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Ashburn, VA, United States

Schoch K.M.,University of Kentucky | Von Reyn C.R.,University of Pennsylvania | Von Reyn C.R.,Janelia Farm Research Campus HHMI | Bian J.,Colorado State University | And 3 more authors.
Journal of Neurochemistry | Year: 2013

The calpain family of calcium-dependent proteases has been implicated in a variety of diseases and neurodegenerative pathologies. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components and membrane receptors, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. Pharmacological and gene-knockout strategies have targeted calpains to determine their contribution to neurodegenerative pathology; however, limitations associated with treatment paradigms, drug specificity, and genetic disruptions have produced inconsistent results and complicated interpretation. Specific, targeted calpain inhibition achieved by enhancing endogenous calpastatin levels offers unique advantages in studying pathological calpain activation. We have characterized a novel calpastatin-overexpressing transgenic mouse model, demonstrating a substantial increase in calpastatin expression within nervous system and peripheral tissues and associated reduction in protease activity. Experimental activation of calpains via traumatic brain injury resulted in cleavage of α-spectrin, collapsin response mediator protein-2, and voltage-gated sodium channel, critical proteins for the maintenance of neuronal structure and function. Calpastatin overexpression significantly attenuated calpain-mediated proteolysis of these selected substrates acutely following severe controlled cortical impact injury, but with no effect on acute hippocampal neurodegeneration. Augmenting calpastatin levels may be an effective method for calpain inhibition in traumatic brain injury and neurodegenerative disorders. Brain injury-induced proteolysis is reduced in a novel calpastatin overexpressing transgenic mouse Following brain injury, the activation of calpain proteases results in protein cleavage and eventual cell death. Calpain inhibition via calpastatin is an advantageous approach due to its highly specific interaction with calpains. Augmenting calpastatin levels in a novel transgenic mouse resulted in decreased breakdown of selected proteins, thereby identifying a potential therapeutic agent for both brain injury and neurodegenerative disorders. © 2013 International Society for Neurochemistry. Source

Parag T.,Janelia Farm Research Campus HHMI | Bahlmann C.,Siemens AG | Shet V.,Siemens AG | Singh M.,Siemens AG
IEEE Computer Society Conference on Computer Vision and Pattern Recognition Workshops | Year: 2012

Modeling objects using formal grammars has recently regained much attention in computer vision. Probabilistic logic programming, such as Bilattice based Logical Reasoning (BLR), is shown to produce impressive results in object detection/recognition. Although hierarchical object descriptions are preferred in high-level vision tasks for several reasons, BLR has been applied to non-hierarchical object grammars (compositional descriptions of object class). To better align logic programs (esp. BLR) with compositional object hierarchies, we provide a formal grammar, which can guide domain experts to describe objects. That is, we introduce a context-sensitive specification grammar or a meta-grammar, the language of which is the set of all possible object grammars. We show the practicality of the approach by an automatic compiler that translates example object grammars into a BLR logic program and applied it for detecting Graphical User Interface (GUI) components. © 2012 IEEE. Source

Gonzalez-Bellido P.T.,Janelia Farm Research Campus HHMI | Gonzalez-Bellido P.T.,National Oceanic and Atmospheric Administration | Wardill T.J.,National Oceanic and Atmospheric Administration
Cold Spring Harbor Protocols | Year: 2012

Neuroscience researchers have long sought methods to describe the neural connectivity of the circuits responsible for specific behaviors. One major obstacle is scale: Neural spines can be <1 μm in diameter, but axons can range from millimeters to centimeters (or larger) in length, making tissue imaging and neuron reconstruction a challenging task. New tissue-clearing agents and long-working-distance objectives offer improved imaging conditions, and here we present a complete protocol for invertebrate tissue that uses these advances. In this protocol, tissue-processing steps previously published in separate articles are combined with recent advances in confocal imaging to visualize invertebrate tissue samples that are >500 μm thick and contain dye-filled neurons. The steps describe dye filling, fixing, antibody labeling, clearing, whole tissue mounting, and confocal imaging with matched refractive indexes. Thus, manual sectioning or "flipping" the tissue to image the whole volume is not required. With matched refractive indexes, loss of resolution and signal is avoided. Tissue volumes are imaged in one stack and nonlinear deformations caused by tissue flipping are prevented. We apply the protocol to whole dragonfly thoracic ganglia (2 × 1 × 0.6 mm) and cephalopod skin samples (20 × 2 × 0.6 mm) with minimal tissue deformation. The resulting images will be used to develop a three-dimensional connectivity atlas of dragonfly ganglia and cephalopod skin innervation. This protocol can be applied to other invertebrate species, and has the advantage that it avoids problems with antigen specificity. © 2012 Cold Spring Harbor Laboratory Press. Source

Chmielewski J.P.,East Carolina University | Henderson L.,Janelia Farm Research Campus HHMI | Smith C.M.,East Carolina University | Christensen T.W.,East Carolina University
Chromosoma | Year: 2012

The condensation state of chromosomes is a critical parameter in multiple processes within the cell. Failures in the maintenance of appropriate condensation states may lead to genomic instability, mis-expression of genes, and a number of disease states. During cell proliferation, replication of DNA represents an ongoing challenge for chromosome packaging as DNA must be unpackaged for replication and then faithfully repackaged. An integral member of the DNA replication machinery is the GINS complex which has been shown to stabilize the CMG complex which is required for processivity of the Mcm2-7 helicase complex during S phase. Through examination of the phenotypes associated with a null mutation in Psf2, a member of the evolutionarily conserved GINS complex, we find that Drosophila Psf2 likely has a role in establishing chromosome condensation and that the defects associated with this mis-condensation impact M phase progression, genomic stability, and transcriptional regulation. © Springer-Verlag 2012. Source

Butler V.J.,University of Cambridge | Butler V.J.,Janelia Farm Research Campus HHMI | Butler V.J.,University of California at San Francisco | Branicky R.,University of Cambridge | And 10 more authors.
Journal of the Royal Society Interface | Year: 2015

Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force-posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control. © 2014 The Authors. Source

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