Activation of Adenylyl Cyclase Reduces TGF-b Profibrotic Response in Osteoarthritic Fibroblast-like Synoviocytes

Purpose: The hallmarks of osteoarthritis (OA) include cartilage degeneration, bone remodeling and synovial fibrosis. Synovial fibrosis is characterized by excessive extracellular matrix (ECM) accumulation due to an imbalance in ECM production, in particular collagen, and its turnover. Transforming growth factor beta (TGF-β) and its associated signaling pathway mediated by ALK5, plays an important role in synovial fibrosis and blocking TGF-β’s effect prevents synovial fibrosis. Increasing intracellular cyclic AMP (cAMP) produces an antifibrotic effect in fibroblasts of multiple origins. Forskolin (FsK) is a naturally occurring diterpene in the roots of the Indian Coleus plant that activates adenylyl cyclase resulting in an elevation in intracellular cAMP levels. We hypothesized that FsK treatment results in an anti-fibrotic effect in TGF-β stimulated fibroblast-like synoviocytes (FLS) from patients with advanced OA. Methods: OA FLS (Cell Applications, USA) were harvested from patients undergoing total knee replacement. Cells were used between the 3rd and 6th passages for all experiments. OA FLS (300,000 cells per well) were treated with TGF-β (1ng/ml; R&D Systems) in the absence or presence of FsK (10μM; Sigma Aldrich) or SB431542, an ALK5 inhibitor (1μM, Sigma Aldrich) for 24 hours followed by RNA extraction using Trizol reagent and RNA concentrations were determined using a NanoDrop ND-2000 spectrophotometer. cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, USA). Quantitative PCR (qPCR) was performed using TaqMan Fast Advanced Master Mix (Lifetechnologies, USA). The cycle threshold (Ct) value of genes of interest were normalized to the Ct value of GAPDH in the same sample, and the relative expression was calculated using the 2−ΔΔCt method. Genes of interest included collagens type 1 (COL1A1) and 3 (COL3A1), α2 smooth muscle actin (ACTA2), proteoglycan-4 (PRG4), matrix metalloproteinases 3, 9 and 13 (MMP3, MMP9 and MMP13), tissue inhibitor of metalloproteinase-1 (TIMP1) and aggrecanase-1 (ADAMTS4). Multiple group comparisons were performed by ANOVA or ANOVA on the ranks followed by pairwise group comparisons using Tukey\u27s test. Data is presented as the average ± S.D. of 3–6 independent experiments. Results:FsK treatment significantly reduced TGF-β induced expression of collagen type I (fig. 1A; p Conclusions: Using a model of TGF-β stimulated OA synovial fibroblasts, FsK treatment resulted in a reduction in the expression of collagen type I, a major component of fibrosis and α2 smooth muscle actin, a marker of fibroblast differentiation to myofibroblasts. To this end, FsK\u27s effect was comparable to the inhibition of intracellular TGF-β signaling. PRG4 regulates synovial proliferation and inflammation and FsK treatment enhanced PRG4 expression by OA fibroblasts. FsK reduced expression of matrix degrading enzymes, especially MMP3 and MMP9 involved in synovial proliferation, and MMP13 and ADAMTS4, involved in cartilage degradation. Increasing intracellular levels of cAMP in synovial fibroblasts may result in antifibrotic and chondroprotective effects in the joint

Arabian Sea mixed layer dynamics experiment : mooring deployment cruise report R/V Thomas Thompson cruise number 46, 14 April-29 April 1995

This report describes in a general manner the work that took place during the R/V Thomas Thompson cruise number 46 which was the mooring turnaround cruise for the moored array program. A detailed description of the WHOI surface mooring and its instrumentation is provided. Information about the XBT and CTD data and near-surface temperature data collected during the cruise is also included.Funding was provided by the Office of Naval Research through Grant No. NOOOl4-94-1-0161

Operational and Technical Updates to the Object Reentry Survival Analysis Tool

The Object Reentry Survival Analysis Tool (ORSAT) has been used in the NASA Orbital Debris Program Office for over 25 years to estimate risk due to uncontrolled reentry of spacecraft and rocket bodies. Development over the last 3 years has included: a major change to the treatment of carbon fiber- and glass fiber-reinforced plastics (CFRP and GFRP, respectively); an updated atmospheric model; a new model for computing casualty area around an impacting debris object; and a newly-implemented scheme to determine the breakup altitude of a reentry object. Software also was written to automatically perform parameter sweeps in ORSAT to allow for uncertainty quantification and sensitivity analysis for components with borderline demisability. These updates have improved the speed and fidelity of the reentry analysis performed using ORSAT, and have allowed for improved engineering understanding by estimating the uncertainty for each components survivability. A statistical model for initial conditions captures the latitude bias in population density, a large improvement over the previous inclination-based latitude-averaged models. A sample spacecraft has been analyzed with standard techniques using ORSAT 6.2.1 and again using all the updated models; we will demonstrate the variation in the total debris casualty area and overall expectation of casualty

Beaufort Gyre Freshwater Experiment : deployment operations and technology 2003

The Beaufort Gyre Freshwater Experiment (BGFE) observational program was designed to measure the freshwater content of the upper ocean and sea ice in the Beaufort Gyre of the Arctic Ocean using bottom-tethered moorings, drifting buoys, and hydrographic stations. The mooring program required the development of a safe and efficient deployment method by which the subsurface system could be deployed in waters surrounded by sea ice. This report documents the mooring procedure used to deploy the three BGFE moorings from the CCGS Louis S. St- Laurent, during the Joint Western Arctic Climate Study – 2003 (August 6 – September 7). The technical details of the instrumentation attached to each mooring and the specific deployment parameters are described. Specifics pertaining to the deployment of four surface-tethered drifters in the ice are also documented.Funding was provided by the National Science Foundation under Grant Number OPP-0230184

Pan American Climate Study (PACS) mooring recovery and deployment cruise report : R/V Thomas Thompson cruise number 73, 28 November to 26 December 1997

Three surface moorings were recovered and redeployed during R/V Thomas Thompson cruise number 73 in the eastern equatorial Pacific as pan of the Pan American Climate Study (PACS). PACS is a NOAA-funded study with the goal of investigating links between sea-surface temperature variability in the tropical oceans near the Americas and climate over the American continents. The three moorings were deployed near 125°W, spanning the strong meridional sea-surface temperature gradient associated with the cold tongue south of the equator and the warmer ocean north of the equator, near the northernmost, summer location of the Intertopical Convergence Zone. The moored array was deployed to improve the understanding of air-sea fluxes and of the processes that control the evolution of the sea surface temperature field in the region. Two surface moorings, located at 3°S, 125°W and lO°N, 125°W, belonging to the Upper Ocean Processes (UOP) Group at the Woods Hole Oceanographic Institution (WHOI), were recovered after being on station for eight months and redeployed. Two eight-month deployments were planned. A third mooring deployed at the equator and 128°W by the Ocean Circulation Group at the University of South Florida (USF) was also recovered and redeployed. The USF mooring, unfortunately, had to be recovered immediately following redeployment due to a problem with the buoy and instrumentation. The buoys of the two WHOI moorings were each equipped with meteorological instrumentation, including a Vector Averaging Wind Recorder (VAWR), and an Improved Meteorological (IMET) system. The WHOI moorings also carried Vector Measuring Current Meters, single point temperature recorders, and conductivity and temperature recorders located in the upper 200 meters of the mooring line. In addition to the instrumentation noted above, a variety of other instruments, including an acoustic current meter, acoustic doppler current meters, bio-optical instrument packages and an acoustic rain gauge, were deployed during the PACS field program. The USF mooring had an IMET system on the surface buoy and for oceanographic instrumentation, two RD Instruments acoustic doppler current profilers (ADCPs), single-point temperature recorders, and conductivity and temperature recorders. Conductivity-temperature-depth (CTD) profiles were made at each mooring site and during the transit between mooring locations. This report describes, in a general manner, the work that took place during R/V Thomas Thompson cruise number 73. A description of the WHOI moored array and instrumentation is provided. Details of the mooring designs and preliminary data from the CTD profies are included.Funding was provided by the National Oceanic and Atmospheric Administration under Contract No. NA66GPO130

Advantages of a Polycentric Approach to Climate Change Policy

Lack of progress in global climate negotiations has led scholars to reconsider polycentric approaches to climate policy. Several examples of subglobal mechanisms to reduce greenhouse-gas emissions have been touted, but it remains unclear why they might achieve better climate outcomes than global negotiations alone. Decades of work conducted by researchers associated with the Vincent and Elinor Ostrom Workshop in Political Theory and Policy Analysis at Indiana University have emphasized two chief advantages of polycentric approaches over monocentric ones: they provide more opportunities for experimentation and learning to improve policies over time, and they increase communications and interactions — formal and informal, bilateral and multilateral — among parties to help build the mutual trust needed for increased cooperation. A wealth of theoretical, empirical and experimental evidence supports the polycentric approach

Incomplete Punishment Networks in Public Goods Games: Experimental Evidence

Abundant evidence suggests that high levels of contributions to public goods can be sustained through self-governed monitoring and sanctioning. This experimental study investigates the effectiveness of decentralized sanctioning institutions in alternative punishment networks. Our results show that the structure of punishment network significantly affects allocations to the public good. In addition, we observe that network configurations are more important than punishment capacities for the levels of public good provision, imposed sanctions and economic efficiency. Lastly, we show that targeted revenge is a major driver of anti-social punishment

The Beaufort Gyre Observing System 2004 : mooring recovery and deployment operations in pack ice

Situated beneath the Arctic perennial ice pack, the principal components of the Beaufort Gyre Observing System are three deep-ocean bottom-tethered moorings with CTD and velocity profilers, upward looking sonars for ice draft measurements, and bottom pressure recorders. A major goal of this project is to investigate basin-scale mechanisms regulating freshwater and heat content in the Arctic Ocean and particularly in the Beaufort Gyre throughout several complete annual cycles. The methods of recovering and re-deploying the 3800 m long instrumented moorings from the Canadian Coast Guard Icebreaker Louis S. St. Laurent in August 2004 are described. In ice-covered regions, deployments must be conducted anchor-first, so heavier wire rope and hardware must be incorporated into the mooring design. Backup buoyancy at the bottom of the mooring is advised for backup recovery should intermediate lengths of the mooring system get tangled under ice floes during recovery. An accurate acoustic survey to determine the exact location of the mooring, adequate ice conditions, and skilled ship maneuvering are all essential requirements for a successful mooring recovery. Windlass (or capstan) procedures could be used for the recovery, but a traction winch arrangement is recommended.Funding was provided by the National Science Foundation under Grant Number OPP-0230184 and Woods Hole Oceanographic Insitution’s Ocean and Climate Change Institute

Symbiont Diversity of Zooxanthellae (Symbiodinium Spp.) In Porities Astreoides and Montastraea Cavernosa from a Reciprocal Transplant in the Lower Florida Keys

In recent years, coral reefs worldwide have suffered high mortality rates due to coral bleaching, a phenomenon contributing to a 40% decrease in coral cover in the Florida Keys since the 1997/98 El Niño event. In the Florida Keys, coral from inshore reefs are known to be more thermotolerant than their conspecifics from offshore reefs but the mechanism behind this difference is unclear. In this study we conducted a two-year, reciprocal transplant of Porites astreoides and Montastraea cavernosa from an inshore and offshore reef in the lower Florida Keys to determine if changes in the dominant symbiotic algae (Symbiodinium spp.) could explain variation in holobiont tolerance as well as to assess the possibility of acclimatization to a changing stress regime. Increased complexity and diversity was demonstrated in the composition of Symbiodinium spp. from both coral species collected at the offshore reef when compared to conspecifics collected inshore. As a result of this complexity, the offshore reef samples displayed higher numbers of transitions of zooxanthellae subclade types between seasons, while inshore fragments demonstrated more stability and may explain previously measured thermotolerance. Additionally, the known thermotolerant subclade type D1 was associated with one M. cavernosa fragment from the inshore reef. When fragments were transplanted, compositional patterns of Symbiodinium spp. were retained from site of collection, indicating a lack of acclimatization to a new environment over the lengthy two-year experiment. These results demonstrate variability in the dominant Symbiodinium spp. of P. astreoides and M. cavernosa conspecifics from inshore and offshore reefs in the lower Florida Keys and point to possible patterns in holobiont thermotolerance. This variability may be key to the continued persistence of these species in the face of climate change, but future studies are needed to determine the mechanisms and range in which these subclade types withstand thermal stress