Methylphenidate (Ritalin) use and abuse

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Development and validation of broad-spectrum magnetic particle labelling processes for cell therapy manufacturing

BACKGROUND: Stem cells are increasingly seen as a solution for many health challenges for an ageing population. However, their potential benefits in the clinic are currently curtailed by technical challenges such as high cell dose requirements and point of care delivery, which pose sourcing and logistics challenges. Cell manufacturing solutions are currently in development to address the supply issue, and ancillary technologies such as nanoparticle-based labelling are being developed to improve stem cell delivery and enable post-treatment follow-up. METHODS: The application of magnetic particle (MP) labelling to potentially scalable cell manufacturing processes was investigated in a range of therapeutically relevant cells, including mesenchymal stromal cells (MSC), cardiomyocytes (CMC) and neural progenitor cells (ReN). The efficiency and the biological effect of particle labelling were analysed using fluorescent imaging and cellular assays. RESULTS: Flow cytometry and fluorescent microscopy confirmed efficient labelling of monolayer cultures. Viability was shown to be retained post labelling for all three cell types. MSC and CMC demonstrated higher tolerance to MP doses up to 100× the standard concentration. This approach was also successful for MP labelling of suspension cultures, demonstrating efficient MP uptake within 3 h, while cell viability was unaffected by this suspension labelling process. Furthermore, a procedure to enable the storing of MP-labelled cell populations to facilitate cold chain transport to the site of clinical use was investigated. When MP-labelled cells were stored in hypothermic conditions using HypoThermosol solution for 24 h, cell viability and differentiation potential were retained post storage for ReN, MSC and beating CMC. CONCLUSIONS: Our results show that a generic MP labelling strategy was successfully developed for a range of clinically relevant cell populations, in both monolayer and suspension cultures. MP-labelled cell populations were able to undergo transient low-temperature storage whilst maintaining functional capacity in vitro. These results suggest that this MP labelling approach can be integrated into cell manufacturing and cold chain transport processes required for future cell therapy approaches

Spatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in McLaughlin, G. A., Langdon, E. M., Crutchley, J. M., Holt, L. J., Forest, M. G., Newby, J. M., & Gladfelter, A. S. (2020). Spatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking. Molecular Biology of the Cell, 31(14), 1498-1511, doi:10.1091/mbc.E20-03-0210.The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm). We developed an image analysis pipeline for 3D imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. We applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations.We would like to thank the 2016 Physiology course and Christina Termini at the Marine Biological Laboratory in Woods Hole, MA, Gregory Brittingham, and Marcus Roper for initial experiments and perspectives on pipeline. We thank David Adalsteinsson for help with DataTank software and many conversations about image analysis on large datasets. We thank Emmanual Levy (Weizmann Institute) for providing plasmids encoding synthetic phase separating peptides. This work was supported by Google Cloud, the National Science Foundation (NSF), the National Institutes of Health (NIH), and the Natural Sciences and Engineering Research Council of Canada (NSERC). ASG, EML, and GAM were supported by the NSF (RoLs: 1840273), HHMI faculty scholar award and the NIH (R01GM081506). JMN was supported by the NSERC (RGPIN-2019-06435, RGPAS-2019-00014, DGECR-2019-00321) and the NSF (DMS-171474). MGF was supported by the NSF (DMS-1816630, DMS-1664645). LJH was supported by the NIH (R01GM132447)

mRNA structure determines specificity of a polyQ-driven phase separation

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here by permission of American Association for the Advancement of Science for personal use, not for redistribution. The definitive version was published in American Association for the Advancement of Science 360 (2018): 922-927, doi:10.1126/science.aar7432.RNA promotes liquid-liquid phase separation (LLPS) to build membrane-less compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here we report that secondary structure allows mRNAs to self-associate and determines if an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.This work was supported by NIH GM R01- GM081506, the HHMI Faculty Scholars program, R35 GM122532, ACS 130845-RSG-17-114- 01-RMC, NIH 1DP2 GM105453, and NIH R01 GM115631

Focused fluid seepage related to variations in accretionary wedge structure, Hikurangi margin, New Zealand

Hydrogeological processes influence the morphology, mechanical behavior, and evolution of subduction margins. Fluid supply, release, migration, and drainage control fluid pressure and collectively govern the stress state, which varies between accretionary and nonaccretionary systems. We compiled over a decade of published and unpublished acoustic data sets and seafloor observations to analyze the distribution of focused fluid expulsion along the Hikurangi margin, New Zealand. The spatial coverage and quality of our data are exceptional for subduction margins globally. We found that focused fluid seepage is widespread and varies south to north with changes in subduction setting, including: wedge morphology, convergence rate, seafloor roughness, and sediment thickness on the incoming Pacific plate. Overall, focused seepage manifests most commonly above the deforming backstop, is common on thrust ridges, and is largely absent from the frontal wedge despite ubiquitous hydrate occurrences. Focused seepage distribution may reflect spatial differences in shallow permeability architecture, while diffusive fluid flow and seepage at scales below detection limits are also likely. From the spatial coincidence of fluids with major thrust faults that disrupt gas hydrate stability, we surmise that focused seepage distribution may also reflect deeper drainage of the forearc, with implications for pore-pressure regime, fault mechanics, and critical wedge stability and morphology. Because a range of subduction styles is represented by 800 km of along-strike variability, our results may have implications for understanding subduction fluid flow and seepage globally