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[Image: see text] α-Synuclein fibrils are considered a hallmark of Parkinson’s disease and other synucleinopathies. However, small oligomers that formed during the early stages of α-synuclein aggregation are thought to be the main toxic species causing disease. The formation of α-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of α-synuclein oligomerization, α-Synuclein–Confocal Nanoscanning (ASYN-CONA), is presented. The α-synuclein A91C single cysteine mutant is modified with a trifunctional chemical tag, which allows simultaneous fluorescent labeling with a green dye (tetramethylrhodamine, TMR) and attachment to microbeads. Beads with bound TMR-labeled α-synuclein are then incubated with a red dye (Cy5)-labeled variant of α-synuclein A91C, and EtOH (20%) to induce aggregation. Aggregation is detected by confocal scanning imaging, below the equatorial plane of the beads, which is known as the CONA technique. On-bead TMR-labeled α-synuclein and aggregated Cy5-labeled α-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or “rings”. α-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 ± 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of α-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening. Baicalein, curcumin, and rifampicin showed concentration-dependent inhibition of the α-synuclein aggregation and the IC(50) (the concentration of the compound at which the maxiumum intensity was reduced by one-half) were calculated.

RNA–protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA–protein interactions have shown promising effects on some diseases that are previously regarded as ‘incurable’. Here, we developed a fluorescent on-bead screening platform, RNA Pull-Down COnfocal NAnoscanning (RP-CONA), to identify RNA–protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7–1. Importantly, miR-7′s primary target is an mRNA of α-synuclein, which contributes to the aetiology of Parkinson’s disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-synuclein. This opens up new therapeutic avenues towards treatment of Parkinson’s disease as well as provides a novel methodology to search for modulators of RNA–protein interaction.

RNA-protein interactions are central to all gene expression processes and contribute to variety of human diseases. Therapeutic approaches targeting RNA-protein interactions have shown promising effects on some diseases that are previously regarded as ‘incurable’. Here we developed a fluorescent on-bead screening platform: RNA pull-down-Confocal Nanoscanning (RP-CONA), to identify RNA-protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7-1. Importantly, miR-7’s primary target is an mRNA of α-Synuclein, which contributes to aetiology of Parkinson’s disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-Synuclein. This opens up new therapeutic avenues towards treatment of Parkinson’s disease as well as provides novel methodology to search for RNA-protein interaction modulators.

Information on how to present visual content in an ergonomic way is crucial in various fields. The current study examines how the gender, race of a person, and picture-slogan relative location influence subjects’ perceived effectiveness about a social advertisement. Relative scores of 45 participants from Wroclaw University of Science and Technology were measured by binary pairwise comparisons and formally analyzed by the loglinear model for frequency data. While making comparisons, subjects’ visual activity was recorded by an eye-tracking system. Mean fixation durations were comprehensively analyzed in the context of examined factors by a standard, five-way analysis of variance: Picture race (black, yellow, white) × Picture gender (man, woman) × Picture location (left, right) × Stimulus location (left, right) × Subject gender (male, female). Subjective results regarding perceived effectiveness of the advertisement variants were compared with objective fixation durations.

Background The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labeled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. Results UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2, and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. Conclusions As a sensitive, quantitative, flexible, and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments. Electronic supplementary material The online version of this article (10.1186/s12915-018-0554-z) contains supplementary material, which is available to authorized users.

Fatigue, as a psychophysiological state, is a normal human condition experienced as a result of mental or physical activity that consumes one's biological resources. In order to relieve its symptoms and to restore human's ability to continue work, a return to a fresh, relaxed state is required. It is usually obtained by an adequate amount of rest and/or sleep, but under the presence of some disadvantageous factors, such as too hot or too cold microclimate, high levels of noise at a workplace, work overload, psychological stress, lack of motivation, and others, e.g., family or health-related problems, this needed amount of rest simply may not fit into a normal 24-hour cycle of activity, resulting in the fatigue built-up. A fatigued worker becomes prone to performance deterioration, loss of concentration, misjudgment of his/her environmental safety, hazardous behaviors, anxiety and sleepiness. Therefore, worker's fatigue management is essential for improving productivity and quality of work, as well as for building worker's satisfaction, motivation and overall wellbeing. To supervise fatigue, appropriate tools for its measurement are required. This paper presents an overview of existing methods of fatigue measurement, including psychological (self-reports and questionnaires), physiological (i.e., changes in various body functions), and performance related (e.g., reaction time, sensory abilities) ones.

The usability of information conveyance is influenced by various factors. It has already been confirmed that verbal stimuli to be more effective should be presented on the right-hand side while non-verbal stimuli on the left-hand side. The aim of this paper is to analyze the impact of three factors: the race, gender and location of the human model picture in relation to a text on people’s perception of visual information promoting social campaign for tolerance. A total of 31 students from the Wroclaw Academy of Art and Design took part in this study. Participants were shown a series of visual banners containing a picture of a human along with the campaign slogan. The subjective evaluation of experimental conditions was conducted by the AHP method involving pairwise comparisons. The obtained results revealed significant effects of the race, gender and the interaction between them.

Summary In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF Skp2 substrate p27 Kip1 . CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

Background Stable transgenesis is an undeniable key to understanding any genetic system. Retrovirus-based insertional strategies, which feature several technical challenges when they are used, are often limited to one particular species, and even sometimes to a particular cell type as the infection depends on certain cellular receptors. A universal-like system, which would allow both stable transgene expression independent of the cell type and an efficient sorting of transfected cells, is required when handling cellular models that are incompatible with retroviral strategies. Results We report here on the combination of a stable insertional transgenesis technique, based on the Tol2 transposon system together with the magnetic cell sorting (MACS) technique, which allows specific selection of cells carrying the transgene in an efficient, reliable and rapid way. Conclusion This new Tol2/MACS system leads to stable expression in a culture of primary chicken erythroid cells highly enriched in cells expressing the transgene of interest. This system could be used in a wide variety of vertebrate species.