Dynamic transport and localization of alpha-synuclein in primary hippocampal neurons.

BACKGROUND: Alpha-synuclein is a presynaptic protein with a proposed role in neurotransmission and dopamine homeostasis. Abnormal accumulation of alpha-synuclein aggregates in dopaminergic neurons of the substantia nigra is diagnostic of sporadic Parkinson's disease, and mutations in the protein are linked to early onset forms of the disease. The folded conformation of the protein varies depending upon its environment and other factors that are poorly understood. When bound to phospholipid membranes, alpha-synuclein adopts a helical conformation that mediates specific interactions with other proteins. RESULTS: To investigate the role of the helical domain in transport and localization of alpha-synuclein, eGFP-tagged constructs were transfected into rat primary hippocampal neurons at 7 DIV. A series of constructs were analyzed in which each individual exon was deleted, for comparison to previous studies of lipid affinity and alpha-helix content. A53T and A30P substitutions, representing Parkinson's disease-associated variants, were analyzed as well. Single exon deletions within the lipid-binding N-terminal domain of alpha-synuclein (exons 2, 3, and 4) partially disrupted its presynaptic localization at 17-21 DIV, resulting in increased diffuse labeling of axons. Similar results were obtained for A30P, which exhibits decreased lipid binding, but not A53T. To examine whether differences in presynaptic enrichment were related to deficiencies in transport velocity, transport was visualized via live cell microscopy. Tagged alpha-synuclein migrated at a rate of 1.85 +/- 0.09 mum/s, consistent with previous reports, and single exon deletion mutants migrated at similar rates, as did A30P. Deletion of the entire N-terminal lipid-binding domain (Delta234GFP) did not significantly alter rates of particle movement, but decreased the number of moving particles. Only the A53TGFP mutant exhibited a significant decrease in transport velocity as compared to ASGFP. CONCLUSIONS: These results support the hypothesis that presynaptic localization involves a mechanism that requires helical conformation and lipid binding. Conversely, the rate of axonal transport is not determined by lipid affinity and is not sufficient to account for differences in presynaptic localization of alpha-synuclein-eGFP variants.This study was funded by the Branfman Family Foundation, including salary support for MLY, LH, and WSW

Fluoroketone inhibition of Ca(2+)-independent phospholipase A2 through binding pocket association defined by hydrogen/deuterium exchange and molecular dynamics.

The mechanism of inhibition of group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA(2) were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA(2) should greatly improve our ability to design new inhibitors with higher potency and selectivity

Cardiac Progenitor Cells from Stem Cells: Learning from Genetics and Biomaterials

Cardiac Progenitor Cells (CPCs) show great potential as a cell resource for restoring cardiac function in patients affected by heart disease or heart failure. CPCs are proliferative and committed to cardiac fate, capable of generating cells of all the cardiac lineages. These cells offer a significant shift in paradigm over the use of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes owing to the latter's inability to recapitulate mature features of a native myocardium, limiting their translational applications. The iPSCs and direct reprogramming of somatic cells have been attempted to produce CPCs and, in this process, a variety of chemical and/or genetic factors have been evaluated for their ability to generate, expand, and maintain CPCs in vitro. However, the precise stoichiometry and spatiotemporal activity of these factors and the genetic interplay during embryonic CPC development remain challenging to reproduce in culture, in terms of efficiency, numbers, and translational potential. Recent advances in biomaterials to mimic the native cardiac microenvironment have shown promise to influence CPC regenerative functions, while being capable of integrating with host tissue. This review highlights recent developments and limitations in the generation and use of CPCs from stem cells, and the trends that influence the direction of research to promote better application of CPCs

Radial Spoke Proteins of \u3cem\u3eChlamydomonas\u3c/em\u3e Flagella

The radial spoke is a ubiquitous component of `9+2\u27 cilia and flagella, and plays an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. The Chlamydomonas reinhardtii radial spoke contains at least 23 proteins, only 8 of which have been characterized at the molecular level. Here, we use mass spectrometry to identify 10 additional radial spoke proteins. Many of the newly identified proteins in the spoke stalk are predicted to contain domains associated with signal transduction, including Ca2+-, AKAP- and nucleotide-binding domains. This suggests that the spoke stalk is both a scaffold for signaling molecules and itself a transducer of signals. Moreover, in addition to the recently described HSP40 family member, a second spoke stalk protein is predicted to be a molecular chaperone, implying that there is a sophisticated mechanism for the assembly of this large complex. Among the 18 spoke proteins identified to date, at least 12 have apparent homologs in humans, indicating that the radial spoke has been conserved throughout evolution. The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the radial spokes

Incorporating basic calibrations in existing machine-learned turbulence modeling

This work aims to incorporate basic calibrations of Reynolds-averaged Navier-Stokes (RANS) models as part of machine learning (ML) frameworks. The ML frameworks considered are tensor-basis neural network (TBNN), physics-informed machine learning (PIML), and field inversion & machine learning (FIML) in J. Fluid Mech., 2016, 807, 155-166, Phys. Rev. Fluids, 2017, 2(3), 034603 and J. Comp. Phys., 2016, 305, 758-774, and the baseline RANS models are the one-equation Spalart-Allmaras model, the two-equation $k$-$\omega$ model, and the seven-equation Reynolds stress transport models. ML frameworks are trained against plane channel flow and shear-layer flow data. We compare the ML frameworks and study whether the machine-learned augmentations are detrimental outside the training set. The findings are summarized as follows. The augmentations due to TBNN are detrimental. PIML leads to augmentations that are beneficial inside the training dataset but detrimental outside it. These results are not affected by the baseline RANS model. FIML's augmentations to the two eddy viscosity models, where an inner-layer treatment already exists, are largely neutral. Its augmentation to the seven-equation model, where an inner-layer treatment does not exist, improves the mean flow prediction in a channel. Furthermore, these FIML augmentations are mostly non-detrimental outside the training dataset. In addition to reporting these results, the paper offers physical explanations of the results. Last, we note that the conclusions drawn here are confined to the ML frameworks and the flows considered in this study. More detailed comparative studies and validation & verification studies are needed to account for developments in recent years

Holocene evolution of halite caves in the Cordillera de la Sal (Central Atacama, Chile) in different climate conditions

Geomorphological studies have been carried out in rapidly evolving salt caves related to small watersheds in the San Pedro de Atacama area, Chile. Radiocarbon ages of bones and wood from cave deposits, combined with the presence of large salt caves, geomorphological and sedimentological observations, and the results of micrometer measurements outside and in some of the caves, suggest a period of speleogenesis in the Cordillera de la Sal during the onset of the Holocene, during which the large halite cave systems developed, followed by an early Holocene hyperarid period.Most smaller caves (i.e. Lechuza del Campanario) most probably formed at the start of the wetter mid-Holocene period (5–4.4 ka), when precipitation was never intense enough to entrain large amounts of sediments, but enough to trigger cave development. A diamicton in Lechuza del Campanario Cave radiocarbon dated at ca. 4.4 ka shows that at least one high intensity rainfall event occurred in this recharge basin during the mid-Holocene wet interval. A wet period with lower intensity rainfall events followed between 4.0 and 2.5 ka, causing the 4.4 kyrs old diamicton in Lechuza del Campanario Cave to be entrenched, and the alluvial fan at the downstream end of Palacio del Sal Cave to be covered with windborne sediments dated by OSL at around 3.6 ka. At ca. 2 ka there was a high-intensity rainfall event documented by the age of a twig stuck in the ceiling of the Palacio del Sal Cave, followed by a period with lower intensity rain events until ca. 1.3 ka, when another intense flood produced a mudflow that deposited a second diamicton in Lechuza del Campanario Cave. From then on clustering of radiocarbon ages forwood and bone recovered fromcaves indicates increased rainfall intensity in the period ca. 0.9–0.5 ka, followed by no registered events until a minor flood at ca. 0.13 ka. The fourcenturies long wetter time interval (0.9–0.5 ka), corresponding to the Medieval Climate Anomaly, has been an archeologically important period in the Atacama Desert (Tiwanaku culture). The observations and a detailed review of paleoclimate literature from this key area have allowed the development of a landscape evolution model related to changing climate conditions during the Late Holocene

The Importance of Hands-on Experience with Telescopes for Students

Proper interpretation and understanding of astronomical data requires good knowledge of the data acquisition process. The increase in remote observing, queue observing, and the availability of large archived data products risk insulating astronomers from the telescope, potentially reducing their familiarity with the observational techniques crucial in understanding the data. Learning fundamental observing techniques can be done in at least three ways: 1) College and university operated observing facilities, 2) Student involvement in national facilities through competitive proposals, 3) Programs at national facilities to increase upper-level undergraduate and graduate student exposure to telescopes. We encourage both national organizations and universities to include programs and funding aimed at supporting hands-on experience with telescopes through the three methods mentioned.Comment: "State of the Profession" white paper for the 2010 Astronomy and Astrophysics Decadal Surve

Predicting blunt cerebrovascular injury in pediatric trauma: Validation of the Utah Score

Risk factors for blunt cerebrovascular injury (BCVI) may differ between children and adults, suggesting that children at low risk for BCVI after trauma receive unnecessary computed tomography angiography (CTA) and high-dose radiation. We previously developed a score for predicting pediatric BCVI based on retrospective cohort analysis. Our objective is to externally validate this prediction score with a retrospective multi-institutional cohort. We included patients who underwent CTA for traumatic cranial injury at four pediatric Level I trauma centers. Each patient in the validation cohort was scored using the “Utah Score” and classified as high or low risk. Before analysis, we defined a misclassification rate <25% as validating the Utah Score. Six hundred forty-five patients (mean age 8.6 ± 5.4 years; 63.4% males) underwent screening for BCVI via CTA. The validation cohort was 411 patients from three sites compared with the training cohort of 234 patients. Twenty-two BCVIs (5.4%) were identified in the validation cohort. The Utah Score was significantly associated with BCVIs in the validation cohort (odds ratio 8.1 [3.3, 19.8], p < 0.001) and discriminated well in the validation cohort (area under the curve 72%). When the Utah Score was applied to the validation cohort, the sensitivity was 59%, specificity was 85%, positive predictive value was 18%, and negative predictive value was 97%. The Utah Score misclassified 16.6% of patients in the validation cohort. The Utah Score for predicting BCVI in pediatric trauma patients was validated with a low misclassification rate using a large, independent, multicenter cohort. Its implementation in the clinical setting may reduce the use of CTA in low-risk patients

Determination of Ice Cloud Models Using MODIS and MISR Data

Representation of ice clouds in radiative transfer simulations is subject to uncertainties associated with the shapes and sizes of ice crystals within cirrus clouds. In this study, we examined several ice cloud models consisting of smooth, roughened, homogeneous and inhomogeneous hexagonal ice crystals with various aspect ratios. The sensitivity of the bulk scattering properties and solar reflectances of cirrus clouds to specific ice cloud models is investigated using the improved geometric optics method (IGOM) and the discrete ordinates radiative transfer (DISORT) model. The ice crystal habit fractions in the ice cloud model may significantly affect the simulations of cloud reflectances. A new algorithm was developed to help determine an appropriate ice cloud model for application to the satellite-based retrieval of ice cloud properties. The ice cloud particle size retrieved from Moderate Resolution Imaging Spectroradiometer (MODIS) data, collocated with Multi-angle Imaging Spectroradiometer (MISR) observations, is used to infer the optical thicknesses of ice clouds for nine MISR viewing angles. The relative differences between view-dependent cloud optical thickness and the averaged value over the nine MISR viewing angles can vary from 0.5 to 0.5 and are used to evaluate the ice cloud models. In the case for 2 July 2009, the ice cloud model with mixed ice crystal habits is the best fit to the observations (the root mean square (RMS) error of cloud optical thickness reaches 0.365). This ice cloud model also produces consistent cloud property retrievals for the nine MISR viewing configurations within the measurement uncertainties