Melatonin and thyroxine response to pollution in white stork nestlings (Ciconia ciconia): aspects of rhythmicity and age

There is growing evidence that ubiquitous environmental contaminants may interfere with vertebrate endocrine systems. The selected endocrine biomarkers are used to indicate the condition of free-ranging populations of wildlife, including avian species. The aim of this study was to determine the impact of environment quality on serum thyroxine (T4) and melatonin (Mel) in white stork nestlings (Ciconia ciconia) living in different locations: small villages in natural areas surrounded by forests and crop fields, near the city and near the copper smelter. We extended our analyses to examine the hormones' day-night changes in conjunction with chicks' age. Total serum T4 and Mel was measured by RIA. T4 level, as a decisive measure of thyroid hormone productivity, was significantly lower in the nestlings exposed to pollutants from the copper smelter. Mel, as a well-known scavenger of free radicals, was elevated in the nestlings in the area near the copper smelter. This study indicates that alteration in T4 and Mel levels could be a useful marker of exposure of nestling wild storks to different toxic substances in field studies. Mel is postulated to be a susceptible defensive molecule as a protective mechanism for organisms.     

Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

A comparison of survey and focal follow methods for estimating individual activity budgets of cetaceans

Activity budget data are essential for determining behavioral responses to physiological and ecological variables. Yet, few studies are available to investigate the robustness, accuracy, and biases of the methods used to estimate activity budgets for cetaceans. In this study, we compare activity budgets of 55 adult female bottlenose dolphins in Shark Bay, Australia derived from two methods: surveys (n = 6,903) and focal follows (n = 1,185, totaling 2,721 h of observation). Activity budgets estimated from survey data differed in all behavioral states compared to focal follow data. However, when controlling for temporal autocorrelation, only time spent socializing and time spent traveling remained disparate between the methods. To control for biases associated with assigning group‐level behavior to individuals, we also compared survey and focal follow activity budgets for lone females. Here we found differences between methods in time spent foraging and traveling regardless of whether we controlled for temporal autocorrelation, which suggests detection biases likely play a role in explaining differences in activity budget estimates between the two methodologies. Our results suggest that surveys are less representative of individual‐level activity budgets, and thus, when individual‐level knowledge about behavior is needed, focal follows are preferred.     

Common functionally important motions of the nucleotide‐binding domain of Hsp70

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate‐binding domain (SBD) that binds client substrates, and the nucleotide‐binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure–function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (PDB 3C7N:B) by all‐atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP‐ and ATP‐unique classes, which reflect conformational trends that are unique to either the ADP‐ or ATP‐bound states, respectively. “Mutual” class motions generally describe “in‐plane” and/or “out‐of‐plane” (scissor‐like) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The “unique” class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the “unique” type, regions of enhanced mobility can be identified; these are termed “hot spots,” and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide‐binding pocket was also found to influence the dynamics of the NBD significantly. Proteins 2015; 83:282–299. © 2014 Wiley Periodicals, Inc.     

Identification of critical residues for transport activity of Acr3p,the Saccharomyces cerevisiae As(III)/H+ antiporter

Acr3p is an As(III)/H⁺ antiporter from Saccharomyces cerevisiae belonging to the bile/arsenite/riboflavin transporter superfamily. We have previously found that Cys151 located in the middle of the fourth transmembrane segment (TM4) is critical for antiport activity, suggesting that As(III) might interact with a thiol group during the translocation process. In order to identify functionally important residues involved in As(III)/H⁺ exchange, we performed a systematic alanine‐replacement analysis of charged/polar and aromatic residues that are conserved in the Acr3 family and located in putative transmembrane segments. Nine residues (Asn117, Trp130, Arg150, Trp158, Asn176, Arg230, Tyr290, Phe345, Asn351) were found to be critical for proper folding and trafficking of Acr3p to the plasma membrane. In addition, we found that replacement of highly conserved Phe266 (TM7), Phe352 (TM9), Glu353 (TM9) and Glu380 (TM10) with Ala abolished transport activity of Acr3p, while mutation of Ser349 (TM9) to Ala significantly reduced the As(III)/H⁺ exchange, suggesting an important role of these residues in the transport mechanism. Detailed mutational analysis of Glu353 and Glu380 revealed that the negatively charged residues located in the middle of transmembrane segments TM9 and TM10 are crucial for antiport activity. We also discuss a hypothetical model of the Acr3p transport mechanism.     

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor–UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5 ng/ml. The detection limit is 0.06 ng/ml for the biosensor with antibody and 0.08 ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20 ng/ml for plasma samples and 0.30 to 0.49 ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.     

Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6 h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ?3 freeze–thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14–17 years of frozen storage (−80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies.     

Influence of native and alien Prunus species and light conditions on performance of the leaf beetle Gonioctena quinquepunctata

The polyphagous beetle Gonioctena quinquepunctata Fabricius (Coleoptera: Chrysomelidae) is a serious leaf pest of the native European bird cherry, Prunus padus L., and the invasive alien black cherry, Prunus serotina Ehrh. (Rosaceae). In the shade, leaf damage is extensive in both species, whereas in full light, it is extensive in P. padus, but very low in P. serotina. We determined the influence of Prunus species and light conditions on differences in performance of both sexes of this folivore. In a laboratory experiment in which larvae were fed with leaves of a single species grown under particular light conditions, we measured larval, pupal, and adult mass, efficiency of conversion of ingested food (ECI), duration of development, total food eaten, and relative growth rate. In the field, we observed differences in beetle mass on shrubs of the two species growing under various light conditions. From the field observations, we hypothesised that leaves of the invasive P. serotina are not an equally good food source as leaves of P. padus for G. quinquepunctata, and the preference of these beetles for shaded shrubs is most favourable for their growth and development. Under laboratory conditions, we found that the beetle growth rate was not affected significantly by Prunus species or light conditions, despite the significant effect of light condition on the structure and chemical composition of Prunus seedlings. The lower ECI value for larvae feeding on sunlit leaves was compensated for by their higher level of consumption. In the field, adult insect mass was higher on P. padus than on P. serotina, and higher on sunlit shrubs of both species than on shaded ones. Under natural conditions, the mass of adult insects is probably also affected by other factors, such as predators and competition among folivores.