Nanocrystalline diamond impedimetric aptasensor for the label-free detection of human IgE

Like antibodies, aptamers are highly valuable as bioreceptor molecules for protein biomarkers because of their excellent selectivity, specificity and stability. The integration of aptamers with semiconducting materials offers great potential for the development of reliable aptasensors. In this paper we present an aptamer-based impedimetric biosensor using a nanocrystalline diamond (NCD) film as a working electrode for the direct and label-free detection of human immunoglobulin E (IgE). Amino (NH(2))-terminated IgE aptamers were covalently attached to carboxyl (COOH)-modified NCD surfaces using carbodiimide chemistry. Electrochemical impedance spectroscopy (EIS) was applied to measure the changes in interfacial electrical properties that arise when the aptamer-functionalized diamond surface was exposed to IgE solutions. During incubation, the formation of aptamer-IgE complexes caused a significant change in the capacitance of the double-layer, in good correspondence with the IgE concentration. The linear dynamic range of IgE detection was from 0.03 μg/mL to 42.8 μg/mL. The detection limit of the aptasensor reached physiologically relevant concentrations (0.03 μg/mL). The NCD-based aptasensor was demonstrated to be highly selective even in the presence of a large excess of IgG. In addition, the aptasensor provided reproducible signals during six regeneration cycles. The impedimetric aptasensor was successfully tested on human serum samples, which opens up the potential of using EIS for direct and label-free detection of IgE levels in blood serum.     

Discovery of INCB10820/PF-4178903, a potent, selective, and orally bioavailable dual CCR2 and CCR5 antagonist

We report the discovery of a potent, selective, and orally bioavailable dual CCR2 and CCR5 antagonist (3S,4S)-N-[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine (19). After evaluation in 28-day toxicology studies, compound 19 (INCB10820/PF-4178903) was selected as a clinical candidate.     

The structural basis for the function of two anti-VEGF receptor 2 antibodies

The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.     

Proteomics reveals that redox regulation is disrupted in patients with ethylmalonic encephalopathy

Deficiency of the sulfide metabolizing protein ETHE1 is the cause of ethylmalonic encephalopathy (EE), an inherited and severe metabolic disorder. To study the molecular effects of EE, we performed a proteomics study on mitochondria from cultured patient fibroblast cells. Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two proteins involved in pathways of detoxification and oxidative/reductive stress were underrepresented in EE patient samples: mitochondrial superoxide dismutase (SOD2) and aldehyde dehydrogenase X (ALDH1B). Sulfide:quinone oxidoreductase (SQRDL), which takes part in the same sulfide pathway as ETHE1, was also underrepresented in EE patients. The other differentially regulated proteins were apoptosis inducing factor (AIFM1), lactate dehydrogenase (LDHB), chloride intracellular channel (CLIC4) and dimethylarginine dimethylaminohydrolase 1 (DDAH1). These proteins have been reported to be involved in encephalopathy, energy metabolism, ion transport, and nitric oxide regulation, respectively. Interestingly, oxidoreductase activity was overrepresented among the regulated proteins indicating that redox perturbation plays an important role in the molecular mechanism of EE. This observation may explain the wide range of symptoms associated with the disease, and highlights the potency of the novel gaseous mediator sulfide.     

Physiological responses during interval training with different intensities and duration of exercise

The purpose of this study was to compare 4 interval training (IT) sessions with different intensities and durations of exercise to determine the effect on mean VO?, total VO?, and duration of exertion ≥95% maximum power output (MPO), and the effects on biomarkers of fatigue such as blood-lactate concentration (BLC) and rating of perceived exertion. The subjects were 12 recreationally competitive male (n = 7, mean ± SD age = 26.2 ± 3.9 years) and female (n = 5, mean ± SD age = 27.6 ± 4.3 years) triathletes. These subjects performed 4 IT sessions on a cycle ergometer varying in intensity (90 and 100% MPO) and duration of exercise (30 seconds and 3 minutes). This study revealed that IT using 30-second duration intervals (30-30 seconds) allows the athlete to perform a longer session, with a higher total and mean VO? HR and lower BLC than 3-minute durations. Similarly, submaximal exertion at 90% of MPO also allows performing longer sessions with a higher total VO? than 100% intensity. Thus, the results of the present study suggested that to increase the total time at high intensity of exercise and total VO? of a single exercise session performed by the athlete, IT protocols of short durations (i.e., 30 seconds) and submaximal intensities (i.e., 90% MPO) should be selected. Furthermore, performing short-duration intervals may allow the athlete to complete a longer IT session with greater metabolic demands (VO?) and lower BLC than longer (i.e., 3 minutes) intervals.     

Home on the range: spatial ecology of loggerhead turtles in Atlantic waters of the USA

Aim Although satellite tracking has yielded much information regarding the migrations and habitat use of threatened marine species, relatively little has been published about the environmental niche for loggerhead sea turtles Caretta caretta in north‐west Atlantic waters. Location North Carolina, South Carolina and Georgia, USA. Methods We tracked 68 adult female turtles between 1998 and 2008, one of the largest sample sizes to date, for 372.2 ± 210.4 days (mean ± SD). Results We identified two strategies: (1) ‘seasonal’ migrations between summer and winter coastal areas (n = 47), although some turtles made oceanic excursions (n = 4) and (2) occupation of more southerly ‘year‐round’ ranges (n = 18). Seasonal turtles occupied summer home ranges of 645.1 km² (median, n = 42; using α‐hulls) predominantly north of 35 ° latitude and winter home ranges of 339.0 km² (n = 24) in a relatively small area on the narrow shelf off North Carolina. We tracked some of these turtles through successive summer (n = 8) and winter (n = 3) seasons, showing inter‐annual home range repeatability to within 14.5 km of summer areas and 10.3 km of winter areas. For year‐round turtles, home ranges were 1889.9 km². Turtles should be tracked for at least 80 days to reliably estimate the home range size in seasonal habitats. The equivalent minimum duration for ‘year‐round’ turtles is more complex to derive. We define an environmental envelope of the distribution of North American loggerhead turtles: warm waters (between 18.2 and 29.2 °C) on the coastal shelf (in depths of 3.0–89.0 m). Main conclusions Our findings show that adult female loggerhead turtles show predictable, repeatable home range behaviour and do not generally leave waters of the USA, nor the continental shelf (< 200m depth). These data offer insights for future marine management, particularly if they were combined with those from the other management units in the USA.     

Former land use affects the nitrogen and phosphorus concentrations and biomass of forest herbs

The colonization rates of understorey plants into forests growing on former agricultural land differ remarkably among species. Different dispersal and recruitment largely account for the contrasting colonization rates, but different effects of the soil legacies of former agricultural land use on plant performance may also play a role. Seven herbaceous forest species were sampled in paired post-agricultural and ancient forest stands to study whether land-use history has an effect on the aboveground nutrient concentrations (N, P and N:P ratios) and biomass of forest herbs and, if so, whether slow and fast colonizing species respond differently. Results showed that P concentrations were significantly affected by former land use with higher concentrations in the post-agricultural stands. N concentrations were unaffected and N:P ratios were significantly higher in the ancient stands. Nutrient concentrations varied considerably among species, but the variation was unrelated to their colonization capacity. Six out of the seven species had higher biomass in the post-agricultural stands relative to the ancient stands, and the degree to which the species increased biomass was positively related to their colonization capacity, i.e., the fast colonizing species showed the strongest increase. Such differential responses to past land use may contribute to the contrasting colonization capacity of forest plants. Land-use history thus affected both the nutrient concentrations and biomass of forest herbs, and only the biomass response was related to colonization capacity.     

A comparison of MS2-based label-free quantitative proteomic techniques with regards to accuracy and precision

The advent of algorithms for fragmentation spectrum-based label-free quantitative proteomics has enabled straightforward quantification of shotgun proteomic experiments. Despite the popularity of these approaches, few studies have been performed to assess their performance. We have therefore profiled the precision and the accuracy of three distinct relative label-free methods on both the protein and the proteome level. We derived our test data from two well-characterized publicly available quantitative data sets.     

A stringent approach to improve the quality of nitrotyrosine peptide identifications

Tyrosine nitration is the consequence of a complex machinery of formation and merging of oxygen and nitrogen radicals, and has been associated with both physiological pathways as well as with several human diseases. The latter turned this posttranslational protein modification into an interesting biomarker, being either a consequence of the disease or a factor contributing to the disease onset. However, the interpretation of MS and MS/MS data of peptides containing nitrotyrosine has proven to be very challenging and consequently, the risk of linking MS/MS spectra to incorrect peptide sequences exists and has been reported. Here, we discuss the causes of data misinterpretation and describe a general method to avoid mistakes of MS/MS spectrum misinterpretation. Central in our approach is the reduction of nitrotyrosine into aminotyrosine and the use of the Peptizer algorithm to inspect MS/MS quality-related assumptions.