A Structural Study of the Cytoplasmic Chaperone Effect of 14-3-3 Proteins on Ataxin-1

Expansion of the polyglutamine tract in the N terminus of Ataxin-1 is the main cause of the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1). However, the C-terminal part of the protein - including its AXH domain and a phosphorylation on residue serine 776 - also plays a crucial role in disease development. This phosphorylation event is known to be crucial for the interaction of Ataxin-1 with the 14-3-3 adaptor proteins and has been shown to indirectly contribute to Ataxin-1 stability. Here we show that 14-3-3 also has a direct anti-aggregation or chaperone effect on Ataxin-1. Furthermore, we provide structural and biophysical information revealing how phosphorylated S776 in the intrinsically disordered C terminus of Ataxin-1 mediates the cytoplasmic interaction with 14-3-3 proteins. Based on these findings, we propose that 14-3-3 exerts the observed chaperone effect by interfering with Ataxin-1 dimerization through its AXH domain, reducing further self-association. The chaperone effect is particularly important in the context of SCA1, as it was previously shown that a soluble form of mutant Ataxin-1 is the major driver of pathology

The identification and structural analysis of potential 14-3-3 interaction sites on the bone regulator protein Schnurri-3

14-3-3 proteins regulate many intracellular processes and their ability to bind in subtly different fashions to their numerous partner proteins provides attractive drug-targeting points for a range of diseases. Schnurri-3 is a suppressor of mouse bone formation and a candidate target for novel osteoporosis therapeutics, and thus it is of interest to determine whether it interacts with 14-3-3. In this work, potential 14-3-3 interaction sites on mammalian Schnurri-3 were identified by an in silico analysis of its protein sequence. Using fluorescence polarization, isothermal titration calorimetry and X-ray crystallography, it is shown that synthetic peptides containing either phosphorylated Thr869 or Ser542 can indeed interact with 14-3-3, with the latter capable of forming an interprotein disulfide bond with 14-3-3σ: a hitherto unreported phenomenon

Visualizing Stem Cells by MRI Labels: the Effect of Paramagnetic Iron Oxide Particles on the Stem Cell Phenotype

status: publishe

Characterization of the Inflammatory Response in a Photothrombotic Stroke Model by MRI: Implications for Stem Cell Transplantation

PURPOSE: The aim of this study was to evaluate the specificity of magnetic resonance imaging (MRI) contrast in a photothrombotic (PT) stroke model with and without engraftment of superparamagnetic iron oxide (SPIO)-labeled stem cells. PROCEDURES: We monitored animals with PT stroke versus animals with PT stroke and stem cell engraftment by T(2)/T(2)*w MRI 4-8 h and 2, 4, 6/7 and 14 days after PT induction. Results were correlated with immunohistochemistry. RESULTS: T(2)*w MRI images showed hypointense contrast due to the accumulation of inflammatory cells and corresponding iron accumulation and glial scar formation in the border zone of the lesion, similar as what was observed for SPIO-labeled cells. Histological analysis was thus indispensable to distinguish between labeled transplanted cells and immune cells. CONCLUSION: These results raise caution regarding the non-invasive monitoring of SPIO-labeled transplanted stem cells by MRI in models that result in a strong inflammatory response.status: publishe

Enabling Regenerative Agriculture Using Remote Sensing and Machine Learning

The emergence of cloud computing, big data analytics, and machine learning has catalysed the use of remote sensing technologies to enable more timely management of sustainability indicators, given the uncertainty of future climate conditions. Here, we examine the potential of “regenerative agriculture”, as an adaptive grazing management strategy to minimise bare ground exposure while improving pasture productivity. High-intensity sheep grazing treatments were conducted in small fields (less than 1 ha) for short durations (typically less than 1 day). Paddocks were subsequently spelled to allow pasture biomass recovery (treatments comprising 3, 6, 9, 12, and 15 months), with each compared with controls characterised by lighter stocking rates for longer periods (2000 DSE/ha). Pastures were composed of wallaby grass (Austrodanthonia species), kangaroo grass (Themeda triandra), Phalaris (Phalaris aquatica), and cocksfoot (Dactylis glomerata), and were destructively sampled to estimate total standing dry matter (TSDM), standing green biomass, standing dry biomass and trampled biomass. We invoked a machine learning model forced with Sentinel-2 imagery to quantify TSDM, standing green and dry biomass. Faced with La Nina conditions, regenerative grazing did not significantly impact pasture productivity, with all treatments showing similar TSDM, green biomass and recovery. However, regenerative treatments significantly impacted litterfall and trampled material, with high-intensity grazing treatments trampling more biomass, increasing litter, enhancing surface organic matter and decomposition rates thereof. Pasture digestibility and sward uniformity were greatest for treatments with minimal spelling (3 months), whereas both standing senescent and trampled material were greater for the 15-month spelling treatment. TSDM prognostics from machine learning were lower than measured TSDM, although predictions from the machine learning approach closely matched observed spatiotemporal variability within and across treatments. The root mean square error between the measured and modelled TSDM was 903 kg DM/ha, which was less than the variability measured in the field. We conclude that regenerative grazing with short recovery periods (3–6 months) was more conducive to increasing pasture production under high rainfall conditions, and we speculate that – in this environment - high-intensity grazing with 3-month spelling is likely to improve soil organic carbon through increased litterfall and trampling. Our study paves the way for using machine learning with satellite imagery to quantify pasture biomass at small scales, enabling the management of pastures within small fields from afar

Effects of MRI Contrast Agents on the Stem cell Phenotype

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell based therapies. To follow the fate of grafted cells in vivo, a number of non-invasive imaging approaches have been developed. Magnetic Resonance Imaging (MRI) is a high resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of 3 different stem cell populations (mouse Embryonic Stem Cells, rat Multipotent Adult Progenitor Cells and mouse Mesenchymal Stem Cells) with three different (ultra) small superparamagnetic iron oxide (U)SPIOs particles (Resovist(R), Endore(R), Sinerem(R)) was compared. Labeling efficiency with Resovist(R) and Endorem(R) differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI, did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.status: publishe