text:
Dynamic compression counteracts IL-1β induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs
DOI: 10.1186/ar2389
© Chowdhury et al.; licensee BioMed Central Ltd. 2008
Received: 27 September 2007
Accepted: 18 March 2008
Published: 18 March 2008
Nitric oxide and prostaglandin E2 (PGE2play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1β and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.
Chondrocyte/agarose constructs were cultured under free-swelling conditions with or without IL-1β and/or SB203580 (inhibitor of p38 MAPK) for up to 48 hours. Using a fully characterized bioreactor system, constructs were subjected to dynamic compression for 6, 12 and 48 hours under similar treatments. The activation or inhibition of p38 MAPK by IL-1β and/or SB203580 was analyzed by western blotting. iNOS, COX-2, aggrecan and collagen type II signals were assessed utilizing real-time quantitative PCR coupled with molecular beacons. Release of nitrite and PGE2 was quantified using biochemical assays. Two-way analysis of variance and the post hoc Bonferroni-corrected t-test were used to examine data.
IL-1β activated the phosphorylation of p38 MAPK and this effect was abolished by SB203580. IL-1β induced a transient increase in iNOS expression and stimulated the production of nitrite release. Stimulation by either dynamic compression or SB203580 in isolation reduced the IL-1β induced iNOS expression and nitrite production. However, co-stimulation with both dynamic compression and SB203580 inhibited the expression levels of iNOS and production of nitrite induced by the cytokine. IL-1β induced a transient increase in COX-2 expression and stimulated the cumulative production of PGE2 release. These effects were inhibited by dynamic compression or SB203580. Co-stimulation with both dynamic compression and SB203580 restored cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which we observed no response with the cytokine and/or SB203580.
These data suggest that dynamic compression directly influences the expression levels of iNOS and COX-2. These molecules are current targets for pharmacological intervention, raising the possibility for integrated pharmacological and biophysical therapies for the treatment of cartilage joint disorders.
The mechanical environment is an important factor that maintains articular cartilage in a healthy state. Mechanical signals generated under normal physiological loading conditions will activate mechanotransduction pathways and drive biochemical events that regulate chondrocyte function and activity [1–4]. It is well established that proinflammatory cytokines such as IL-1β act as the key mediators of cartilage breakdown and stimulate the release of nitric oxide (NO) and prostaglandin (PG)E2, via induction of inducible isoforms of the nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes [5–9]. There is growing evidence that mechanical stimulation inhibits the release of NO and PGE2 by articular chondrocytes [10–18]. Thus, mechanical strain acts in an anti-inflammatory manner that may influence the progression of osteoarthritis (OA). However, the molecular mechanisms that underlie specific mechanotransduction pathways are complex and vary depending on the type of mechanical stimuli and pathological environment of the tissue.
The fundamental pathways that play a role in increasing the release of NO and PGE2 by IL-1β involve activation of members of the mitogen-activated protein kinase (MAPK) pathway, namely extracellular signal-regulated kinase (ERK)-1/2, p38 and c-Jun amino-terminal kinase (JNK) families, and the transcription factors activator protein-1 and nuclear factor-κB (NF-κB) [19–27]. These studies demonstrated strong stimulation of p38 MAPK by IL-1β and the subsequent induction of iNOS and COX-2 expression in articular chondrocytes. Thus, the potential of p38 MAPK as drug target in cartilage disease has led to the development of several inhibitors by pharmaceutical companies. However, no study has examined the involvement of the p38 MAPK pathway in response to both IL-1β and mechanical loading.
Mechanical stimulation in the form of static or intermittent compression of varying loading modalities, including shear stress or tension, may influence the signal transduction pathways activated by IL-1β [28–37]. For instance, loading studies that apply physiological levels of compression to chondrocytes have demonstrated a role for the integrins in mediating the mechanotransduction process, including downregulation of NO and PGE2 release, both in the presence and absence of IL-1β, utilizing iNOS and COX-2 specific inhibitors [16–18, 38, 39]. The nature of the mechanical loading regimen and model system will therefore determine whether mechanical signals will prevent or induce these inflammatory mediators. Accordingly, the present study examines the interplay between mechano-sensitive and cytokine-sensitive pathways, and determines the effects of IL-1β and dynamic compression on the expression levels of iNOS and COX-2 and involvement of the p38 MAPK pathway.
Materials and methods
Isolation of chondrocytes and culture in agarose constructs
This study involves bovine cells procured from a local abbatoir with authorization from the relevant meat inspectors (Dawn Cardington, Bedfordshire, UK). It does not involve humans, human tissue, or experimentation on animals. Full-depth slices of articular cartilage were dissected from the proximal surface of the metacarpalphalangeal joints of 18-month-old cattle [40, 41] and diced. The tissue was then incubated on rollers for 1 hour at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% (vol/vol) foetal calf serum plus 2 μmol/l l-glutamine, 5 μg/ml penicillin, 5 μg/ml streptomycin, 20 mmol/l Hepes buffer and 0.85 μmol/l l-ascorbic acid, plus 700 units/ml pronase. It was subsequently incubated for a further 16 hours at 37°C in medium supplemented with 100 units/ml collagenase type XI (All Sigma Chemical Co., Poole, UK). The chondrocyte suspension was washed and cell viability assessed using trypan blue. Chondrocytes were finally re-suspended in media at a cell concentration of 8 × 106 cells/ml. The cell suspension was added to an equal volume of molten 6% (weight/vol) agarose type VII (Sigma-Aldrich, Poole, UK) in Earle's Balanced Salt Solution (Sigma Chemical Co., Poole, UK) to yield a final cell concentration of 4 × 106 cells/ml in 3% (weight/vol) agarose. The cell/agarose suspension was transferred into a sterile stainless steel mould, containing holes 5 mm in diameter and 5 mm in height and allowed to gel at 4°C for 20 minutes to yield cylindrical constructs. Chondrocyte/agarose constructs were equilibrated in culture in 1 ml DMEM plus 1 × ITS liquid media supplement (Sigma-Aldrich) at 37°C in 5% carbon dioxide for 24 hours.
Temporal effects of IL-1β under free-swelling conditions
Constructs were cultured in 1 ml DMEM + 1 × ITS supplemented with 0 or 10 ng/ml IL-1β (Peprotech EC Ltd, London, UK) and/or 10 μmol/l SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]1H-imidazole; a selective inhibitor of p38 MAPK) [24, 42] under free-swelling conditions for 0, 0.75, 1.5, 3, 6, 12, 24, or 48 hours (Merck Chemicals, Nottingham, UK). At the specified time points, representative constructs were snap frozen in liquid nitrogen and stored at -80°C before extraction of mRNA. The corresponding media were stored at -20°C before biochemical analysis.
Application of dynamic compression
A fully characterised bioreactor system (Zwick Testing Machines Ltd, Leominster, UK) was used to apply physiological levels of dynamic compressive strain to chondrocyte/agarose constructs, as detailed previously [38–41]. Equilibrated constructs were transferred into individual wells of a 24-well culture plate (Costar, High Wycombe, UK) and mounted within the bioreactor apparatus. One millilitre of DMEM plus 1 × ITS was introduced into each well. The media were supplemented with 0 or 10 ng/ml IL-1β and/or 10 μmol/l SB203580. Control constructs were unstrained but were maintained within the bioreactor system. Strained constructs were subjected to a dynamic compressive strain ranging from 0% to 15% in a sinusoidal waveform at a frequency of 1 Hz, as previously described [38–41]. The constructs were cultured at 37°C/5% carbon dioxide for 6, 12, or 48 hours. At the end of each experiment, the constructs were snap frozen in liquid nitrogen and stored at -80°C before extraction of mRNA. The corresponding media were stored at -20°C before biochemical analysis.
Protein extraction and analysis by Western blotting
Following IL-1β stimulation, chondrocyte/agarose constructs (n = 3) were washed with ice-cold phosphate-buffered saline containing 100 μmol/l Na3VO4 and pulverized in ice-cold lysis buffer containing 1% Igepal, 1 mmol/l Na3VO4 (both from Sigma Aldrich) and protease inhibitor cocktail (Roche Diagostics, Lewes, UK). The cell/agarose lysis solution was left on ice for 30 minutes and centrifuged at 13,000 rpm for 15 minutes. Supernatants were collected and protein concentration was determined by Folin-Lowry method on the Dynatech MR5000 microplate reader (Dynatech, Alexandria, VA, USA). Equal amounts of protein (400 μg) were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore Immobilon-P; Sigma Aldrich). Membranes were blocked in buffer containing 1 × Tris-buffered saline (TBS) plus 0.1% Tween-20, and 1% nonfat milk for 2 hours at room temperature, and then washed five times with TBS plus 0.1% Tween-20.
Membranes were incubated with a polyclonal rabbit antibody for phospho-p38 MAPK (Thr180/Tyr182) at a dilution of 1:1,000 (New England Biolabs Ltd, Hitchin, UK). After washing extensively with TBS plus 0.1% Tween-20, membranes were incubated with horseradish peroxidase-linked secondary antibody for 1 hour and the binding was detected using Enhanced Chemiluminescence Plus Western blotting detection system, in accordance with the manufacturer's instructions (Amersham, Buckinghampshire, UK). Membranes were stripped with a solution containing 62.5 mmol/l Tris (pH 6.8), 2% SDS and 100 mmol/l β-mercaptoethanol for 30 minutes at 50°C, and then reprobed using a phosphorylation state-independent antibody for p38 MAPK and α-tubulin, which served as an internal control (Cell Signalling).
RNA extraction, cDNA synthesis and real-time PCR
Total RNA was isolated from individual constructs using protocols described in the QIAquick® Spin gel extraction and Rneasy® kits (Qiagen, West Sussex, UK), as previously described [43]. Following the manufacturer's instructions, Ambion's DNA-free DNase treatment and removal reagents were used to eliminate any contaminating DNA from the RNA sample (Ambion Applied Biosystems, Warrington, UK). RNA was quantified using the Nanodrop ND-1000 spectrophotometer (LabTech, East Sussex, UK) and stored in 40 μl RNase-free water at -80°C until reverse transcription could be performed using manufacturer's protocols from the Stratascript™ First-Strand cDNA synthesis kit (Stratagene, Amsterdam, The Netherlands). Briefly, 200 ng of total RNA was reverse transcribed in a 20 μl reaction volume using the manufacturer-supplied oligo(dT) primers. Minus reverse transcriptase (NoRT) control reactions were prepared for each sample by omitting the Stratascript™ reverse transcriptase.
Description of the Beacon designer sequences used to quantify gene expression and real-time reaction efficiencies of PCR assays
Accession number
Product size (base pairs)
Probe: 5'-FAM-CGCGATCCCTGCTTGGTGGCGAAGATGAGCGATCGCG-DABCYL-3' Forward: 5'-GTAACAAAGGAGATAGAAACAACAGG-3' Reverse: 5'-CAGCTCCGGGCGTCAAAG-3'
1.98 ± 0.06
Probe: 5'-FAM-CGCGATCGTCAGAAATTCGGGTGTGGTACAGTTGATCGCG-DABCYL-3' Forward: 5'-CGAGGTGTATGTATGAGTGTAGG-3' Reverse: 5'-GTTGGGAGTGGGTTTCAGG-3'
1.99 ± 0.03
Probe: 5'-FAM-CGCGATCCACTCAGCGAGTTGTCAGGTTCTGAGATCGCG-DABCYL-3' Forward: 5'-TGGTGTTTGTGACTCTGAGG-3' Reverse: 5'-GATGAAGTAGCAGGGGATGG-3'
1.97 ± 0.05
Collagen type II
Probe: 5'-FAM-CGCGATGCGTCAGGTCAGGTCAGCCATATCGCG-DABCYL-3' Forward: 5'-AAACCCGAACCCAGAACC-3' Reverse: 5'-AAGTCCGAACTGTGAGAGG-3'
2.00 ± 0.05
Probe: 5'-HEX-CGCGATCCACCATCTTCCAGGAGCGAGATCCGATCGCG-DABCYL-3' Forward: 5'-TTCAACGGCACAGTCAAGG-3' Reverse: 5'-TTCAACGGCACAGTCAAGG-3'
2.03 ± 0.01
Molecular beacon design and characterization for real-time PCR
Molecular beacons were introduced for real-time detection of PCR products and were synthesized from oligonucleotides (Sigma Genosys Limited, Cambridge, UK) using the Beacon Designer software (Premier Biosoft International, California, USA). Probes have a hairpin structure and contain fluorescein (FAM) or 6-carboxyhexafluorescein (HEX) as the 5'-reporter dye and 4-(4'-dimethylaminophenylazo)benzoic acid as the 3'-quencher (Table 1). Sequences were designed to avoid regions of cross homology and analyzed using the Basic Local Alignment Search Tool to verify specificity. Additionally, templates were folded and secondary structures avoided using MFold programme and beacon hairpin melting temperatures were calculated using the Zucker software. Primer accession numbers (EMBL [European Molecular Biology Laboratory]; Table 1) were obtained from previously published studies utilizing bovine chondrocytes [44–46]. Primers used in PCR experiments with molecular beacons produced amplicons, which were between 70 and 82 base pairs (Table 1).
PCR efficiencies for optimal primer pair and probe concentrations were derived from standard curves (n = 3) by preparing a 10-fold serial dilution of cDNA from a sample that represented the untreated control at time zero conditions. The real-time PCR efficiencies (E) of amplification for each target was defined according to the following relationship: E = 10(-1/slope). The R2 value of the standard curve exceeded 0.9998 and revealed efficiency values presented in table 1.
Data normalization and statistical analyses
Fluorescence data was collected during the annealing stage of amplification and data was analyzed using the MxPro™ QPCR software (version 3.0; Stratagene). Baselines and thresholds were automatically set by the software and used after manual inspection. The cycle threshold (Ct) value for each duplicate reaction was expressed as the mean value and the results were exported as tab-delimited text files into Microsoft Excel for further analysis. The data obtained by PCR assay for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was validated as a reference gene by displaying the Ct values as box and whisker plots, and the distribution examined under free swelling and mechanical loading conditions (data not shown). The Ct values for GAPDH remained stable with no changes detected under all culture conditions, suggesting its suitability as a reference gene.
E represents the efficiencies obtained for the target and reference gene. ΔCttarget represents the difference in Ct values for the mean calibrator or sample for the target gene. ΔCtReferencerepresents the difference in Ct values for the mean calibrator or sample for the reference gene GAPDH. Ratios were expressed on a logarithmic scale (arbitrary units).
Nitrite and prostaglandin E2 analysis
Quantification of nitrite and PGE2 release were previously described in detail [16–18, 38]. Absolute concentrations of nitrite (μmol/l), a stable end-product of NO, were determined in the media using a spectrophotometric method based on the Griess reaction. PGE2 production was measured in the culture media using an enzyme immunoassay kit (Amersham Biosciences, Buckinghamshire, UK).
Statistical analysis
For the time course studies under free-swelling conditions, data represent the mean and standard error of the mean values of six replicates from three separate experiments. Two-way analysis of variance and the post hoc Bonferroni-corrected t-test was used to examine data for constructs cultured in the presence or absence of IL-1β and/or SB203580. For the mechanical loading experiments, data represent the mean and standard error of the mean values of replicates indicated in the individual figure legend. Statistical analysis was performed by a two-way analysis of variance and the post hoc Bonferroni-corrected t-tests to compare differences between unstrained and strained constructs cultured under the different treatment conditions. Data were also examined between unstrained constructs cultured in the absence and presence of the cytokine and/or inhibitor. In all cases, a level of 5% was considered statistically significant (P < 0.05).
IL-1β activates the phosphorylation of p38 MAPK
IL-1β induced iNOS and COX-2 and inhibited aggrecan expression
In the absence of the cytokine, there was a downregulation of aggrecan expression up to 6 hours of culture when compared with time zero (Figure 2c). Aggrecan expression levels peaked at 12 hours (threefold increase; P < 0.01; Figure 2c) when compared with time zero and was not significantly detected at any other time point. At 12 hours, aggrecan expression was inhibited by the presence of the cytokine (P < 0.001) but was not significantly influenced by the addition of SB203580 (Figure 2c). In contrast, the expression levels of collagen type II appeared to be consistently downregulated in the presence or absence of the cytokine or with the addition of the inhibitor (Figure 2d). However, the level of inhibition under the various treatment conditions did not differ significantly relative to that at time zero.
IL-1β stimulates the production of nitrite and PGE2release
Dynamic compression inhibited IL-1β induced iNOS and COX-2 expression, production of nitrite and PGE2release
Dynamic compression restored IL-1β induced inhibition of aggrecan expression
IL-1β plays a pivotal role in both the pathogenesis of OA and as a potent mediator of catabolic processes in articular chondrocytes. The inflammatory process involves the excessive production of NO, PGs, matrix metalloproteinases, aggrecanases, and cytokines associated with the IL-1 and tumour necrosis factor families [5–9]. The net result is a tissue milieu with increased levels of inflammatory mediators that lead to cellular stress, chondrocyte apoptosis and loss of matrix tissue [5–9]. There is clinical evidence to support the beneficial effects of controlled moderate exercise in relieving adults with OA, in combination with chondroprotective agents [49, 50]. However, the success of these treatments largely relies on the validation of the drugs and their pathophysiological interactions within the OA affected joint. Consequently, in vitro bioreactor systems have been developed as model systems to investigate the mechano-sensitive and cytokine-sensitive intracellular pathways. More specifically, a number of studies have shown that the application of controlled loading regimens at physiological magnitude prevents the harmful effects of IL-1β by blocking the release of NO, PGE2 and matrix metalloproteinases, and restoring extracellular matrix production [10–18, 38, 39, 45, 46]. Consequently, the tissue maintains a balance in matrix turnover and regulates cytokine-induced pathways during articular cartilage loading.
In the present study, we utilized real-time quantitative PCR assays coupled with a novel fluorescent probe known as the molecular beacon to detect the catabolic (iNOS and COX-2) and anabolic (aggrecan and collagen type II) genes utilizing the chondrocyte/agarose model in conjunction with a well characterized bioreactor system [40, 41]. The data provide striking evidence that dynamic compression antagonizes the IL-1β induced expression of iNOS and COX-2 levels and production of NO and PGE2 release. Additionally, we provide evidence to support restoration of aggrecan levels by both types of stimuli. These data support our previous studies that show that dynamic compression counteracts the IL-1β induced production of NO and PGE2 release by full-depth chondrocytes and superficial zone chondrocytes, and restores cell proliferation and proteoglycan synthesis [16, 17].
The free-swelling studies were undertaken to determine how the cytokine influenced gene expression levels with time, in the absence of the bioreactor system. IL-1β activated the phosphorylation of p38 MAPK and was inhibited by the p38 MAPK inhibitor (Figure 1). The cytokine additionally induced a transient increase in the expression levels of both iNOS and COX-2 (Figure 2). COX-2 expression appeared to be more sensitive to the cytokine and to the presence of SB203580 (Figure 2b), suggesting that COX-2 activation may occur independently of NO and primarily involves a p38 MAPK dependent pathway. We observed a similar effect with the release of NO (Figure 3a) and PGE2 production (Figure 3b) in response to IL-1β, in which SB203580 had a more potent effect on PGE2 release than on NO production. It could be argued that the p38 MAPK inhibitor may have nonspecific effects. Previous studies have shown inhibition of NO release by SB203580 at a concentration of 1 and 10 μmol/l in IL-1β treated chondrocytes [20–22, 24, 51]. However, at concentrations at 10 μmol/l or greater, SB203580 was demonstrated to inhibit JNK in chondrocyte monolayers, suggesting less specificity at a higher dose [52]. Accordingly, iNOS induction and NO release may therefore involve activation of multiple MAPK pathways, in particular JNK. A number of previous studies have reported differential mechanisms through which IL-1β upregulates iNOS and COX-2 expression that involve activating transcription factor-2, NF-κB, IL-6, cAMP responsive element binding protein-1, or activator protein-2 transcription factors and activation of all members of the MAPKs [20, 23, 53]. Nevertheless, NF-κB appears to be the primary transcription factor that influences iNOS expression and NO production, and has been shown to strongly inhibit PGE2 synthesis in OA affected cartilage [52]. Our studies utilizing human chondrocyte/agarose constructs cultured with selective inhibitors of iNOS (1400W) and COX-2 (NS-398), also suggest that NO could have a negative influence on PGE2 production [18]. However, whether NO induces or suppresses PGE2 release in the presence of IL-1β remains controversial [9, 18, 54].
In separate experiments, constructs were subjected to dynamic compression or remained unstrained within a bioreactor system. The present data shows that unstrained culture within the bioreactor system induced a significant enhancement in the levels of NO release in the presence and absence of IL-1β (Figure 4d), when compared with free-swelling culture conditions (Figure 2a), whereas PGE2 release (Figures 2b and 5d) was largely unaffected, a phenomenon that concurs with our previous studies [16, 17]. A similar effect was noted for iNOS induction in response to IL-1β, with a sevenfold increase in free-swelling conditions (Figure 2a), as compared with a 145-fold increase in unstrained constructs cultured within the bioreactor without the application of dynamic compression (Figure 4a). In accordance with the PGE2 release data, the fold increase in expression of COX-2 was broadly similar under both culture conditions (Figures 2b and 5). There are marked differences in the diffusional constraints in unstrained constructs in the bioreactor, involving contact of the upper surface with a fluid impermeable loading pin. Accordingly, the mass transport of oxygen into the construct may be impaired, leading to increased hypoxia in the bioreactor system, compared with constructs cultured under free-swelling conditions. A number of recent studies support the hypothesis that oxygen tension can influence the production of inflammatory mediators in cartilage. For example, hypoxia dramatically increased iNOS expression and NO production by bovine chondrocytes in response to IL-1β, whereas COX-2 expression was not strongly influenced by oxygen tension [44, 55, 56].
Co-stimulation with dynamic compression and inhibitor for 6 hours completely abolished iNOS induction in response to IL-1β (Figure 4a). Interestingly, neither dynamic compression or the inhibitor alone were able to abolish the IL-1β induced iNOS expression. The effect was more striking for COX-2 with levels returning to basal values after application of dynamic compression or the p38 MAPK inhibitor alone for 12 hours (Figure 5b). The data suggest interactions between mechano-sensitive and p38 MAPK dependent pathways in mediating the inhibitory effect of dynamic compression on iNOS and COX-2 expression. The importance of these findings is supported by recent studies utilizing monolayer chondrocytes, which showed sustained levels of iNOS and COX-2 expression in response to IL-1β for up to 24 hours, with a dramatic reduction by cyclic tensile strain for 8 hours over a 24-hour loading regimen [15]. Additionally, the mechanism was shown to involve NF-κB activation, which could act downstream of p38 MAPK [13]. Nonetheless, static or intermittent compression of different magnitudes applied to cartilage explants increased NO and PGE2 production [28, 29]. These differences may potentially be attributed to both the nature of the mechanical stimulus and the level of oxygen tension, as indicated in recent studies [56–58].
We initially examined the involvement of the p38 MAPK because this kinase was influenced by either IL-1β or mechanical loading [24, 52]. We demonstrated activation of the p38 MAPK following 20 minutes of stimulation with the cytokine (Figure 1). However, a major limitation of the bioreactor system is the time taken to retrieve constructs at the end of an experiment (up to 10 minutes). This causes difficulties associated with analyzing transient phosphorylation events, because alterations in activation state may occur after the end of the mechanical loading regimen. Accordingly, the time courses of phosphorylation events in response to both IL-1β and dynamic compression are not presented here, but work is actively ongoing. In addition, we feel that the regulation of the IL-1β pathways may be through many factors, and dynamic compression may actually target a global mechanism involving JNK or NF-κB, as indicated in previous studies [13, 51, 52].
In addition to the regulation of iNOS and COX-2 by dynamic compression, co-stimulation with the inhibitor was important in restoring the cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which no response to IL-1β or dynamic compression was observed. It is highly likely that matrix gene expression is controlled by transient factors, and could involve rapid activation of multiple transcription factors that are influenced by a combination of mechanical loading and IL-1β [19, 22, 31, 32]. Consequently, the multiple events activated in a time-dependent manner will influence the rate of expression and assembly of the extracellular matrix molecules, causing flare-ups in gene expression levels. This phenomenon may start as early as 1 hour after mechanical stimulation, as reported with aggrecan and collagen type II expression [37, 59].
Overall, these observations indicate a hypothetical mechanism for the activation of iNOS by IL-1β that are, respectively, dependent on p38 MAPK signalling and could involve activation of other MAPKs. In free-swelling conditions the p38 independent pathway is predominantly activated, as indicated by the partial effect of the p38 inhibitor on iNOS expression at later time points. However, at early time points the p38 inhibitor blocked activation of the p38 MAPK. Incorporation of the construct into the bioreactor system, with its inherent diffusional constraints, dramatically enhances iNOS induction, potentially via preferential activation of the p38 MAPK dependent pathway and possibly through a hypoxia-driven mechanism. Accordingly, the inhibitor has a greater effect on iNOS expression under these conditions. Dynamic compression substantially reduces iNOS expression and co-stimulation with both dynamic compression and the p38 MAPK inhibitor abolished iNOS expression when compared with each in isolation, suggesting that dynamic compression targets the p38 MAPK dependent pathway. By contrast, COX-2 activation by IL-1β appears to be primarily mediated by a p38 MAPK dependent pathway that is largely unaffected by transfer to the bioreactor system but is highly susceptible to the p38 MAPK inhibitor or dynamic compression. In addition to the regulation of iNOS and COX-2 expression by dynamic compression, co-stimulation with the inhibitor was important in restoring the cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which we observed no response. It is probable that matrix gene expression is controlled by transient factors and could involve rapid activation of multiple MAPKs and transcription factors. Ultimately, elucidation of the intracellular pathways will enable the identification of appropriate pharmacological agents and provide a clinical rationale for promoting the benefits of controlled physical activity to manage and treat OA.
It is important to define the mechanistic pathways induced by physiologically relevant mechanical signals in the presence of proinflammatory mediators, because this information will provide key parameters for the safe application of pharmacological therapies, in conjunction with biophysical treatments for OA.
- Ct:
cycle threshold
glyceraldehyde 3-phosphate dehydrogenase
- iNOS:
inducible isoforms of the nitric oxide synthase
c-Jun amino-terminal kinase
mitogen-activated protein kinase
- NF-κB:
nuclear factor-κB
nitric oxide
polymerase chain reaction
This study was supported by The Wellcome Trust (project grant: 073972). Dr Chowdhury would like to thank Drs Kerry Elliot, Lindsay Ramage and Ying Zhou for their excellent support at the Queens Medical Research Institute, Edinburgh University.
Authors' Affiliations
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- Fermor B, Weinburg JB, Pisetsky DS, Misukonis MA, Fink C, Guilak F: Induction of cyclooxygenase-2 by mechanical stress through a nitric oxide-regulated pathway. Osteoarthritis Cartilage. 2002, 10: 792-798. 10.1053/joca.2002.0832.View ArticlePubMedGoogle Scholar
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- Fitzgerald JB, Jin M, Dean D, Wood DJ, Zheng MH, Grodzinsky AJ: Mechanical compression of cartilage explants induces multiple time-dependent gene expression patterns and involves intracellular calcium and cyclic AMP. J Biol Chem. 2004, 279: 19502-19511. 10.1074/jbc.M400437200.View ArticlePubMedGoogle Scholar
- Hung CT, Henshaw DR, Wang CC, Mauck RL, Raia F, Palmer G, Chao PH, Mow VC, Ratcliffe A, Valhmu WB: Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization. J Biomech. 2000, 33: 73-80. 10.1016/S0021-9290(99)00176-1.View ArticlePubMedGoogle Scholar
- Murata M, Bonassar LJ, Wright M, Mankin HJ, Towle CA: A role for the interleukin-1 receptor in the pathway linking static mechanical compression to decreased proteoglycan synthesis in surface articular cartilage. Arch Biochem Biophys. 2003, 413: 229-235. 10.1016/S0003-9861(03)00129-2.View ArticlePubMedGoogle Scholar
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- Mathy-Hartert M, Burton S, Deby-Dupont G, Davel P, Reginster JY, Henrotin Y: Influence of oxygen tension on nitric oxide and prostaglandin E2 synthesis by bovine chondrocytes. Osteoarthritis Cartilage. 2005, 13: 74-79. 10.1016/j.joca.2004.09.009.View ArticlePubMedGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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subset: commoncrawl


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Search Classified Ads
- Year: 1999
- Mileage (Km): 150000-160000
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- Listed: August 3, 2015 2:05 am
- Expires: This ad has expired
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This is
lower than
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Two homes on one lot. House on the right is 1440 Sq ft built in 1961 - 3 bedroom 1 bath, living rm, dining rm, kitchen and add on room in rear. House on left is 964 Sq ft built in 1954 - 3 bedrooms 3/4 bath - Great opportunity for guest house or rental.
Real Living Success Realty
8687 E. Via de Ventura Suite 308
Scottsdale, AZ 85258
Email Us | Visit our Real Estate Website
www.realliving.com | Real Living Real Estate is a network brand of HSF Affiliates LLC, which is majority owned by HomeServices of America, Inc.TM, a Berkshire Hathaway affiliate.
2016 Real Living Real Estate, LLC. Real Living is a registered service mark. Equal Opportunity Housing Provider. Privacy | Terms of Use | Accessibility


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Photos: The Bachelor Sean Lowe's Dance Partner Peta Calls Sean 'Her Brother' at OK Party
OK! magazine threw its annual "So Sexy" event last night in Los Angeles at Skybar in the Mondrian Los Angeles. Hosted by Real Housewives of Beverly Hills star, Brandi Glanville, the party highlighted Hollywood's sexiest and most glamorous celebrities including cast members of Dancing With the Stars, MTV's Awkward and Vanderpump Rules.
Brandi was the perfect host. She was super nice and accommodating and filled everyone in on her super hectic life. She dished on all the details of her book, Drinking and Tweeting, and confirmed the rights were bought to make it into a movie. Brandi also coyly affirmed that she was dating someone special but wasn't ready to give his name. She also wasn't ready to tell major news but said she'd be ready to announce something big in three days. One thing she did want to get across was how excited she was to be teaming up with OK! for the party.
"I was really honored. As a single mother at 40 I do try to be sexy. I like to be pretty so when they asked I knew it was an honor," she gushed.
One person who was willing to spill kissing secrets of Brandi's was Jesse Heiman. Do you remember him? He's the guy who locked lips with supermodel, Bar Refaeli, in the now infamous Superbowl ad. It turns out he also locked lips with Brandi and said she was "very aggressive."
"She licked me and then we made out. It was good. Everybody's got their own style of kissing," Jesse joked. "I prefer sweet and gentle but I'm not going to say it was bad."
Two platonic Brandi chums and fellow Housewives regulars Marisa Zanuck and Jennifer Gimenez were also at the OK! party to show their support for their friend.
"She and I really hit it off the first time we met," Marisa said. She, like Brandi, came under hot water for comments she made in the past season. But she still hopes the network invites her back for another round of the ladies. "There's a lot of things I wish I didn't say but I had fun with it," she laughed.
In addition to a strong Bravo contingent, Dancing With the Stars alums made a good showing last night at the event. Sharna Burgess and Peta Murgatroyd dished on their partners, Andy Dick and Sean Lowe. Sharna explained that everyone came to her with an "Andy Dick" story but the guy she met the first day of practice was nothing but hard-working and serious about his commitment to their partnership.
Peta, on the other hand, has been plagued with are they or aren't they whispers concerning Lowe.
Keep reading on page 2.


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Penn Offers Chance to Win Chrysler Sebring
For a limited time this summer, specially marked four-packs of Penn Championship and Pro Penn tennis balls will include a chance to win a 2008 Chrysler Sebring convertible and other prizes from HEAD Penn. Also included in the pack is a digital carabiner sports watch.
Chrysler and Penn partnered through their mutual sponsorship of USTA's League Tennis program — the largest grassroots league program in the country with more than 600,000 tennis players. Chrysler is the presenting sponsor of the program while Penn serves as the official ball.
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Grocers Day at the Capitol
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CGA actively promotes and protects the legislative and regulatory interests of the retail food industry throughout California.Read More
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| was $2800 |
32% off estimated retail price
Cash on delivery | Cash On Location (*) | Free shipping
Debuting as part of Chanel's 2011 Cruise Collection, this elegantly shaped Just Mademoiselle bowling bag is crafted from python and features double chain-link shoulder straps and a 'CC' logo charm. The black leather lined interior features three compartments, a zipped pocket and leather Chanel label. Original Retail Price: US $2,800
Estimated retail price US $2800
Condition:New (Unused item)
Gender: Women
Includes: Original Box, Original Dustbag
Dimensions: Width: 16.5 CM, Height: 14 CM,
Handle Drop: 18 CM
Exterior Material: Python
Interior Material: Leather
Hardware: Silver-tone
Condition:New (Unused item)
Within UAE/Dubai
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Brand: Chanel
You can also see all the Chanel Shoulder Bags


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Website by:ASYSTech
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text:
Fresh Applesauce
|Mcintosh apples
|1 Pound, peeled, cored, and chunked (About 5 In Number)
|Lemon juice/25 milligram ascorbic acid tablet, crushed
|4 Tablespoon
|1 Pinch
|3 Drop
|1⁄4 Teaspoon
1.Take the container of a blender, put apple chunks, lemon juice and crushed ascorbic acid tablet in it.
2.Blend the mixture.
3.Add salt, vanilla and cinnamon in it and blend further.
4.Serve chilled in a bowl.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 711 Calories from Fat 0
% Daily Value*
Total Fat 0.02 g0.02%
Saturated Fat 0 g0.02%
Trans Fat 0 g
Cholesterol 0 mg
Sodium 256.5 mg10.7%
Total Carbohydrates 188 g62.5%
Dietary Fiber 27.6 g110.3%
Sugars 134.8 g
Protein 6 g11.2%
Vitamin A 11% Vitamin C 81.6%
Calcium 12.4% Iron 0.69%
*Based on a 2000 Calorie diet


text:
Whether you want to freely download Aras Innovator and try it yourself, looking to get projects or applications developed by the community or want tutorials and self study materials, we've got you covered.
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Download and run the Aras Update utility to enable installation of all applications that run inside of Aras Innovator, such as Aras Technical Documentation, Aras Quality Management System, and more.
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12. Making Lemon Bars Out of Lemons: Using the Power of Teamwork to Transform Concepts to Reality
- Jacqueline H. Beckley,
- M. Michele Foley,
- Elizabeth J. Topp,
- J. C. Huang and
- Witoon Prinyawiwatkul
Published Online: 30 NOV 2007
DOI: 10.1002/9780470277621.ch12
© 2007 Blackwell Publishing and the Institute of Food Technologists
Book Title
Accelerating New Food Product Design and Development
Additional Information
How to Cite
Wagner, M. K. and Herzog, L. J. (2007) Making Lemon Bars Out of Lemons: Using the Power of Teamwork to Transform Concepts to Reality, in Accelerating New Food Product Design and Development (eds J. H. Beckley, M. M. Foley, E. J. Topp, J. C. Huang and W. Prinyawiwatkul), Blackwell Publishing, Ames, Iowa, USA. doi: 10.1002/9780470277621.ch12
Publication History
- Published Online: 30 NOV 2007
- Published Print: 19 MAR 2007
ISBN Information
Print ISBN: 9780813808093
Online ISBN: 9780470277621
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Aspect Estate Agents Pty Ltd
Investment Property, Rental Properties, Residential Real Estate
The motivation behind this business was borne through years of experience working in other real-estate agencies which proved that in business, bigger is not always better and that branding does not automatically equal a better quality service.
These experiences highlighted that investors and tenants are too often ignored by their real estate agency in favour of chasing property sales or placed in the hands of property managers already trying to manage hundreds of other investment properties. It became obvious that those agencies priority for profit and focus on sales meant that property management was clearly lowest in their pecking order, with sales the priority.
THE SOLUTION - Develop an agency solely dedicated to property management determined to deliver a quality property management solution to Sydney's Eastern Suburbs. Provide a service that is not dominated by a sales department but rather is specialised in property management services.
OUR ANSWER – Aspect Estate Agents Pty Ltd, created to challenge the local real estate industry and to finally give property owners and reputable tenants the service and attention they really deserve.
Our unique boutique management style allows us to limit the number of properties the agency handles, thereby guaranteeing that you get the personal attention you deserve.
We embrace change and are constantly challenging tradition because we have a strong desire to improve the way the real estate industry operates and enhance the service delivered to our clients.
Print this page
Aspect Estate Agents is an agency solely dedicated to property management, determined to deliver a quality property management solution to investors with properties in Greater Sydney. We provide a service that is not dominated by a sales department but rather is specialised in property management services.
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Unilever says more than half of its 252 facilities worldwide divert no waste to landfills, including all in the U.S., the U.K., France, Japan, Canada, Germany and the Netherlands. Procter & Gamble recently announced that 45 of its facilities have achieved zero manufacturing waste to landfill.
Full Story:Retailing Today, Consumer Goods Technology News, PlasticsToday.com


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Next Batman Arkham Could be Batman Arkham Insurgency, Announcement in March?
Warner Bros. is about to make an important announcement on March 8, 2017. There's no official...
Just like the other three terminals in Final Fantasy 13-2, New Town North terminal (City of Academia) also has some questions which can be answered to earn rewards.
Answer 10 consecutive questions and you will win reward in the form of CP, Fragments and a Treasure box. This guide will walk you through all the questions that will be asked at North Terminal.
For more help read our Captain Cryptic, Grand Square Avenue Terminals, Entrance Terminal and Southwest Terminal Quiz Answers.
Q. According to the philosopher Denrith, humans are merely an intellectual version of a certain monster, with the ability of looking innocent on the outside, but being conniving on the inside. Which monster was he referring to?
A. Cactuar
Q. After the fall of Cocoon, the public became more and more aware of the truth behind the fal'Cie and their actions. What part of the truth was fudged in history textbooks?
A. The intent of the fal'Cie
Q. Around 100 AF, a TV show based on the battle of the l'Cie was in production, but got canceled at the last minute. What was the name of the show?
A. 'Don't Mess with the 'Fro'
Q. Around 100 AF, which test was proved invalid and superfluous by the Academy?
A. Role-based personality test
Q. Around 400 AF, the '??? male' became a popular phrase. What goes in the blank?
A. Armored
Q. BBQ Ben's is famous for their cheap, but delicious behemoth meat. Rumor has it they have close ties with certain government officials. How are they able to serve meat at such a reasonable price?
A. They got leftover beasts from research labs
Q. Before Cocoon's aerial railway, people rode the trains that ran along the ground. What was the name of Cocoon's old terminal?
A. Nautilus and Bodhum
Q. Chocobo riding used to be mandatory at all private schools. However, it was canceled indefinitely due to what kind of complaint from the parents?
A. The chocobo smell would rub off on the students
Q. During the festival of the Day of Rebirth, which food stand gets the most business from children?
A. Microchu mousse
Q. How do people refer to the day of Cocoon's fall?
A. The Day of Rebirth
Q. How does Hope refer to Lightning?
A. Light
Q. How does Hope's time capsule work?
A. It slows down time within the device
Q. In the children's song 'Chelsea's Bubbles,' she is able to blow a bubble as high as the biggest, roundest thing one can see. How high was she able to blow her bubble?
A. To the skies of Cocoon
Q. In the popular children's show 'Cocoon Five,' what is the name of the five heroes' ultimate attack?
A. Cocoon's Final Attack
Q. Spells can be sealed into cards and sent as gifts. Which spell makes for a popular birthday card?
A. Cure
Q. Stem is the new recreational drug of choice among the young and is quickly becoming a social issue. The ingredient can be easily found in the Sunleth Waterscape. What is it?
A. Dried habanero leaves
Q. The chocobos first used by Chocobo Delivery Services were based on the colors of nature. What colors were they?
A. Green and yellow
Q. The lumeritz singing competition began 100 years after the first paradox. What year was that?
Q. The monster movie 'The Imp' was a big hit among the niche crowd that raved about its cuteness. What did half of the movie consist of?
A. Dancing imps
Q. The New Bodhum urban legend about a man with a knife is based on who?
A. Tonberry
Q. The popular DJ DK sports thick sideburns and a mohawk. What is the name of this hairstyle?
A. Chocobo hair
Q. What accessory is considered to grant wishes, but is seldom used because of its smell?
A. Chocobo feather
Q. What accessory is recommended for those who want to boost their physical attack power?
A. Power Wristband
Q. What advertising media was widely seen in Palumpolum as a result of energy conservation?
A. Blimps
Q. What coarse substance is known as a fast-acting cold reliever?
A. Microchu baby teeth
Q. What company's business has been booming due to the numerous deliveries required between Cocoon and Gran Pulse?
A. Chocobo Delivery Service
Q. What determines the altitude of flights from Cocoon to Gran Pulse?
A. The arm's length of fal'Cie Titan
Q. What does one call cooked behmoth meat that's hung out to dry for three days?
A. Cured meat
Q. What does the cactuar sign seen at amusement parks imply?
A. Emergency exits
Q. What happens to a l'Cie once they complete their Focus?
A. They turn to crystal
Q. What happens to a l'Cie that's unable to complete their Focus?
A. They become a Cie'th
Q. What is a baby moogle called?
A. A moogling
Q. What is someone who sits at home all day generally called?
A. Bear
Q. What is the acryonym for the Academia Central Broadcasting Station?
Q. What is the behemoth king incapable of doing?
A. Shooting a thousand needles from its body
Go to next page for answers to the rest of questions.


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Regional North America United States California Localities F Fresno Recreation and Sports
This category is for sites about Fresno outdoor hobbies, sports, and recreational activities. This includes sites about activities, events, clubs, groups, services, and facilities. Exception: Businesses that sell sporting goods and products used in various recreational activities are listed under Shopping.
Subcategories 7
Related categories 1
Sites 17
Airways Municipal Golf Course
A public course for the beginning to experienced golfer. Has a short course with tight fairways and small greens.
Blackbeard's Family Fun Center
Offers batting cages, bumper boats, paintball, miniature golf, sprint cars, water slides and arcade games. Provides map with photographs and details of the park, rates, and birthday party information.
Copper River Country Club
Information about the fitness center, golf course, restaurants, clubhouse, and membership.
Fig Garden Golf Club
Semi-private club located in northwest section of Fresno. Includes photographs of the course, greens fees and membership information.
Fresno Amateur Radio Club
Offers monthly meetings, social events, ARRL exam session, monthly newsletter. Site has links to many other sites of interest.
Fresno Bass Club
Members enjoy monthly meetings and fishing tournaments at local area lakes. Tournaments feature cash prizes to the winners.
Fresno Dolphins Swim Team
A swimming association designed to help young people reach their full potential as competitive swimmers. For ages four to eighteen.
Fresno Field Archers
Fresno's archery club. Offers members monthly meetings, club competitions, and participation in local charity events. CBH/SAA and NFAA affiliated.
Fresno Rugby Club
Club activities include scheduled practices, league games, social events and fundraisers. 1992 Pacific Coast Rugby Champions.
Fresno Ski Club
Sponsors numerous social, sporting and travel events throughout the year. Also offers biweekly seasonal meetings and an annual Ski Swap.
Gateway Ice Center
Ice skating rink, with information about public sessions, skating and hockey lessons, birthday party packages and corporate rental.
Ice Skating Club of Fresno
Offering a chance for skaters to socialize and qualify for amateur skating competitions. Associated with Gateway Ice Center. Lists upcoming events, results, session schedule, photos, and club information.
Island Water Park
Water slide park. Includes ticket information, map of the park, photographs and events calendar.
Mazzei Flying Service
Flight training. Details of facilities, financial aid, staff profiles, fleet, and student housing.
Riverside Golf Course
Public course lists green fees, club and pro shop information, photographs of the course, and driving directions.
San Joaquin Worthogs
Home brewing club. Includes recipes, glossary, upcoming events, and membership information.
Valley British Auto Club
Dedicated to the restoration, preservation and the driving enjoyment of all British vehicles. Club activities include monthly meetings, tours on the two lane roads of the Sierra Nevada Mountains, car shows and social events.
Last update:December 18, 2016 at 2:18:08 UTC
All Languages


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This is the ninth installment of our 12-part series breaking down the Cowboys roster. Today we look at outside linebacker.
Players: DeMarcus Ware (signed through 2015), Anthony Spencer (unrestricted free agent), Victor Butler (signed through 2012), Alex Albright (signed through 2013)
Top free agents: Mario Williams, Houston Texans; Anthony Spencer, Dallas Cowboys; Ahmad Brooks, San Francisco 49ers; Jarret Johnson, Baltimore Ravens; Manny Lawson, Cincinnati Bengals.
Top draft prospects: Courtney Upshaw, Alabama; Melvin Ingram, South Carolina; Chandler Jones, Syracuse; Nick Perry, USC; Ronnell Lewis, Oklahoma; Zach Brown, North Carolina.
2011 review: Ware put together another Pro Bowl season, ranking second in the NFL with 19.5 sacks. You can question how many of those sacks were game-changing plays, but there's no doubt that Ware is one of the most dominant pass-rushers of this era, if not NFL history. Spencer, on the other hand, had another average season. He was solid against the run, tying for the team lead with eight tackles for losses, but wasn't a difference-maker as a pass-rusher again (six sacks). Butler, who had three sacks in limited playing time, shows flashes as a pass rusher but apparently hasn't earned the coaches' trust to play a significant role.
Offseason review: Whether to use the franchise tag on Spencer might be the most important decision the Cowboys make this offseason. That would mean paying Spencer $8.8 million in 2012. That's too much for a non-impact player, but letting Spencer walk in free agency would create a big hole opposite Ware. Signing Spencer to a long-term contract would be rewarding mediocrity, a mistake the Cowboys would likely regret down the road, considering he's likely to be overpaid in a weak free agency crop of outside linebackers. Even if the Cowboys use the franchise tag on Spencer, they would be wise to use an early-round draft pick to find the long-term solution at this position. If they let Spencer go, this becomes arguably the Cowboys' biggest need in the draft.
Bryan Broaddus' Scout's Eye: Maybe the position that has the most question marks. Will the Cowboys use the franchise tag on Anthony Spencer, or will they use the 14th overall selection in the draft to fill the need, or will they do both? Regardless, the position opposite Ware needs to be addressed. Too many times, Ware was a one-man show this past season. When the Cowboys needed pressure in the passing game, it fell to Ware to provide it.
The decision to potentially franchise Spencer tells me a couple of things: that the coaches and the front office like what they have in Spencer and don't like the current group of outside linebackers on the current free agent market; and they have questions about those players available in the draft. The tag wouldn't surprise me one bit because I have always believed that coaches love the known more than the unknown. As fans you might not feel that Spencer is worth the tag, but it allows the Cowboys to buy time and go with a player that has had some productive moments.
I have always liked Victor Butler and have seen a productive player when given the chance to take snaps. Why not give him a shot? Like at inside linebacker, the depth at this position has been killed by the draft or the lack of it. Alex Albright made the 53-man roster last season, and was a nice player on special teams and did see snaps during the season in some packages. However, he was on the field when the defense gave up some big plays.
The team can best fix this position through the draft with Ingram or Upshaw AND by keeping Spencer in the mix. That'd allow the Cowboys an opportunity to get that corner, guard or safety that could also make a difference.
Need meter (0-5): 4


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Learning Center Search Results
Showing 1-3 of 3 results
Doing Research in Real Time: A Collaborative Approach
This video is adapted from a presentation given at the 2010 National Genealogical Society Conference entitled "Doing Research in Real Time-An Exhilarating Collaboration Experience!" Its purpose is to demonstrate that genealogical research can be conducted by forming a collaborative team that can work together online. By utilizing this type of approach, a small team of researchers can produce more artifacts and results in less time than a single researcher working alone. The Wiki Article/Handout link found above provides more detailed information about the process and tools that can be selected and utilized.
Find Your Ancestors at the BYU Family History Library
Brigham Young University has become known for its Christian higher education, sports teams, performing groups, and other program. There is one important trait that is often neglected: many people don't think of BYU as an excellent place to do genealogical research. This webinar will introduce you to the many collections and services available to genealogical researchers at the BYU Family History Library and in other departments of the Harold B. Lee Library.
Research in Iroquois County Illinois
Learn about the resources available at the Iroquois County Genealogical Society in Illinois.


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Printing & Distribution Services (PDS) is the digital print and direct mail marketing department located at the Hawkins Building on South Campus. We process over 1.5 million pieces of mail each year, to domestic, international and campus destinations. We have sent more than 1.4 million emails and offer creative services for .html design for social media communications. We provide a wide range of fulfillment services to the university community. PDS can also act as a liaison for your projects with Printing Services, Office of Publications and the Schine
Copy Center.
Tour our website to find helpful information or contact us directly at 443-2240 or email@example.com to allow PDS to provide cost effective solutions for your marketing and communication needs.
Printing & Distribution Services
1600 Jamesville Ave, Syracuse, NY 13244
Email: email@example.com


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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
I was hurt at work and had to have spinal surgery. I had 2 discs replaced. Two weeks after surgery I started hurting and had the feeling something was wrong. I had what felt like cords running down my neck and a bulge started running down my neck. I called the doctors office several times about it finally they sent me to an xray. They never even looked at me. They called me and said the discs were fine and that I still had a few weeks of healing before they wanted to see me. Since then they sent me for a mylogram. when I asked them if they could tell what was wrong they stated they did it to cover their end. The bulge is huge now 7 months later and shows up in pics and it hurts like hell it is now all through my right side. I can not function and do they things I used to do.
Can you please clarify your question?
I just want to know if there is anything I can do legally. I can not do the things i used to. I lost my route at frito lay. I suffer from headaches and my right side hurts bad every day. My neck frequently swells. My surgeon told me that he has no other ideas and left it at that? he does not seem to want to fix what has happened. I even told him there is not a minute that goes by that i do not regret having this surgery
Ethics / Professional Responsibility Lawyer
Medical malpractice cases arising from spinal surgery are particularly difficult to win for many reasons. One of them is that a certain percentage of back surgeries fail, even when performed flawlessly. That said, your outcome does not sound like a garden variety failed back surgery. Something more seems to be wrong and you need a second medical opinion. Don't ask the second opinion doctor if there was malpractice, that is liable to scare him away. Just get a diagnosis and a medical plan. Then see a medical malpractice lawyer. There are several good ones in your area and they offer free consultation. Don't bother trying to get our own records before you see a lawyer. If the lawyer is interested in the case, he or she will do that.
You would need to have a medical malpractice lawyer order the medical records and have a physician expert review. Search Avvo for a medical malpractice lawyer in your city to discuss.
Click on name or picture to see profile page.
Thank you for your answer. Can u tell me does it sound like I might have a case.
You should also consult a workers compensation lawyer even if you are now being paid benefits. Whether you have a malpractice claim depends upon whether you have suffered a serious injury as a result of delay in diagnosis and treatment. Has anyone recommended treatment? You need to focus on your health. Get a second opinion. You may require surgery.
I am not a workers compensation lawyer.
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Contact: email@example.com
The UF|IFAS School of Forest Resources & Conservation is proud to offer enriching spaces at the Austin Cary Forest for successful conferences, courses, meetings, and events. We strive to create an atmosphere conducive to learning, growth and collaboration and at the same time to provide visitors with a serene experience in the forest, emphasizing the importance of natural resources to our lives.
About the Austin Cary Facilities
About Austin Cary Forest
215 Dauer: No charge, not open for student use. Must be reserved by UF College of Liberal Arts and Sciences faculty.
Contact: Jesse Killinger (email@example.com)
219 Dauer: No charge, not open for student use.
Contact: Jesse Killinger (firstname.lastname@example.com)
About Dauer Hall Meeting Rooms.
About Emerson Alumni Hall.
Emerson Alumni Hall Rental Information.
The historic Florida Gymnasium provides the perfect location for any organization's event—whether academic, athletic, or even charitable in nature. Recent events held in the gym include the following: seminars, lectures, luncheons, exhibitions, orientation programs, award ceremonies, student group meetings, academic competitions, holiday parties, and benefit dinners; basketball, volleyball, and ping-pong tournaments. Click this link to see pictures of previous Florida Gym events.
Chairs, tables (rectangle or round), staging, audio/video equipment, and other items are available to rent, making the planning and preparation for your event seamless.
Reserve the Florida Gym for your organization's event today. Navigate to this web page (http://gym.hhp.ufl.edu), completely fill out the Facility Request Form, and submit the form. General rules, rental rates, and available rental items are also listed on the web page. Keep in mind that events are scheduled on a first come, first serve basis: the earlier you submit the form, the better your chance of securing the gym on your preferred date.
Florida Gym Measurements
Basketball courts: 2
Volleyball courts: 3
91 feet x 121 feet
Maximum persons allowed in gym: 595
Functional Area: ~11,000 sq. ft.
Auditorium-style seating: ~550
Cafeteria-style seating: ~550
Comfortable round table seating: ~300
If you have any questions or need further assistance, contact Michael Murphy at firstname.lastname@example.com or 352.294.1621.
About the Florida Museum of Natural History.
FLMNH Rental Information.
Contact: Vicki Tyson 392-9826, x2153 (email@example.com)
Harn Museum of Art Rental Information
Go to http://www.harn.ufl.edu/rentals/ to learn more about rental opportunities.
Conference Services' Event Coordination Website:
Contact Information
352-392-2161 x.16400
Reservation Request Forms:
On-Campus Event Spaces: http://www.housing.ufl.edu/conferenceservices/yearround/event/oncampus/
Infinity Hall Event Spaces: http://www.housing.ufl.edu/conferenceservices/yearround/event/infinityevent/
About the J. Wayne Reitz Union.
JWRU Event Services.
The Keene Faculty Center (103 Dauer Hall) rents for $100 for College of Liberal Arts and Sciences (CLAS) faculty and $200 for non-CLAS faculty. Student groups cannot use this room. No furniture can be moved. Cleanup is the renter's responsibility.
Contact: Jesse Killinger (firstname.lastname@example.com)
About Lake Wauburg.
Lake Wauburg Rental Information.
Must obtain approval from the Department of Student Activities and Involvement.
Contact: (352) 392-1671
This space may be rented after 5pm on weekdays.
There is no charge for usage.
Contact: Sherry Feagle, firstname.lastname@example.com
All Pugh Hall Meeting Rooms must be reserved by UF College of Liberal Arts and Sciences faculty. Not available for student use.
210 Pugh Hall: Conference room. Holds 30 people. No charge.
Contact: Jorge Quintana (email@example.com)
150 Pugh Hall: Small room with tables. Holds 15 people. No charge.
Contact: Jorge Quintana (firstname.lastname@example.com)
160 Pugh Hall: Small room with tables. Holds 15 people. No charge.
Contact: Jorge Quintana (firstname.lastname@example.com)
About the Stephen C. O'Connel Center.
O'Connell Center Reservation Information.
The Straughn IFAS Extension Professional Development Center aims to provide outstanding venue for workshops, trainings and meetings but is also available for other types of events. The center consists of a large, covered outdoor pavilion and a large ballroom which can seat up to 300 people or be divided with one or two air walls to create up to three separate meeting spaces. Each room includes state of the art, built-in audio visual equipment.
Facilty Web site
About the TREEO Center
Request for Use of TREEO Facility (.pdf)
About the University Athletic Association (UAA).
UF groups only. Any caterer may be used, with permission from UAA.
Holloway Touchdown Terrace
Rental: $2000, but additional charges may apply.
Contact: Matt Goss at 375-4683 x6028 firstname.lastname@example.com.
University Women's Club
Rental: $1000, but additional charges may apply.
Contact: Matt Goss at 375-4683 x6028 firstname.lastname@example.com.
Individuals/groups that reserve Room 100 (Previously Room 1A) are responsible for the following:
About George A. Smathers Libraries.
Contact: Smathers Library Administration, (firstname.lastname@example.com)
About the UF Performing Arts (UFPA).
UFPA Rental Information.
UF President's House Rental Information.
About Ustler Hall.
Contact: Donna Tuckey, email@example.com
About Whitney Marine Laboratory.
Whitney Lab Rental Information.


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What if we told you there was a way to obtain a divorce or separation without going to court? Can you imagine an alternative that focuses on the family first and allows you to customize your agreement? Would you believe that there is a process that may cost less than litigation (aka going to court), and enables both parties to express their interests in a non-abrasive, positive and respectful approach? The Collaborative Law Practice does this.
The Collaborative Process is ideal for partners who:
- are seeking an alternative to traditional, court-based approaches to divorce and separation.
- have children together and want to maintain the best possible family relationships, now and in the future.
- recognize that they're the best at making decisions about their family and finances, and not someone else.
- place a high value on taking personal responsibility for handling conflicts with integrity.
Get started
Talk with your partner about Collaborative Practice and share the information on this website. When you're ready, contact one of our professionals and discuss your situation.


text: If you're not familiar, Kuchen is a German coffee cake. It has a dense crust topped with nut filling and it's usually loaded with fruit. It's a hearty confection that improves on standing. I think it's best the day after it's made, but sometimes it's rare thing to have leftover Kuchen. There are several steps to this scratch-made recipe, but it's not much trouble to put together.


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Hotel EXTENDED STAY AMERICA FAYETTEV
Service & facilities
|Earliest check-in
Service & facilities
Hotel facilities
- Elevator
- Hotel-owned car park
- Safety deposit boxes at reception
Room facilities
- Internet access
In-house services
- Pets permitted
- Laundry service
Style of hotel and ambience
Property description-, at extended stay america guests find a friendly, home away from home whether for a day a week or a, month. Only 9 miles from fort bragg our rates are, a great value often lower than per diems. Those, who serve for us save with us each of our 120, spacious suites offers a fully equipped kitchen, free wifi and a flat screen tv with premium, channels with free grab and go breakfast and, laundry facilities onsite. Only 5 miles from, fayetteville and close to restaurants our hotel is, the convenient affordable choice for savvy, business and leisure travelers. Hotel requires a, valid credit card on file upon check in for, incidental charges or damages to guest rooms. If, no valid credit card or guest is paying cash a, cash room deposit in the amount of 100usd per stay, per room is collected. After satisfactory, inspection at check out room deposit will be, refunded. Refunds over 100usd will be refunded by, check within 14 days of departure. ...Visit, esa.Com-extendedperks to join the extended perks, rewards program. It is free to join and includes, exclusive offers from extended stay america and, hundreds of your favorite brands no points, required, ., -Year built 1997, -public area refurbished 2014, -guest rooms refurbished 2014, -total rooms 120, -number of floors 3
Hotel data
Trade fair/conference hotel, child-friendly hotel
Service times
Earliest check-in:
Accepted payment methods
- Diners Club
- Visa
- Eurocard/MasterCard
- American Express
- Japan Credit Bureau (JCB Intl.)
408 OWENS DRIVE, 28304 Fayetteville -
- 4.97 km
- 94.28 km