Õpetajakoolituse üliõpilaste oskus märgata ja analüüsida psühholoogilisi baasvajadusi toetavat õpetamist

Selle uuringu eesmärk oli kohandada situatsioonispetsiifiliste ehk PID-oskuste (perception, interpretation, decision-making skills) hindamisvahend isemääramisteooria konteksti, et analüüsida õpetajakoolituse üliõpilaste suutlikkust märgata ja tõlgendada psühholoogilisi baasvajadusi toetavaid tegevusi klassiruumi kontekstis. Andmeid koguti aineõpetajate õpetajakoolituse magistriõpingute esimese aasta üliõpilastelt (N = 97), kasutades tunnivideote analüüsi. Uuringu tulemuste põhjal võib öelda, et üliõpilased märkavad tunnivideotest psühholoogiliste baasvajadustega seotud aspekte. Tõlgendamisel ning otsuste tegemisel olid üliõpilaste vastused pigem kas kirjeldavad või hinnangulised ega olnud seotud teoreetiliste teadmistega. Töökogemuse põhjal olulisi erinevusi tulemustes ei täheldatud. Kuigi õpetajakoolituse üliõpilaste puhul on tulemused ootuspärased, tuleks PID-oskuste arendamisele õpetajakoolituses senisest teadlikumalt tähelepanu pöörata, toetamaks paremini teoreetiliste teadmiste ülekannet praktikasse. Summar

Rapid Structural, Kinetic, and Immunochemical Analysis of Alpha-Synuclein Oligomers in Solution.

Oligomers comprised of misfolded proteins are implicated as neurotoxins in the pathogenesis of protein misfolding conditions such as Parkinson's and Alzheimer's diseases. Structural, biophysical, and biochemical characterization of these nanoscale protein assemblies is key to understanding their pathology and the design of therapeutic interventions, yet it is challenging due to their heterogeneous, transient nature and low relative abundance in complex mixtures. Here, we demonstrate separation of heterogeneous populations of oligomeric α-synuclein, a protein central to the pathology of Parkinson's disease, in solution using microfluidic free-flow electrophoresis. We characterize nanoscale structural heterogeneity of transient oligomers on a time scale of seconds, at least 2 orders of magnitude faster than conventional techniques. Furthermore, we utilize our platform to analyze oligomer ζ-potential and probe the immunochemistry of wild-type α-synuclein oligomers. Our findings contribute to an improved characterization of α-synuclein oligomers and demonstrate the application of microchip electrophoresis for the free-solution analysis of biological nanoparticle analytes

On-chip measurements of protein unfolding from direct observations of micron-scale diffusion.

Investigations of protein folding, unfolding and stability are critical for the understanding of the molecular basis of biological structure and function. We describe here a microfluidic approach to probe the unfolding of unlabelled protein molecules in microliter volumes. We achieve this objective through the use of a microfluidic platform, which allows the changes in molecular diffusivity upon folding and unfolding to be detected directly. We illustrate this approach by monitoring the unfolding of bovine serum albumin in solution as a function of pH. These results show the viability of probing protein stability on chip in small volumes

Targeting nucleic acid phase transitions as a mechanism of action for antimicrobial peptides.

Acknowledgements: The research leading to these results has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant MicroREvolution (agreement no. 101023060; T.S.) and the ERC grant DiProPhys (agreement ID 10100161, T.P.J.K.); the Royall Scholarship (N.A.E.); Global Research Technologies Novo Nordisk A/S (H.A., T.P.J.K.); the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013) through the ERC grants PhysProt (agreement no. 337969; T.P.J.K.); the Frances and Augustus Newman Foundation (T.S.); the Schmidt Science Fellowship programme in partnership with the Rhodes Trust (K.L.S.); St. John’s College Junior Research Fellowship (K.L.S.), the Harding Distinguished Postgraduate Scholar Programme (T.J.W.); Boehringer Ingelheim Fonds (K.K.G.); and Wellcome Trust (213437/Z/18/Z, A.B.).Funder: Frances and Augustus Newman Foundation; doi: https://doi.org/100007898Funder: The European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013) through the ERC grants PhysProt (agreement no. 33796) ERC grant DiProPhys (agreement ID 10100161)Funder: Boehringer Ingelheim Fonds (Stiftung für medizinische Grundlagenforschung); doi: https://doi.org/501100001645Antimicrobial peptides (AMPs), which combat bacterial infections by disrupting the bacterial cell membrane or interacting with intracellular targets, are naturally produced by a number of different organisms, and are increasingly also explored as therapeutics. However, the mechanisms by which AMPs act on intracellular targets are not well understood. Using machine learning-based sequence analysis, we identified a significant number of AMPs that have a strong tendency to form liquid-like condensates in the presence of nucleic acids through phase separation. We demonstrate that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. These results suggest that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. The development of antimicrobials that target nucleic acid phase transitions may become an attractive route to finding effective and long-lasting antibiotics

Surface Electrostatics Govern the Emulsion Stability of Biomolecular Condensates.

Liquid-liquid phase separation underlies the formation of biological condensates. Physically, such systems are microemulsions that in general have a propensity to fuse and coalesce; however, many condensates persist as independent droplets in the test tube and inside cells. This stability is crucial for their function, but the physicochemical mechanisms that control the emulsion stability of condensates remain poorly understood. Here, by combining single-condensate zeta potential measurements, optical microscopy, tweezer experiments, and multiscale molecular modeling, we investigate how the nanoscale forces that sustain condensates impact their stability against fusion. By comparing peptide-RNA (PR25:PolyU) and proteinaceous (FUS) condensates, we show that a higher condensate surface charge correlates with a lower fusion propensity. Moreover, measurements of single condensate zeta potentials reveal that such systems can constitute classically stable emulsions. Taken together, these results highlight the role of passive stabilization mechanisms in protecting biomolecular condensates against coalescence

Liquid–liquid phase separation underpins the formation of replication factories in rotaviruses

RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent proteinRNA condensates that are formed via liquid–liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches

The Chromatin Regulator HMGA1a Undergoes Phase Separation in the Nucleus.

Funder: European Research Council (ERC)The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through electrostatic interactions via AT hooks 2 and 3. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure

α-Synuclein Oligomers Displace Monomeric α-Synuclein from Lipid Membranes.

Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD

Direct digital sensing of protein biomarkers in solution.

Funder: Please see main manuscript file.The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches

Direct digital sensing of protein biomarkers in solution.

Funder: Please see main manuscript file.The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches