of different sampling intervals (1–10 days) by estimating the number of successful samples, success rate of individual identification and laboratory costs per successful sample. Cost per successful sample increased and success and efficiency declined as the sampling interval increased. Results indicate NDS of faecal pellets is a feasible method for individual identification, population estimation and demographic monitoring of Sonoran pronghorn. We recommend collecting samples >7 days old and estimate that a sampling interval of 4–7 days in summer conditions (i.e. extreme heat and exposure to UV light) will achieve desired sample sizes for mark–recapture analysis while also maximizing efficiency.     

Altered neurochemical profile in the McGill‐R‐Thy1‐APP rat model of Alzheimer's disease: a longitudinal in vivo 1H MRS study

We investigated metabolite levels during the progression of pathology in McGill‐R‐Thy1‐APP rats, a transgenic animal model of Alzheimer's disease, and in healthy age‐matched controls. Rats were subjected to in vivo ¹H magnetic resonance spectroscopy (MRS) of the dorsal hippocampus at age 3, 9 and 12 months and of frontal cortex at 9 and 12 months. At 3 months, a stage in which only Aβ oligomers are present, lower glutamate, myo‐inositol and total choline content were apparent in McGill‐R‐Thy1‐APP rats. At age 9 months, lower levels of glutamate, GABA, N‐acetylaspartate and total choline and elevated myo‐inositol and taurine were found in dorsal hippocampus, whereas lower levels of glutamate, GABA, glutamine and N‐acetylaspartate were found in frontal cortex. At age 12 months, only the taurine level was significantly different in dorsal hippocampus, whereas taurine, myo‐inositol, N‐acetylaspartate and total creatine levels were significantly higher in frontal cortex. McGill‐R‐Thy1‐APP rats did not show the same changes in metabolite levels with age as displayed in the controls, and overall, prominent and complex metabolite differences were evident in this transgenic rat model of Alzheimer's disease. The findings also demonstrate that in vivo ¹H MRS is a powerful tool to investigate disease‐related metabolite changes in the brain.     

Regional registration of [6‐14C]glucose metabolism during brain activation of α‐syntrophin knockout mice

α‐Syntrophin is a component of the dystrophin scaffold‐protein complex that serves as an adaptor for recruitment of key proteins to the cytoplasmic side of plasma membranes. α‐Syntrophin knockout (KO) causes loss of the polarized localization of aquaporin4 (AQP4) at astrocytic endfeet and interferes with water and K⁺ homeostasis. During brain activation, release of ions and metabolites from endfeet is anticipated to increase perivascular fluid osmolarity, AQP4‐mediated osmotic water flow from endfeet, and metabolite washout from brain. This study tests the hypothesis that reduced levels of endfoot AQP4 increase retention of [¹⁴C]metabolites during sensory stimulation. Conscious KO and wild‐type mice were pulse‐labeled with [6‐¹⁴C] glucose during unilateral acoustic stimulation or bilateral acoustic plus whisker stimulation, and label retention was assayed by computer‐assisted brain imaging or analysis of [¹⁴C]metabolites in extracts, respectively. High‐resolution autoradiographic assays detected a 17% side‐to‐side difference (p < 0.05) in inferior colliculus of KO mice, not wild‐type mice. However, there were no labeling differences between KO and wild‐type mice for five major HPLC fractions from four dissected regions, presumably because of insufficient anatomical resolution. The results suggest a role for AQP4‐mediated water flow in support of washout of metabolites, and underscore the need for greater understanding of astrocytic water and metabolite fluxes.     

Vinpocetine and α‐tocopherol prevent the increase in DA and oxidative stress induced by 3‐NPA in striatum isolated nerve endings

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3‐nitropropionic acid (3‐NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum‐isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC), and that 3‐NPA increased DA, reactive oxygen species (ROS), DA‐quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3‐NPA‐induced increase in DA, ROS, lipid peroxidation, and DA‐quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α‐tocopherol. Results show that the increase in DA induced by 3‐NPA was inhibited by both 25 μM vinpocetine and 50 μM α‐tocopherol. Vinpocetine, as α‐tocopherol, also inhibited 3‐NPA‐induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA‐RS formation), and DA‐quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3‐NPA, 3‐NPA increases DA‐oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α‐tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA‐rich structures.     

Light measurement for entomology in the field and laboratory

ABSTRACT. Light units weighted for human vision (e.g. lux) are unsuitable for work on insects which have good sensitivity in the blue and UV. We have built and calibrated two light meters - a portable field unit and a mains operated laboratory device-which measure illumination in physical units over a wavelength range suitable for insect vision. Both meters share a single photocell, so field and laboratory measurements are strictly comparable. The field meter covers a range from starlight to tropical sunlight on a single scale, and can be used in conditions when the operator has no effective vision. The laboratory meter is linear over five decade ranges, of which the most sensitive has a full-scale deflection of 1 mW m^-2.     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

Evaluation of the Origin of a Sample of <Emphasis Type="Italic">Sporothrix Schenckii</Emphasis> that Caused Contamination of a Researcher in Southern Brazil

We report a case of a researcher from a laboratory of Mycology in Rio Grande do Sul, Brazil that presented a clinical evidence of sporotrichosis. The researcher had an accident while manipulating the microculture slides of chromoblastomycosis agents and presented a clinical evidence of sporotrichosis. As the laboratory has some cultures of Sporothrix schenckii, it was suggested that it might be a laboratory contamination. In order to test this hypothesis, the genotypic characterization of the samples was performed by means of the random amplified polymorphic DNA (RAPD) analysis method. In addition, we evaluated the in vitro antifungal activity of four antifungal agents against the isolated fungus. The sample obtained from the researcher was not genetically similar to any of the samples kept in the laboratory and showed the minimum inhibitory concentrations of 0.5 μg/mL for itraconazole and ketoconazole, >64 μg/mL for fluconazole and 0.125 μg/mL for terbinafine. It is suggested that the contamination had an environmental origin.     

A high-throughput method for liquid chromatography–tandem mass spectrometry determination of plasma alkylresorcinols,biomarkers of whole grain wheat and rye intake

Plasma alkylresorcinols are increasingly analyzed in cohort studies to improve estimates of whole grain intake and their relationship with disease incidence. Current methods require large volumes of solvent (>10 ml/sample) and have relatively low daily sample throughput. We tested five different supported extraction methods for extracting alkylresorcinols from plasma and improved a normal-phase liquid chromatography coupled to a tandem mass spectrometer method to reduce sample analysis time. The method was validated and compared with gas chromatography–mass spectrometry analysis. Sample preparation with HybridSPE supported extraction was most effective for alkylresorcinol extraction, with recoveries of 77–82% from 100 μl of plasma. The use of 96-well plates allowed extraction of 160 samples per day. Using a 5-cm NH₂ column and heptane reduced run times to 3 min. The new method had a limit of detection and limit of quantification equivalent to 1.1–1.8 nmol/L and 3.5–6.1 nmol/L plasma, respectively, for the different alkylresorcinol homologues. Accuracy was 93–105%, and intra- and inter-batch precision values were 4–18% across different plasma concentrations. This method makes it possible to quantify plasma alkylresorcinols in 100 μl of plasma at a rate of at least 160 samples per day without the need for large volumes of organic solvents.     

Field‐compatible protocol for detecting tetracyclines with bioluminescent bioreporters without pipetting steps

Whole‐cell bioreporters are living organisms and thus using them for detecting environmental contaminants would reflect biological effects of these pollutants. However, bioreporters are not widely used in field studies. Many of the bioreporter field protocols are suitable for liquid samples or include pipetting steps, which is a demanding task outside the laboratory. We present a bioreporter protocol without pipetting or sample type requirements. The protocol utilizes polyester swabs, commonly used in cleanroom technology. As an example contaminant, we used tetracycline and generated test samples with known concentrations up to the maximum tetracycline residue limit of milk set by the European Union (EU) regulation. The matrices of the test samples were Milli‐Q water, milk and soil. The swabs were first dipped in the bioreporter cell cultures and then to test samples and luminescence was measured after incubation. The standard deviation of measurements from ten replicate swabs was in the same range as commonly in pipetting protocols (4–19%). The test samples with lowest tetracycline concentration (5 ng mL^?1) were distinguished from the control samples (0 ng mL^?1 tetracycline). Our results show that swabs can be used together with luminescent whole cell bioreporters, making it possible to conduct the measurements in field conditions.     

Rapid molecular methods for in‐field and laboratory identification of the yellow‐legged Asian hornet (Vespa velutina nigrithorax)

The yellow‐legged Asian hornet (Vespa velutina nigrithorax) is an invasive species that presents a threat to apiculture in Europe; first introduced into France in 2004, it has subsequently spread into neighbouring European countries. There is a risk of invasion and establishment in the UK, and in 2016, nests were found and destroyed in Alderney in the Channel Islands, and in Tetbury, Gloucestershire, illustrating a need for screening of suspect specimens so that invading hornets can be rapidly identified, and their nests destroyed. In this study, loop‐mediated isothermal amplification (LAMP) and real‐time PCR assays were developed to enable both in‐field and laboratory testing. Species‐specific identification assays and generic invertebrate control assays were developed. All the assays were validated according to the European Plant Protection Organisation standard PM 7/98. The assays were tested successfully against V. velutina nigrithorax obtained from France, Asia and the UK. Eight non‐target species, that were closely related or morphologically similar to the Asian hornet, gave negative results with the species‐specific assays, and positive results with the control assays. The assays could be used to detect target DNA at concentrations as low as 5 pg per reaction. LAMP was rapid, and cable of generating positive results within 10 min. Using simplified sample homogenization protocols that could be performed in the field, the LAMP assay was successful when tested against all developmental stages and nest samples, assisting with identification of samples that cannot be determined morphologically and allowing detection away from the laboratory. These assays provide a valuable tool for fast and reliable detection of this invasive species, offering the ability to identify damaged/incomplete specimens and immature life‐stages.