Direct Visualization of Protease Action on Collagen Triple Helical Structure

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease

NMDA and Dopamine Converge on the NMDA-Receptor to Induce ERK Activation and Synaptic Depression in Mature Hippocampus

The formation of enduring internal representation of sensory information demands, in many cases, convergence in time and space of two different stimuli. The first conveys the sensory input, mediated via fast neurotransmission. The second conveys the meaning of the input, hypothesized to be mediated via slow neurotransmission. We tested the biochemical conditions and feasibility for fast (NMDA) and slow (dopamine) neurotransmission to converge on the Mitogen Activated Protein Kinase signaling pathways, crucial in several forms of synaptic plasticity, and recorded its effects upon synaptic transmission. We detected differing kinetics of ERK2 activation and synaptic strength changes in the CA1 for low and high doses of neurotransmitters in hippocampal slices. Moreover, when weak fast and slow inputs are given together, they converge on ERK2, but not on p38 or JNK, and induce strong short-term synaptic depression. Surprisingly, pharmacological analysis revealed that a probable site of such convergence is the NMDA receptor itself, suggesting it serves as a detector and integrator of fast and slow neurotransmission in the mature mammalian brain, as revealed by ERK2 activation and synaptic function

Cost-Effectiveness of New Cardiac and Vascular Rehabilitation Strategies for Patients with Coronary Artery Disease

Objective: Peripheral arterial disease (PAD) often hinders the cardiac rehabilitation program. The aim of this study was evaluating the relative cost-effectiveness of new rehabilitation strategies which include the diagnosis and treatment of PAD in patients with coronary artery disease (CAD) undergoing cardiac rehabilitation. Data Sources: Best-available evidence was retrieved from literature and combined with primary data from 231 patients. Methods: We developed a Markov decision model to compare the following treatment strategies: 1. cardiac rehabilitation only; 2. ankle-brachial index (ABI) if cardiac rehabilitation fails followed by diagnostic work-up and revascularization for PAD if needed; 3. ABI prior to cardiac rehabilitation followed by diagnostic work-up and revascularization for PAD if needed. Quality-adjusted-life years (QALYs), life-time costs (US $), incremental cost-effectiveness ratios (ICER), and gain in net health benefits (NHB) in QALY equivalents were calculated. A threshold willingness-to-pay of$75 000 was used. Results: ABI if cardiac rehabilitation fails was the most favorable strategy with an ICER of $44 251 per QALY gained and an incremental NHB compared to cardiac rehabilitation only of 0.03 QALYs (95$75 000/QALY. After sensitivity analysis, a combined cardiac and vascular rehabilitation program increased the success rate and would dominate the other two strategies with total lifetime costs of $30 246 a quality-adjusted life expectancy of 3.84 years, and an incremental NHB of 0.06 QALYs (95%CI:−0.24, 0.46) compared to current practice. The results were robust for other different input parameters. Conclusion: ABI measurement if cardiac rehabilitation fails followed by a diagnostic work-up and revascularization for PAD if needed are potentially cost-effective compared to cardiac rehabilitation only

Mixed Feelings of Children and Adolescents with Unilateral Congenital Below Elbow Deficiency: An Online Focus Group Study

The existing literature is inconsistent about the psychosocial functioning of children and adolescents with Unilateral Congenital Below Elbow Deficiency (UCBED). The objective of this qualitative study was to explore the psychosocial functioning of children and adolescents with UCBED in terms of their feelings about the deficiency and what helps them to cope with those feelings. Additionally, the perspectives of prosthesis wearers and non-wearers were compared, as were the perspectives of children, adolescents, parents and health professionals. Online focus group interviews were carried out with 42 children and adolescents (aged 8–12, 13–16 and 17–20), 16 parents and 19 health professionals. Questions were asked about psychosocial functioning, activities, participation, prosthetic use or non-use, and rehabilitation care. This study concerned remarks about psychosocial functioning. Children and adolescents with UCBED had mixed feelings about their deficiency. Both negative and positive feelings were often felt simultaneously and mainly depended on the way people in the children’s environment reacted to the deficiency. People staring affected the children negatively, while support from others helped them to cope with the deficiency. Wearing a prosthesis and peer-to-peer contact were also helpful. Non-wearers tended to be more resilient than prosthesis wearers. Wearers wore their prosthesis for cosmetic reasons and to prevent them from negative reactions from the environment. We recommend that rehabilitation teams make parents aware of their great influence on the psychosocial functioning of their child with UCBED, to adjust or extend the currently available psychosocial help, and to encourage peer-to-peer contact

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions

Cross-Modal Prediction in Speech Perception

Speech perception often benefits from vision of the speaker's lip movements when they are available. One potential mechanism underlying this reported gain in perception arising from audio-visual integration is on-line prediction. In this study we address whether the preceding speech context in a single modality can improve audiovisual processing and whether this improvement is based on on-line information-transfer across sensory modalities. In the experiments presented here, during each trial, a speech fragment (context) presented in a single sensory modality (voice or lips) was immediately continued by an audiovisual target fragment. Participants made speeded judgments about whether voice and lips were in agreement in the target fragment. The leading single sensory context and the subsequent audiovisual target fragment could be continuous in either one modality only, both (context in one modality continues into both modalities in the target fragment) or neither modalities (i.e., discontinuous). The results showed quicker audiovisual matching responses when context was continuous with the target within either the visual or auditory channel (Experiment 1). Critically, prior visual context also provided an advantage when it was cross-modally continuous (with the auditory channel in the target), but auditory to visual cross-modal continuity resulted in no advantage (Experiment 2). This suggests that visual speech information can provide an on-line benefit for processing the upcoming auditory input through the use of predictive mechanisms. We hypothesize that this benefit is expressed at an early level of speech analysis

The intermuscular 3–7 Hz drive is not affected by distal proprioceptive input in myoclonus-dystonia

In dystonia, both sensory malfunctioning and an abnormal intermuscular low-frequency drive of 3–7 Hz have been found, although cause and effect are unknown. It is hypothesized that sensory processing is primarily disturbed and induces this drive. Accordingly, experimenter-controlled sensory input should be able to influence the frequency of the drive. In six genetically confirmed myoclonus-dystonia (MD) patients and six matched controls, the low-frequency drive was studied with intermuscular coherence analysis. External perturbations were applied mechanically to the wrist joint in small frequency bands (0–4, 4–8 and 8–12 Hz; ‘angle protocol) and at single frequencies (1, 5, 7 and 9 Hz; ‘torque’ protocol). The low-frequency drive was found in the neck muscles of 4 MD patients. In these patients, its frequency did not shift due to the perturbation. In the torque protocol, the externally applied frequencies could be detected in all controls and in the two patients without the common drive. The common low-frequency drive was not be affected by external perturbations in MD patients. Furthermore, the torque protocol did not induce intermuscular coherences at the applied frequencies in these patients, as was the case in healthy controls and in patients without the drive. This suggests that the dystonic 3–7 Hz drive is caused by a sensory-independent motor drive and sensory malfunctioning in MD might rather be a consequence than a cause of dystonia

The Neuronal EGF-Related Gene Nell2 Interacts with Macf1 and Supports Survival of Retinal Ganglion Cells after Optic Nerve Injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01)