A Novel Testing Platform for Assessing Knee Joint Mechanics: A Parallel Robotic System Combined with an Instrumented Spatial Linkage

Assessing joint function following trauma and its inter-relation with degenerative changes requires an understanding of the normal state of structural loading in the joint. Very few studies have attempted to reproduce joint specific in vivo motions in vitro to quantify the actual loads carried by different tissues within the knee joint. The most significant challenge in this area is the very high sensitivity of the loads in joint structures to motion reproduction accuracy. A novel testing platform for assessing knee joint mechanics is described, comprised of a highly accurate (0.3 ± 0.1 mm, 0.3 ± 0.1°) six-degree-of-freedom (6-DOF) instrumented spatial linkage (ISL) for in vivo joint kinematic assessments and a unique 6-DOF parallel robotic manipulator. A position feedback system (ISL and position controller) is used for accurate reproduction of in vivo joint motions and estimation of “in situ” joint/tissue loads. The parallel robotic manipulator provides excellent stiffness and repeatability in reproducing physiological motions in 6-DOF, compared to the commonly used serial robots. The position feedback system provides real-time feedback data to the robot to reproduce in vivo motions and significantly enhances motion reproduction accuracy by adjusting for robot end-effector movements. Using this combined robot-ISL system, in vivo motions can be reproduced in vitro with very high accuracy (0.1 mm, 0.1°). Our results indicate that this level of accuracy is essential for meaningful estimation of tissue loads during gait. Using this novel testing platform, we have determined the normal load-carrying characteristics of different tissues within the ovine knee joint. The application of this testing system will continue to increase our understanding of normal and pathological joint states.     

Anatomy of hierarchy: Feedforward and feedback pathways in macaque visual cortex

The laminar location of the cell bodies and terminals of interareal connections determines the hierarchical structural organization of the cortex and has been intensively studied. However, we still have only a rudimentary understanding of the connectional principles of feedforward (FF) and feedback (FB) pathways. Quantitative analysis of retrograde tracers was used to extend the notion that the laminar distribution of neurons interconnecting visual areas provides an index of hierarchical distance (percentage of supragranular labeled neurons [SLN]). We show that: 1) SLN values constrain models of cortical hierarchy, revealing previously unsuspected areal relations; 2) SLN reflects the operation of a combinatorial distance rule acting differentially on sets of connections between areas; 3) Supragranular layers contain highly segregated bottom‐up and top‐down streams, both of which exhibit point‐to‐point connectivity. This contrasts with the infragranular layers, which contain diffuse bottom‐up and top‐down streams; 4) Cell filling of the parent neurons of FF and FB pathways provides further evidence of compartmentalization; 5) FF pathways have higher weights, cross fewer hierarchical levels, and are less numerous than FB pathways. Taken together, the present results suggest that cortical hierarchies are built from supra‐ and infragranular counterstreams. This compartmentalized dual counterstream organization allows point‐to‐point connectivity in both bottom‐up and top‐down directions. J. Comp. Neurol. 522:225–259, 2014. © 2013 Wiley Periodicals, Inc.     

Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1

Plants and animals use innate immunity as a first defense against pathogens, a costly yet necessary tradeoff between growth and immunity. In Arabidopsis, the regulatory leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1 combines with the LRR-RLKs FLS2 and EFR in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and the LRR-RLK BRI1 in brassinosteroid (BR)-mediated growth. Therefore, a potential tradeoff between these pathways mediated by BAK1 is often postulated. Here, we show a unidirectional inhibition of FLS2-mediated immune signaling by BR perception. Unexpectedly, this effect occurred downstream or independently of complex formation with BAK1 and associated downstream phosphorylation. Thus, BAK1 is not rate-limiting in these pathways. BRs also inhibited signaling triggered by the BAK1-independent recognition of the fungal PAMP chitin. Our results suggest a general mechanism operative in plants in which BR-mediated growth directly antagonizes innate immune signaling.     

Structural insights into Helicobacter pylori oncoprotein CagA interaction with β1 integrin

Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag-T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer β-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1–4), which represent putative hot-spots for protein–protein interactions. The proximal part of the single-layer β-sheet, covering CSP4, is involved in specific binding of CagA to the β1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind β1 integrin to mediate its own translocation.     

X-ray crystal structure of the streptococcal specific phage lysin PlyC

Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria.