Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes

Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells

Alpha-induced reactions for the astrophysical p-process: the case of 151Eu

The cross sections of 151Eu(alpha,gamma)155Tb and 151Eu(alpha,n)154Tb reactions have been measured with the activation method. Some aspects of the measurement are presented here to illustrate the requirements of experimental techniques needed to obtain nuclear data for the astrophysical p-process nucleosynthesis. Preliminary cross section results are also presented and compared with the predictions of statistical model calculations.Comment: Accepted for publication in Journal of Physics Conference Series, proceeding of the Nuclear Physics in Astrophysics IV. conferenc

Diurnal variations in the expression of core-clock genes correlate with resting muscle properties and predict fluctuations in exercise performance across the day

OBJECTIVES: In this study, we investigated daily fluctuations in molecular (gene expression) and physiological (biomechanical muscle properties) features in human peripheral cells and their correlation with exercise performance. METHODS: 21 healthy participants (13 men and 8 women) took part in three test series: for the molecular analysis, 15 participants provided hair, blood or saliva time-course sampling for the rhythmicity analysis of core-clock gene expression via RT-PCR. For the exercise tests, 16 participants conducted strength and endurance exercises at different times of the day (9h, 12h, 15h and 18h). Myotonometry was carried out using a digital palpation device (MyotonPRO), five muscles were measured in 11 participants. A computational analysis was performed to relate core-clock gene expression, resting muscle tone and exercise performance. RESULTS: Core-clock genes show daily fluctuations in expression in all biological samples tested for all participants. Exercise performance peaks in the late afternoon (15-18 hours for both men and women) and shows variations in performance, depending on the type of exercise (eg, strength vs endurance). Muscle tone varies across the day and higher muscle tone correlates with better performance. Molecular daily profiles correlate with daily variation in exercise performance. CONCLUSION: Training programmes can profit from these findings to increase efficiency and fine-tune timing of training sessions based on the individual molecular data. Our results can benefit both professional athletes, where a fraction of seconds may allow for a gold medal, and rehabilitation in clinical settings to increase therapy efficacy and reduce recovery times

Corrigendum to "European contribution to the study of ROS:A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)" [Redox Biol. 13 (2017) 94-162]

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed

Magnetic anisotropies in ultrathin iron films grown on the surface-reconstructed GaAs substrate

Magnetic anisotropies of epitaxial ultrathin iron films grown on the surface-reconstructed GaAs substrate were studied. Ferromagnetic resonance technique was exploited to determine magnetic parameters of the films in the temperature range of 4-300 K. Extraordinary angular dependence of the FMR spectra was explained by the presence of fourfold and twofold in-plane anisotropies. A strong in-plane uniaxial anisotropy with magnetic hard axis along the [1 (1) over bar0] crystallographic direction is present at the GaAs/Fe(001) interface while a weak in-plane uniaxial anisotropy for the Fe grown on Au has its easy axis oriented along [1 (1) over bar0]. A linear dependence of the magnetic anisotropies as a function of temperature suggests that the strength of the in-plane uniaxial anisotropy is affected by the magnetoelastic anisotropies and differential thermal expansion of contacting materials. (c) 2007 American Institute of Physics

Effect of dietary methionine on performance, carcase characteristics and breast meat composition of heterozygous naked neck (Na/na+) birds under spring and summer conditions

PubMed ID: 106706831. Heteroxygous naked neck (Na/na+) birds and their normally feathered counterparts (na+/ na+) were fed from 0 to 7 weeks on 3 diets differing in methionine concentrations. From 0 to 3 and 3 to 7 weeks, respectively, the concentrations were: low containing 4.3 and 3.3 g/kg; optimum containing 5.0 and 3.8 g/kg and; high with 5.7 and 4.4 g/kg under spring (optimum ambient temperature) and summer conditions (high ambient temperature). Performance, carcase characteristics and breast meat chemical composition were determined. 2. Summer rearing resulted in a decrease in body weight, body weight gain, food consumption, and yields of carcase and breast. The summer temperature effect was more pronounced in males. Under summer temperatures, the protein content of the breast decreased while the fat content increased compared to birds reared in spring. 3. By 7 weeks of age, both genotypes reached similar body weights in the spring experiment while, in summer Na/na+ birds were 3.3% heavier and gained more in the period from 3 to 7 weeks than na+/na+ birds. Carcase and breast yields of Na/na+ birds were greater than in na+/na+ birds. 3. Second order polynomial coefficients of the dietary methionine effect were found to be significant for body weight at 3 and 7 weeks. Daily body weight gain between 3 and 7 weeks was linearly affected by the dietary methionine concentration. There was no interaction between genotype and methionine. 4. Methionine had no significant effect on carcase yield. Second order polynomial coefficients of the dietary methionine effect were found to be significant for breast yield while the methionine effect on abdominal fat was linear. Na/na+ females fed on the low methionine diet had lower protein content than the Na/na+ males. 5. It is concluded that the methionine requirement of Na/na+ birds did not differ from that of their normally feathered counterparts under either spring or summer ambient temperature conditions. © 1999 British Poultry Science Ltd

The effect of harvest time on the bioactive properties and volatile components of lavender (Lavandula officinalis)

This study was designed to determine the effect of harvest time on the bioactive properties and volatile components of flowers, leaves and stems of Lavandula officinalis. The bioactive properties were examined in terms of total phenolic content, antiradical activity and antioxidant capacity by the Folin-Ciocalteu, 1.1-diphenyl-2-picrylhydrazyl scavenging and phosphomolybdenum methods, respectively. Volatile components were detected with gas chromatography-mass spectrometry. Water and methanol were used for extraction solvent and three different parts of the day (morning, midday and evening) were selected for harvest time. Water extracts had higher total phenolic contents (337.2-771.5 mg gallic acid equivalents (GAE)/g dry extract) than methanolic extracts (170.3-320.8 mg GAE/g dry extract), and in general, midday values were less than morning and evening rates and had a significant difference depending on harvest time. A similar feature was observed in respect to antioxidant properties. The inhibition rates (%) of flowers and leaves were higher than those of the stems for either extract. In terms of the volatile components linalool, camphor, 1,8-cineole, terpinen-4-ol and isoborneol were the most representative compounds in the samples