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Neurons are vulnerable to injury, and failure to activate self-protective systems after injury leads to neuronal death. However, sensory neurons in dorsal root ganglions (DRGs) mostly survive and regenerate their axons. To understand the mechanisms of the neuronal injury response, we analyzed the injury-responsive transcriptome and found that Stc2 is immediately upregulated after axotomy. Stc2 is required for axon regeneration in vivo and in vitro, indicating that Stc2 is a neuronal factor regulating axonal injury response. The application of the secreted stanniocalcin 2 to injured DRG neurons promotes regeneration. Stc2 thus represents a potential secretory protein with a feedback function regulating regeneration. Finally, the in vivo gene delivery of STC2 increases regenerative growth after injury in peripheral nerves in mice. These results suggest that Stc2 is an injury-responsive gene required for axon regeneration and a potential target for developing therapeutic applications.

DLK is a key regulator of axon regeneration and degeneration in response to neuronal injury. To understand the molecular mechanisms controlling the DLK function, we performed yeast two-hybrid screening analysis and identified FKBPL as a DLK-binding protein that bound to the kinase domain and inhibited the kinase enzymatic activity of DLK. FKBPL regulated DLK stability through ubiquitin-dependent DLK degradation. We tested other members in the FKBP protein family and found that FKBP8 also induced DLK degradation as FKBPL did. We found that Lysine 271 residue in the kinase domain of DLK was a major site of ubiquitination and SUMO3-conjugation and responsible for FKBP8-mediated degradation. In vivo overexpression of FKBP8 delayed progression of axon degeneration and neuronal death following axotomy in sciatic and optic nerves, respectively, although axon regeneration efficiency was not enhanced. This research identified FKBPL and FKBP8 as new DLK-interacting proteins that regulated DLK stability by MG-132 or bafilomycin A1-sensitive protein degradation.

Objective Cerebral hemodynamic and metabolic changes may occur during the rewarming phase of targeted temperature management in post cardiac arrest patients. Yet, studies on different rewarming rates and patient outcomes are limited. This study aimed to investigate post cardiac arrest patients who were rewarmed with different rewarming rates after 24 hours of hypothermia and the association of these rates to the neurologic outcomes. Methods This study retrospectively investigated post cardiac arrest patients treated with targeted temperature management and rewarmed with rewarming rates of 0.15°C/hr and 0.25°C/hr. The association of the rewarming rate with poor neurologic outcomes (cerebral performance category score, 3 to 5) was investigated. Results A total of 71 patients were analyzed (0.15°C/hr, n=36; 0.25°C/hr, n=35). In the comparison between 0.15°C/hr and 0.25°C/hr, the poor neurologic outcome did not significantly differ (24 [66.7%] vs. 25 [71.4%], respectively; P=0.66). In the multivariate analysis, the rewarming rate of 0.15°C/hr was not associated with the 1-month neurologic outcome improvement (odds ratio, 0.54; 95% confidence interval, 0.16 to 1.69; P=0.28). Conclusion The rewarming rates of 0.15°C/hr and 0.25°C/hr were not associated with the neurologic outcome difference in post cardiac arrest patients.

The effects of ultra-high pressure (UHP, 100 MPa, 40, 24 h) processing and enzyme treatment on the physicochemical properties and anticoagulant activity of fucoidan obtained from Undaria pinnatifida sporophyll (UPS) were investigated. Crude UPS fucoidan (FUPS) with an average MW of 877 kDa was slightly depolymerized by UHPassisted Tunicase treatment, yielding fucoidan with a lower MW (600–800 kDa). UHP-enzyme treatment decreased the sulfate, fucose and galactose contents of FUPS but increased its glucose content. From FT-IR spectrum, UHP-enzyme treatment was found not to cause a structural change on S=O and C-O-S. After UHP-enzyme treatment, the sulfate content and average MW of FUPS decreased with the increase of Tunicase concentration. It was found that among the UHP-enzyme-treated fucoidans, FUPS-T0.3-U (Tunicase 0.3% treatment+UHP treatment) possessed the highest anticoagulant activity. FUPS-T0.3-U appeared to inhibit blood coagulation via intrinsic pathway. With the increase of sulfate content in FUPS, activated partial thromboplastin time (APTT) and thrombin time (TT) showed a tendency of increase. APTT and TT had the highest values in FUPS-T0.3-U in which the sulfate concentration was 24%, but on the contrary decreased at a sulfate concentration of above 24%. This result indicates that there is an optimum sulfate concentration for the anticoagulant activity of fucoidan. Consequently, UHP-assisted enzymatic treatment was found to be helpful for the improvement of anticoagulant activities of fucoidan.

Chondrocytes are the only cell type present in the articular cartilage and their response to mechanical stimuli influences the maintenance and remodeling of the cartilage. Numerous studies have shown that the balance between anabolic and catabolic responses of the chondrocytes to mechanical loading is dependent on the loading intensities (reviewed in ref. [1]). Moderate, physiological loading, for instance, increases synthetic activity of the extracellular matrix (ECM) such as collagen type II, aggrecan, and proteoglycan, while decreasing the catabolic activity of degradative enzymes such as matrix metalloproteinases (MMPs) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) [2,3]. In contrast to moderate loading, static or high-intensity loading has been shown to degrade the cartilage resulting from inhibition of matrix synthesis and up-regulation of catabolic activities [3,4]. Therefore, the importance of these load-dependent signaling pathways involved in the maintenance and remodeling of the cartilage is widely accepted. However, the underlying mechanisms as to how varying magnitudes of mechanical stimuli trigger differential signaling activities that consequently lead to selective gene expression are not clear. FAK and Src are considered to be the main mechanotransduction signaling proteins at the cell-ECM adhesion sites and their activities influence various structural and signaling changes within the cell, including cytoskeletal organization, migration, proliferation, differentiation, and survival [5]. Accumulating evidence has shown that Src and FAK play crucial roles in regulating cartilage maintenance and degeneration and their activation stimulates matrix catabolic genes and activity [6,7]. Rho family GTPases such as RhoA and Rac1 play critical roles in fundamental processes including morphogenesis, polarity, movement, and cell division [8]. They also contribute to cartilage development and degradation [9,10]. Despite these studies, much remains to be elucidated on how load-induced Src and FAK participate in chondrocyte functions, and how their interactions are linked and regulated in connection to the activities of RhoA and Rac1 under different loading conditions. In this study, we use fluorescence resonance energy transfer (FRET)-based biosensors to monitor activities of Src and FAK as well as individual GTPases and evaluate the potential linkage of a network of these signaling molecules under different loading conditions.Copyright © 2013 by ASME

Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate that both Rac1 and Cdc42 GTPases are critical regulators in shear stress-driven β-catenin signaling in osteoblasts.