Daily activity patterns of coyotes (Canis latrans) in a tropical deciduous forest of western Mexico

We studied the activity patterns of the coyote (Canis latrans) in a tropical deciduous forest in the Mexican Pacific coast over 3 years. Fifteen coyotes (six females, nine males) were fitted with radio-collars equipped with activity sensors to determine the influence of seasonality (dry vs. wet), gender (males vs. females) and diel intervals (dusk, night, dawn, and day) on activity patterns. We found differences in activity patterns between diel intervals, but the only pair of diel intervals that showed significant differences was dawn (more active) vs. day (less active). We found no differences due to sex or season on any of the four studied diel intervals. Coyote activity patterns in this tropical forest could be responding to prey availability, human avoidance or thermoregulation.     

HERG is protected from pharmacological block by alpha-1,2-glucosyltransferase function

The HERG (human ether-à-go-go-related gene) protein, which underlies the cardiac repolarizing current I(Kr), is the unintended target for many pharmaceutical agents. Inadvertent block of I(Kr), known as the acquired long QT syndrome (aLQTS), is a leading cause for drug withdrawal by the United States Food and Drug Administration. Hence, an improved understanding of the regulatory factors that protect most individuals from aLQTS is essential for advancing clinical therapeutics in broad areas, from cancer chemotherapy to antipsychotics and antidepressants. Here, we show that the K(+) channel regulatory protein KCR1, which markedly reduces I(Kr) drug sensitivity, protects HERG through glucosyltransferase function. KCR1 and the yeast alpha-1,2-glucosyltransferase ALG10 exhibit sequence homology, and like KCR1, ALG10 diminished HERG block by dofetilide. Inhibition of cellular glycosylation pathways with tunicamycin abrogated the effects of KCR1, as did expression in Lec1 cells (deficient in glycosylation). Moreover, KCR1 complemented the growth defect of an alg10-deficient yeast strain and enhanced glycosylation of an Alg10 substrate in yeast. HERG itself is not the target for KCR1-mediated glycosylation because the dofetilide response of glycosylation-deficient HERG(N598Q) was still modulated by KCR1. Nonetheless, our data indicate that the alpha-1,2-glucosyltransferase function is a key component of the molecular pathway whereby KCR1 diminishes I(Kr) drug response. Incorporation of in vitro data into a computational model indicated that KCR1 expression is protective against arrhythmias. These findings reveal a potential new avenue for targeted prevention of aLQTS.     

Spatial and temporal distribution of archaeal diversity in meromictic,hypersaline Ocnei Lake (Transylvanian Basin,Romania)

Saline, meromictic lakes with significant depth are usually formed as a result of salt mining activity. Ocnei Lake is one of the largest Transylvanian (Central Romania) neutral, hypersaline lake of man-made origin. We aimed to survey the seasonal dynamics of archaeal diversity in the water column of Ocnei Lake by employing microbiological methods as well as molecular techniques based on the sequence analysis of the 16S rRNA gene. We found that archaeal diversity in the water column increased with depth and salinity, with 8 OTUs being detected in the epilimnion compared to 21 found in the chemocline, and 32 OTUs in the monimolimnion. Down to 3.5 m depth, the archaeal community was markedly dominated by the presence of an unclassified archaeon sharing 93 % sequence identity to Halogeometricum spp. At the chemocline, the shift in archaeal community composition was associated with an increase in salinity, the main factor affecting the vertical distribution of archaeal assemblages. It appears that the microoxic and hypersaline monimolimnion is populated by several major haloarchaeal taxa, with minor fluctuations in their relative abundances throughout all seasons. The culturable diversity was reasonably correlated to the dominant OTUs obtained by molecular methods. Our results indicate that Ocnei Lake represents a relatively stable extreme habitat, accommodating a diverse and putatively novel archaeal community, as 30 % of OTUs could not be classified at the genus level.     

Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture

Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with a novel molecular architecture among family 9 enzymes that comprises a catalytic domain (GH9), a family 3c cellulose-binding domain (CBM3c), a fibronectin III-like domain repeat (Fn3_1,2), and a C-terminal family 3b cellulose-binding domain (CBM3b). A series of truncated derivatives of endoglucanase Cel9B have been constructed and characterized. Deletion of CBM3c produced a notable reduction in hydrolytic activity, while it did not affect the cellulose-binding properties as CBM3c did not show the ability to bind to cellulose. On the contrary, CBM3b exhibited binding to cellulose. The truncated forms devoid of CBM3b lost cellulose-binding ability and showed a reduced activity on crystalline cellulose, although activity on amorphous celluloses was not affected. Endoglucanase Cel9B produced only a small ratio of insoluble products from filter paper, while most of the reducing ends produced by the enzyme were released as soluble sugars (91%), indicating that it is a processive enzyme. Processivity of Cel9B resides in traits contained in the tandem of domains GH9–CBM3c, although the slightly reduced processivity of truncated form GH9–CBM3c suggests a minor contribution of domains Fn3_1,2 or CBM3b, not contained in it, on processivity of endoglucanase Cel9B.     

Integrating sign surveys and telemetry data for estimating brown bear (Ursus arctos) density in the Romanian Carpathians

Accurate population size estimates are important information for sustainable wildlife management. The Romanian Carpathians harbor the largest brown bear (Ursus arctos) population in Europe, yet current management relies on estimates of density that lack statistical oversight and ignore uncertainty deriving from track surveys. In this study, we investigate an alternative approach to estimate brown bear density using sign surveys along transects within a novel integration of occupancy models and home range methods. We performed repeated surveys along 2‐km segments of forest roads during three distinct seasons: spring 2011, fall‐winter 2011, and spring 2012, within three game management units and a Natura 2000 site. We estimated bears abundances along transects using the number of unique tracks observed per survey occasion via N‐mixture hierarchical models, which account for imperfect detection. To obtain brown bear densities, we combined these abundances with the effective sampling area of the transects, that is, estimated as a function of the median (± bootstrapped SE) of the core home range (5.58 ± 1.08 km²) based on telemetry data from 17 bears tracked for 1‐month periods overlapping our surveys windows. Our analyses yielded average brown bear densities (and 95% confidence intervals) for the three seasons of: 11.5 (7.8–15.3), 11.3 (7.4–15.2), and 12.4 (8.6–16.3) individuals/100 km². Across game management units, mean densities ranged between 7.5 and 14.8 individuals/100 km². Our method incorporates multiple sources of uncertainty (e.g., effective sampling area, imperfect detection) to estimate brown bear density, but the inference fundamentally relies on unmarked individuals only. While useful as a temporary approach to monitor brown bears, we urge implementing DNA capture–recapture methods regionally to inform brown bear management and recommend increasing resources for GPS collars to improve estimates of effective sampling area.     

Nanostructured digital microfluidics for enhanced surface plasmon resonance imaging

The advances in genomics and proteomics have unveiled an exhaustive catalogue of biomarkers that can potentially be used as diagnostic and prognostic indicators of genetic and infectious diseases. Current thrust in biosensor development is towards rapid, real-time, label-free and highly sensitive detection of the indicative biomarkers. While surface plasmon resonance imaging (SPRi) biosensors could potentially be the best suited candidate for biomarker-based diagnosis, important milestones need to be reached. Commercially available SPRi instrumentation is currently limited by the flow-cell technology to serial-sample processing and has limited sensitivity for the detection of markers present at low concentration. In this paper, we have implemented an approach to enhance sample handling and increase the sensitivity of the SPRi detection technique. We have developed a digital microfluidic platform with an integrated nanostructured biosensor interface that allows for rapid, ultra-low volume, sensitive, and automated on-chip SPRi detection of DNA hybridization reactions. Through the exploitation of electromagnetic properties of nanofabricated periodic gold nanoposts, SPRi signal was increased by 200% with the estimated limit of detection of 500 pM (90 attomoles). Using the versatile fluidic manipulation provided by the digital microfluidics, rapid and parallel target identification was achieved on multiple array elements within 1 min using 180 nL sample volume. By delivering multiple target analytes in individually addressable low volume droplets, without external pumps and fluidic interconnects, the overall assay time, cost and complexity was reduced. The proposed platform allows extreme versatility in the manipulation of precious low volume samples which makes this technology very suitable for diagnostic applications.     

Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry in aqueous solution

The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.     

Microbial community succession during lactate amendment and electron acceptor limitation reveals a predominance of metal-reducing Pelosinus spp

The determination of the success of in situ bioremediation strategies is complex. By using controlled laboratory conditions, the influence of individual variables, such as U(VI), Cr(VI), and electron donors and acceptors on community structure, dynamics, and the metal-reducing potential can be studied. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI)-contaminated groundwater from the Hanford, WA, 100-H area, amended with lactate, and incubated for 95 days to obtain stable, enriched communities. The reactors were kept anaerobic with N(2) gas (9 ml/min) flushing the headspace and were fed a defined medium amended with 30 mM lactate and 0.05 mM sulfate with a 48-h generation time. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp., with low levels of other organisms, including methanogens. Four new strains of Pelosinus were isolated, with 3 strains being capable of Cr(VI) reduction while one also reduced U(VI). Under limited sulfate, it appeared that the sulfate reducers, including Desulfovibrio spp., were outcompeted. These results suggest that during times of electron acceptor limitation in situ, organisms such as Pelosinus spp. may outcompete the more-well-studied organisms while maintaining overall metal reduction rates and extents. Finally, lab-scale simulations can test new strategies on a smaller scale while facilitating community member isolation, so that a deeper understanding of community metabolism can be revealed.     

Quartz crystal microbalance bioaffinity sensor for rapid identification of glycosyldisulfide lectin inhibitors from a dynamic combinatorial library

Carbohydrate-lectin interactions were probed with dynamic combinatorial libraries, using the plant lectin Concanavalin A as target species. The dynamic combinatorial libraries were generated from a pool of thiol components through reversible thiol-disulfide interchange, and screened using a simple and efficient method based on a quartz crystal microbalance setup. It was found that dimers based on 1-thio- and 6-thio-mannose analogues were the most active inhibitors. Furthermore, the results clearly show that the 6-thio-mannose possess unique characteristics compared to its oxygen-containing counterpart.