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Osteoporosis is a common skeletal disease characterized by low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. We previously demonstrated that Col1a1-SOX9 transgenic mice, in which SOX9 specifically expresses in osteoblasts driven by a 2.3kb Col1a1 promoter, display osteopenia during the early postnatal stage. In this study, to further analyze the osteopenia phenotype and especially the effect of the osteoblast-specific expression of SOX9 on bone mechanical properties, we performed bone geometry and mechanical property analysis of long bones from Col1a1-SOX9 transgenic mice (TG) and wild type littermates (WT) at different time points. Interestingly, after body weight adjustment, TG mice have similar whole-bone strength as WT mice, but exhibit significantly thinner cortical bone, lower elastic modulus, and higher moment of inertia. Thus, osteoblast-specific SOX9 expression results in altered bone structure and material properties. Furthermore, the expression levels of Pcna, Col1a1, Osteocalcin, and the Opg/Rankl ratio in TG mice are significantly lower until 4 months of age compared with WT mice, suggesting that TG mice have dysregulated bone homeostasis. Finally, bone marrow stromal cells (MSCs) isolated from TG mice display enhanced adipocyte differentiation and decreased osteoblast differentiation in vitro, suggesting that osteoblast-specific expression of SOX9 can lead to altered mesenchymal stem cell differentiation potentials. In conclusion, our study implies that SOX9 activity has to be tightly regulated in adult skeleton to ensure optimal bone quality.
Many factors, including genetic elements, influence the risk of osteoporosis. Among the large number of candidate genes for the regulation of bone mass and osteoporotic fracture, runt-domain transcription factor Runx2 plays an essential role in regulating osteoblast differentiation and function [1, 2]. Runx2 has been shown to regulate all the major genes expressed by osteoblasts in tissue culture [3]. Mouse genetic studies show that homozygous Runx2 null mice have a complete lack of all mineralized skeletal elements due to the absence of osteoblast differentiation [4, 5]. Transgenic Runx2 over-expressing mouse models reveal that Runx2 also regulates postnatal bone formation and bone remodeling [6-8]. Importantly, mutations in RUNX2 result in cleidocranial dysplasia (CCD), a dominantly inherited skeletal dysplasia [9-12]. Furthermore, increasing evidence suggests that subtle polymorphic variations in RUNX2 could also regulate bone mineral density (BMD) in the general population [13, 14]. Thus, RUNX2 itself has been proposed as one of the candidate genes for osteoporosis [15].
HMG-domain transcription factor Sox9 is a potent transcriptional activator of chondrocyte-specific genes such as Col2a1, Col11a1, and Aggrecan [16]. Sox9 is essential for successive stages of chondrocyte differentiation and cartilage formation as demonstrated by human and mouse genetic studies [17-19]. Interestingly, both Sox9 and Runx2 are expressed in osteo-chondroprogenitor cells, and our previous study demonstrated the dominance of SOX9 function over RUNX2 during skeletogenesis [20]. SOX9 and RUNX2 directly interact with each other via their respective DNA binding domains. SOX9 decreases RUNX2 binding to its target sequences and drastically inhibits RUNX2 transactivation of osteoblast-specific enhancers. Importantly, SOX9 expression diminishes when osteo-chondroprogenitor cells further differentiate into osteoblasts, and the significance of down-regulating SOX9 expression in osteoblasts remains speculative [21]. Interestingly, the transgenic mice—in which either the full-length or RUNX2-interacting domain of SOX9 specifically expresses in osteoblasts driven by a 2.3kb Col1a1 promoter—display dwarfism and osteopenia at 6 weeks of age, with significantly reduced bone volume with a decreased number of osteoblasts and bone formation rate, defective osteoblast function and mineralization, and down-regulation of osteoblast differentiation markers [20]. However, the effect of SOX9 expression in osteoblasts on bone mechanical properties remains unknown.
Bone marrow is constituted of two separate and distinct stem cell populations: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs, also termed marrow stromal cells) [22, 23]. While mesenchymal stem cells can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts as determined by different cell-type specific transcription factors [24-28], recent report shows that human perivascular cells give rise to adherent, multi-lineage progenitor cells that exhibit MSC features [29]. However, the direct effects of osteoblasts on MSC differentiation remain poorly understood. Interestingly, there appears to be antagonism between osteoblastic versus adipogenic cell lineage differentiation in MSCs. Indeed, besides being a transcriptional activator essential for osteoblast differentiation and chondrocyte maturation, Runx2 is also a repressor of adipocyte differentiation. Runx2 deficient chondrocytes and osteoblasts can differentiate into adipocytes at increased frequency [30] [31].
In this study, to further analyze the osteopenia phenotype, especially at the adult stage in Col1a1-SOX9 transgenic mice, we performed a detailed mechanical property analysis of adult long bone from Col1a1-SOX9 transgenic mice (TG) and wild type littermates (WT). To explore the underlying molecular mechanisms of SOX9 function in regulating bone mechanical properties, we evaluated the expressions of bone-related markers in TG and WT adult long bones at different time points. Finally, to investigate the potential effect of SOX9-expressing osteoblasts on MSC differentiation, we isolated MSCs and analyzed their differentiation potential in Col1a1-SOX9 transgenic mice.
Col1a1-SOX9 FL or Col1a1-SOX9 NT transgenic mice were generated and genotyped as previously reported and maintained on a FVB/N background (Fig. 1A) [20]. All mice were maintained and housed at the Case Western Reserve University animal facility under standard conditions. All animal protocols have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University. At ages 1, 2, 4, and 6 months, age- and sex-matched transgenic mice and wild type littermates were sacrificed for mechanical tests (see Supplemental Table 1 for the numbers of mice used) and molecular analysis (additional 3–6 mice per genotype per time point). The whole skeletons and the dissected hindlimbs were analyzed by contact radiography taken at the same time for transgenic and wild-type littermates by a radiographic inspection unit (Faxitron, McMinnville, Oregon).
Femurs were dissected and cleaned of soft tissue, wrapped in saline soaked gauze, sealed in airtight containers, and stored at -20°C until mechanical testing. The left femurs were tested in three-point bending; the right femurs were used for geometric analysis (as below). Specimens were thawed in saline and placed posterior side down on the bottom pins of a bending fixture (5-mm span between lower pins, 0.75-mm diameter pins) within a materials testing device (Electroforce 3400, Bose, Eden Prairie, MN, USA). The upper pin was aligned and loading was applied at 0.1 mm/sec. The stiffness of the specimen, K, was determined by fitting a linear regression line to the force-displacement curve. Yield strength was evaluated as the point on the force displacement curve where the secant stiffness declined by 10% from the original stiffness [32]. The bending strength was evaluated as the maximum moment applied to the specimen (ultimate load * span length/4; note that maximum moment is directly proportional to ultimate load but accounts for the span length to allow comparisons to other studies).
The right femurs were embedded undecalcificied in methyl methyacrylate, and mid-diaphyseal cross-sections were cut using a low speed diamond saw. The cross sections were hand polished, mounted on glass slides, and observed at 40X magnification. Digital images of the cross-sections under UV illumination were collected to visualize the entire bone cross-section (4.6 micrometers/pixel). The periosteal and endosteal diameters in the anterior-posterior (minor) axis and medial-lateral (major) axis were determined by manual measurement from the image using ImageJ (http://rsbweb.nih.gov/ij/). Average thickness of the cross section was calculated directly using a thresholded image and a distance transform method. The second moment of inertia, I, was determined from the thresholded image using custom Matlab software. The elastic modulus, E, of the bone tissue was estimated using the following equation from beam theory:
where L is the span length (5mm), I is the second moment of inertia, and K is the stiffness obtained from the force-displacement curve.
Using TRIzol reagent (Invitrogen) and Purelink Micro-To-Midi RNA Kit (Invitrogen), total RNA was extracted from fresh femur samples at different time points (n=3-6/group/time point). The epiphyses at both ends were cut off and the bone marrows were flushed out with PBS as described [33]. cDNAs were prepared from 1μg total RNA with iScript cDNA Synthesis Kit (Bio-Rad). The Power SYBR Green PCR Master Mix (Applied Biosystems) and the 7500 Real Time PCR system (Applied Biosystems) were used for real-time RT-PCR with gene-specific primers listed in Table 1. The Gapdh gene was used as an internal control of the quantity and quality of the cDNAs. The relative gene expression levels were quantified in triplicate and calculated by the 2-ΔΔCT method for fold induction as described previously [34]. For each gene at each time point, WT values were arbitrarily designated as 1 and gene expression levels in TG were compared with WT samples to calculate the relative-fold changes of gene expression at each time points. A single peak was observed for all qPCR primers used in this study. The slopes of the qPCR reactions were between −3.16 and −3.40, indicating efficient amplification.
For bone marrow stromal cells (MSCs) isolation, mice were sacrificed at 2 months of age. Bone marrows were immediately flushed out with cold PBS from the femurs and tibias, followed by centrifugation at 400 × g for 10 min and cell counting. MSCs were then re-suspended to a final concentration of 5 × 106 cells/ml in DMEM medium (HyClone, Utah, USA) with 10% FBS (Invitrogen, USA) as described [35, 36]. MSCs were cultured in a humidified 5% CO2 incubator at 37°C for 72 hours before non-adherent cells were removed by changing the medium. MSCs were then cultured in 6-cm dishes at 1 × 107 cells/dish for 10 days. After washing with PBS twice, dishes were stained with toluidine blue for fibroblast colony-forming unit (CFU-F).
At 50-70% confluence, MSCs were further passaged for 2-3 times before differentiation induction as described [35, 36]. For adipocyte differentiation, after cells reached confluence, adipogenesis was stimulated by three induction/maintenance cycles. Each cycle consisted of 3-day culturing of MSCs with adipogenesis induction DMEM medium, which contains 10% FBS, 10 μg/ml ITS (Insulin-Transferrin-Sodium Selenite Supplement, Roche, Germany), 0.2 mM Indomethacine, and 1 μM Dexamethasone. Cells were then cultured for 3-4 days in adipogenesis maintenance DMEM medium, which contains 10% FBS and 1 μM Dexamethasone. Cells were then fixed, washed with PBS, and stained with 0.5% Oil red O (Sigma) in isopropyl alcohol for 1 h, followed by PBS washing. Osteogenesis differentiation was induced by culturing MSCs for up to 4 weeks in DMEM containing 10% FBS, 50 mg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10-8 M Dexamethasone. To observe calcium deposition, cells were washed once with PBS and stained with alizarin red S solution for 5 min at room temperature.
For histological analysis, tibiae were collected from 8-week-old Col1a1-SOX9 transgenic mice and wild-type littermates, decalcified and then embedded in paraffin, followed by hematoxylin and eosin staining. For GFP transgenic MSC analysis, MSCs were collected as described above from 8-week-old osteoblast-specific 2.3 Col1a1-GFP transgenics (a kind gift from Dr. David Rowe) [37], CAG-EGFP transgenics that ubiquitously express GFP (The Jackson Laboratory # 006053), and wild-type littermates. After 12-day culture, MSCs were subjected to fluorescence microscopy analysis as described [37].
Results were given as mean ± standard deviations (SD). Statistical analysis was performed by Student's t-test to compare the differences in gene expression between age-matched Col1a1-SOX9 transgenic mice and wild-type littermates (n=3-6). P<0.05 was considered statistically significant. The linear correlations between geometry, mechanical property data, and body weight were calculated by Microsoft Excel. To compare the difference in linear correlation, statistical analysis was performed by ANCOVA test with XLSTAT 2009 to detect differences in stiffness and strength after accounting for body weight. The ANCOVA was implemented using a generalized least squares model with body weight as a covariate [38]. P<0.05 was considered statistically significant.
Whole body X-ray analysis confirmed the dwarfism phenotype in 2-month-old Col1a1-SOX9 full length (SOX9 FL) (Fig. 1B) and Col1a1-SOX9 N-terminal (SOX9 NT) (Fig. 1C) transgenic mice [20]. X-ray photography of dissected femurs showed that both the length and the bone mineralization were reduced in Col1a1-SOX9 FL transgenic mice at age 1 month and 2 months (Fig. 1D). Col1a1-SOX9 FL transgenic mice had more translucent and wider femurs until 6-months of age, suggesting compromised bone mineralization and altered bone geometry (Fig. 1D). The body weight was also significantly lower (Fig. 1E), and the femur length was significantly shorter until age 4 months in Col1a1-SOX9 FL transgenic mice (TG) compared with wild-type littermates (WT) (Fig. 1F). The differences in the femur length between TG and WT appeared to be much reduced at later time points. Indeed, at age 6 months, TG mice had femur lengths similar to those of WT mice (Fig. 1F). There were similar linear correlations between femur length and body weight in WT and TG mice (WT vs TG, p=0.854) (Fig. 1G). Thus, for the same body weight, there was no significant difference in the femur length between WT and TG mice.
At 1 month of age, Col1a1-SOX9 FL transgenic mice (TG) exhibited severely decreased cortical bone thickness compared with wild-type littermates (WT) (Fig. 2A). Although the cortical bone thickness increased in TG in 2-month- and 4-month-olds, it was still lower than that of WT (Fig. 2A). Additionally, both the femoral periosteal diameter and the endosteal diameter of TG mice were wider than those of WT littermates at all time points (Fig. 2B and 2D). The average cortical bone thickness of TG mice was also significantly lower than that of WT littermates until 4 months of age (Fig. 2F). Importantly, after body weight adjustment, the difference in periosteal diameter, endosteal diameter, and average cortical bone thickness was even more dramatic. There were significant differences in the linear correlations between WT and TG mice (p<0.01) (Fig. 2C, 2E, and 2G). Thus, for the same body weight, TG mice had wider and thinner cortical bone than WT mice.
To evaluate the effect of the osteoblast-specific expression of SOX9 on bone mechanical strength, we performed a standard three-point bending assay. Stiffness, yield moment, and maximum moment were all significantly decreased in SOX9 FL transgenic (TG) mice compared with those in wild-type (WT) littermates at all time points we tested (Fig. 3A, 3B, and 3C). However, with body weight adjustment, there was no significant difference in maximum moment between WT and TG mice (Fig. 3D). Thus, for the same body weight, transgenic and wild-type mice had similar whole-bone strength.
In both TG and WT mice, there was a linear correlation between the elastic modulus and the body weight (Fig. 3F). However, the elastic modulus was significantly lower in TG than in WT mice at all time points we tested (Fig. 3E). Importantly, for the same body weight, TG mice appear to have more compliant bone tissue material with significantly lower elastic modulus than WT mice (P<0.01) (Fig. 3F). Interestingly, transgenic mice had a moment of inertia similar to that of wild type littermates at the same age (Fig. 3G). There was also a linear correlation between the moment of inertia and the body weight in both TG and WT mice (Fig. 3H). In contrast with the elastic modulus, the slope of the moment of inertia was significantly higher for TG than for WT after body weight adjustment (p<0.01). Thus, femurs from TG mice showed wider bone than WT mice at the same body weight, consistent with the geometry data which showed that TG mice have wider and thinner cortical bone (Fig. 2C, 2E, and 2G). Together, our data show that despite their more compliant bone tissue, at the same body weight, Col1a1-SOX9 FL transgenic mice appear to achieve whole-bone biomechanical performance similar to that of WT by compensating with altered bone geometry, namely an increased second moment of inertia through wider and thinner cortices.
We have previously shown that—similar to Col1a1-SOX9 full length (SOX9 FL), which contains a full-length SOX9 construct—another transgenic mouse line, Col1a1-SOX9 N-terminal (SOX9 NT), which harbors only the N-terminal RUNX2-interacting domain of SOX9, also exhibits osteopenia with defective osteoblast function (Fig. 1C and [20]). To confirm our findings in Col1a1-SOX9 FL transgenic mice and to investigate whether the bone biomechanical changes in mutant mice depend on the RUNX2-interacting domain of SOX9, we performed the same mechanical test in the Col1a1-SOX9 NT transgenic mouse line (Fig. 4A). The bone parameter data in Col1a1-SOX9 NT and wild-type littermates at 2 months of age—including the body weight, femur length, stiffness, yield moment, maximum moment, moment of inertia, and elastic modulus—were consistent with those in the Col1a1-SOX9 FL line (Fig. 4B-4H). Hence, Col1a1-SOX9 NT transgenic mice had a similar biomechanical performance as Col1a1-SOX9 FL transgenic mice. This suggests that the SOX9 effect on bone material and geometry is likely due to its RUNX2-interacting N-terminal that harbors the HMG DNA-binding domain of SOX9.
To uncover the underlying molecular mechanisms for the altered bone mechanical properties in Col1a1-SOX9 FL transgenic mice (TG), we extracted RNAs from femurs and performed quantitative real-time RT-PCR analysis for osteoblast proliferation and function. We first confirmed the transgene expression in Col1a1-SOX9 FL transgenic mice at all time points (data not shown). To evaluate osteoblast proliferation status, we quantified the expression level of Pcna (proliferating cell nuclear antigen), a proliferation maker. Compared with wild-type femurs, Pcna expression in TG femurs was significantly lower until 4 months of age but significantly higher at 6 months of age (Table 2). This suggests that osteoblasts in TG mice might be deficient in proliferation at an early stage but compensate by over-proliferating at a later stage. Type I collagen is the most abundant extracellular protein in bone, essential for maintaining skeletal strength. In TG mice, Col1a1 expression was significantly lower at 1, 2, and 4 months of age but was significantly higher at 6 months of age than WT (Table 2). Osteocalcin is a non-collagenous protein, secreted specifically by osteoblasts, critical for bone mineralization and calcium ion homeostasis. Interestingly, the expression dynamics of Osteocalcin were similar to those of Col1a1 in TG mice. Osteocalcin expression level was significantly lower at 1 and 2 months of age, increasing to similar levels at age of 4 months, and significantly higher at age 6 months in TG than in WT (Table 2). Thus, the expression profiles of Pcna, Col1a1, and Osteocalcin suggest that Col1a1-SOX9 FL transgenic mice might have delayed proliferation and deregulated bone mineralization.
Osteoblasts and osteoclasts are intimately coupled during bone formation and bone remodeling. To assess the effect of osteoblast-specific SOX9 expression on adult bone remodeling, we quantified the bone metabolism markers Opg and Rankl by real-time RT-PCR. Opg is a major negative regulator of bone resorption as an osteoclastogenesis inhibitory factor, while Rankl is a key stimulator of bone resorption. The Opg/Rankl ratio is commonly used as a prognostic indicator of bone remodeling [39]. The Opg/Rankl ratio was significantly lower at age 1 month in TG than in WT. But afterwards, the Opg/Rankl ratio kept on increasing in TG mice and reached a level similar to that of WT littermates at 2 and 4 months of age. Eventually, the Opg/Rankl ratio was significantly higher at 6 months of age in TG compared with WT. The Opg/Rankl ratio changes hint at the dysregulated bone homeostasis in Col1a1-SOX9 FL transgenic mice. Thus, TG mice might have delayed bone formation and active bone resorption at an early postnatal stage, but switch to increased bone formation and decreased bone resorption at later stages. These osteoblast-related gene expression profiles correlate well with the bone mechanical and geometry data, such as the lower elastic modulus and higher moment of inertia in Col1a1-SOX9 FL transgenic mice compared with WT mice.
In bone marrow, osteoblasts might play important roles in regulating mesenchymal stem cell (MSC, also termed marrow stromal cell) maintenance and differentiation, probably as part of the MSC niche. To analyze the MSC function in Col1a1-SOX9 FL transgenic mice, we first performed the in vitro colony formation assay of MSCs derived from Col1a1-SOX9 FL transgenic mice and wild type littermate controls. There was no significant difference in the total colony numbers (CFU-F) (Fig. 5A), suggesting no significant change in MSC numbers in the bone marrow of TG mice.
To investigate the effect of osteoblast-specific SOX9 expression on MSC differentiation capability, we performed in vitro adipogenesis and osteogenesis induction assays. Interestingly, for both wild type and transgenic mice, MSCs from males exhibited higher osteogenic activity than those of females by alizarin red staining (Fig. 5B). This suggests a gender difference in murine MSCs differentiation potential, which correlates with a previous report showing that mouse skeletal muscle-derived stem cells from males exhibited higher osteogenic activity than those derived from females [40]. Compared with WT, for both male and female, Col1a1-SOX9 FL MSCs appeared to exhibit modestly decreased osteogenic differentiation (Fig. 5B). However, MSCs from Col1a1-SOX9 FL mice displayed drastically enhanced adipocyte differentiation as assayed by oil red O staining (Fig. 5C). In addition, histological analysis revealed significantly increased numbers of adipocytes in the bone marrow of Col1a1-SOX9 transgenic mice compared with WT (Fig. 5D and 5E). Thus, ectopic expression of SOX9 in osteoblasts seems to tip the MSC differentiation potential towards adipogenesis. Importantly, 2.3Col1a1 promoter is a well-characterized promoter that targets only well-differentiated osteoblasts, not BMSCs [41] [37]. Consistent with a previous report that 2.3 Col1a1-GFP transgene was inactive in human BMSCs [42], our 2.3 Col1a1-GFP transgenic MSC culture experiment indicates that 2.3 Col1a1-targeted osteoblastic lineage cells were not a major direct contributor to the MSC population in cell culture (Fig. 6A), suggesting that the changes in ex vivo differentiation potential in MSCs derived from Col1a1-SOX9 transgenic mice is unlikely due to the direct expression of SOX9 transgene itself in MSCs.
Bone is a living tissue capable of self-repair, with complex mechanical properties. Bone strength is determined by structural and material properties. Structural properties include features such as bone size, bone geometry, and bone microstructure. Material properties include features such as bone mineral density, chemical composition, and the size of hydroxyapatite crystals [43]. Our previous studies revealed that osteoblasts from Col1a1-SOX9 FL transgenic mice were less active than those from wild-type mice. Bone histomorphometric analysis showed that osteoblast numbers and bone formation rates were significantly reduced in transgenic mice [20]. In the current study, we have investigated the mechanical properties of long bone in Col1a1-SOX9 FL transgenic mice. Interestingly, Col1a1-SOX9 FL transgenic mice exhibit a lower elastic modulus and higher moment of inertia than WT after body weight adjustment. This indicates that for the same body weight, TG mice have wider bone diameters than WT mice to compensate for the decreased elastic modulus. The average cortical bone thickness of SOX9 FL transgenic mice is also significantly lower than that of WT mice until 4 months of age, even after the body weight adjustment (Fig. 2F and 2G). Thus, these results collectively demonstrate the altered bone structure and bone material in TG mice (Fig. 6B).
In previous work, we uncovered the down-regulation of the osteoblast markers Col1a1 and Osteocalcin in Col1a1-SOX9 transgenic mice at both the newborn and 6-week-old time points [20]. In our present work, we further evaluate the expression of various bone-related markers in Col1a1-SOX9 FL transgenic mice to explore the underlying molecular mechanisms of SOX9 in regulating bone mechanical properties. Compared with WT, proliferation marker Pcna expression in TG is significantly lower until age 4 months but increasing to a significantly higher level at age 6 months, indicating delayed bone growth in TG. Col1a1, Osteocalcin, and the Opg/Rankl ratio all display dynamic expression profiles similar to those of Pcna, suggesting delayed bone mineralization and deregulated bone remodeling. These molecular changes are consistent with the inhibitory effect of Sox9 on Runx2 in adult transgenic mice [20].
Genetic factors play an important role in the pathogenesis of osteoporosis [15]. Among them, Runx2 is a master transcription factor essential for osteoblast differentiation and bone remodeling. The Runx2 gene produces two major transcripts driven by alternative promoters, designated P1 and P2, respectively. Interestingly, Runx2-II deficient mice, which have P1 promoter and exon 1 deleted, exhibited a low-turnover osteopenia phenotype similar to Col1a1-SOX9 FL transgenic mice [44, 45]. Runx2-II deficient mice displayed significantly reduced trabecular bone volume, decreased cortical thickness, and decreased bone mineral density. They also exhibited down-regulated osteoblastic and osteoclastic marker expression, decreased bone formation, significant reductions in bone mechanical properties, and impaired MSC differentiation in vitro. Our current study demonstrates that Col1a1-SOX9 FL transgenic mice also exhibit a significant reduction in cortical bone thickness, stiffness, yield moment, and maximum moment, as well as altered MSC differentiation (Fig. 6B). Our previous study demonstrated that SOX9 decreases RUNX2 binding to its target sequences and drastically inhibits RUNX2 transactivation of osteoblast-specific enhancers, indicating the dominance of SOX9 function over RUNX2 during skeletogenesis [20]. Another recent study further demonstrated that Sox9 also directly induced Runx2 protein degradation [46]. Interestingly, the phenotypes in Col1a1-SOX9 mice were also very similar to those observed in Col1a1-Sox8 mice, in which the expression of a highly related HMG transcription factor Sox8 was driven by the same Col1a1 promoter element [47]. Cola1-Sox8 mice displayed impaired bone-formation characterization by low bone mass, defective osteoblast function, and increased spontaneous fracture occurrence. Furthermore, Col1a1-Sox8 also had decreased expression of Runx2 downstream targets such as Col1a1 and Osteocalcin in osteoblasts [47]. Collectively, these three different mouse models show that compromised Runx2 activity leads to osteopenia with altered bone mechanical properties and likely leads to altered MSC differentiation potential. Thus, tight regulation of Runx2 function is not only critical for early embryogenesis, it is also essential for proper adult bone remodeling.
The osteoblast is part of the well-established HSC niche due to its function in maintaining a quiescent HSC microenvironment in bone marrow. However, the effect of osteoblasts on MSCs residing in bone marrow remains poorly understood. Recently, a new factor was reported as regulating stem cell fate: the elasticity of the matrix microenvironment, as a rigid matrix stimulates osteogenic differentiation with elevated Osteocalcin expression [48]. Another report indicates that MSCs respond to ECM rigidity in bone through an integrin-ROCK-FAK-ERK1/2 axis during osteogenic differentiation [49]. However, the mechanism of osteoblast impact on MSC differentiation is still largely unclear. In our current study, although in vitro CFU-F assay showed no significant difference in MSC numbers, MSCs from Col1a1-SOX9 FL transgenic mice exhibited modestly decreased osteogenic potential compared with WT mice. The decreased osteogenesis potential correlated with the decreased elastic modulus in TG mice. Col1a1-SOX9 FL MSCs also displayed enhanced adipocyte differentiation in vitro. There were also more adipocytes in the bone marrow of TG mice compared with WT mice. Our results correlate with previous reports that MSCs derived from osteoporotic patients exhibited important functional differences compared to MSCs obtained from control donors: decreased osteoblast differentiation, less Type I collagen synthesis and increased adipocyte differentiation [50, 51]. The underlying molecular mechanism for the changes in MSC differentiation in Col1a1-SOX9 transgenic mice is still unclear and is likely to be complex. It may reflect the above-mentioned effect of the elasticity of the matrix microenvironment on MSCs, the direct interaction between osteoblasts and MSCs, or the effect of some osteoblast-secreted paracrine factor(s) on MSCs. These possibilities don't necessarily exclude each other.
Although cell lineage analysis revealed that Sox9-expressing progenitor cells contribute not only to chondrocytes but also to osteoblasts during embryogenesis, Sox9 expression is extinguished during osteoblast differentiation [21]. However, the importance of down-regulating Sox9 expression in osteoblasts is still poorly understood. Interestingly, Sox9 was identified to be associated with Col1a1 promoter in a recent chromatin immunoprecipitation (ChIP)-on-chip study, raising the intriguing possibility that Sox9 can directly inhibit Col1a1 expression in vivo [52]. Additionally, the loss of Sox9 in differentiated chondrocytes resulted in eptopic Col1a1 and Runx2 expression [53]. Thus, Sox9 is likely to repress Col1a1 and Runx2 in differentiated chondrocytes at the transcriptional level. Our current study reveals the adverse effect of mis-expressing Sox9 in osteoblasts during mouse postnatal skeletal development and underscores the importance of inhibiting Sox9 activity in osteoblastic cell linage. Thus, it is important to identify the negative regulators of Sox9 in osteo-chondroprogenitor cells and osteoblasts. In summary, our study shows that tight regulation of SOX9 activity may be critical for maintaining the material and structural integrity of adult bone. A better understanding of SOX9 function in adult skeleton might lead to improved diagnosis of and therapeutic options for osteoporosis. Furthermore, SOX9 mis-expression has been associated with various pathological conditions including fibrosis and cancers [54]. Thus, our study may also shed new light for understanding the mechanism of SOX9-associated disease.
This work is supported by the NIH grant DE15139 and Case start-up fund (G. Zhou) and the fellowship support from the Department of Anatomy, Case Western Reserve University (M. M. Cotter). We thank Chiderah Okoye and Justin Daggett for assistance with image analysis, and Teresa Pizzuto for expert histology work, Dr. David Rowe for 2.3 Col1a1-GFP mice. We also thank Valerie Schmedlen for editorial assistance and Dr. Shunichi Murakami for critical reading of the manuscript.
The authors have stated that they have no conflict of interest.
Disclosures None.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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8 comments:
- Evdokia Tsiligiridou said...
What a lovely green company :)
Wish you will feel better soon!
- 5.4.16
- cococita said...
Thinking of you, dear Claudia!
The first few months of year aren't treating you well :(
Like your living room jungle a lot: luckily it brings some natural beauty inside ...
Take good care: may the fever disappear soon so that you can enjoy the season and life in a more proper way again!
- 5.4.16
- Mi Joya said...
Hope you'll feel better soon!
And i love your jungle!!!!!!!
- 5.4.16
- jabbott said...
Get better soon x
- 5.4.16
- Sandra Dunn said...
Sending peaceful healing prayers, dear Claudia.
Your jungle is amazing! I have a cactus farm like this. lol
- 7.4.16
- wide-eyed-tree said...
@Evdokia: Thank you!
@Cococita: no, it's definitely not the best year so far. Thank you! It is a tenacious virus...
@Mi Joya: Thank you!!
@jabbott: Thank you!!
@Sandra: Thank you! Cactuses are amazing! My mum has a little cactus farm also ;)
- 11.4.16
- coco said...
I have been enjoying in the garden lately and
repotted sacculents and herbs for them to have
plenty of space to grow!
Your plants looks happy as always ❤
- 23.4.16
- emmel-prutsemieke said...
wow your pileas are great!!
- 4.5.16

subset: commoncrawl


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News from IFTD - Day Two of the Fly Fishing Show
This is a dedicated thread for discussing article: News from IFTD - Day Two of the Fly Fishing Show
Best of Show award winners are listed 'hot off the press' and there is a bunch more photos from today.
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Walton Park Damaged
(Walton) The city of Walton is offering a reward for information. It wants to find the person or persons responsible for damaging the city's park.
It happened sometime between Tuesday and Wednesday at the park on Stephenson Mill Road. Police found spray painted graffiti on the playground equipment, bathroom, picnic tables and shelter areas.
Anyone with information is asked to contact Boone County investigators.


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Smartphones and tablets make it possible to receive and even interact with information on the goer. With Iteris' mobile apps for Android® and iOS®, you can bring all the information you need to make crucial decisions into a single tool.
Traveler Information
Iteris' ATIS/511 and ICM apps provide travelers with critical information and timely updates in an easy-to-use map representation using icons and road highlighting to show:
- Traffic and road conditions
- Construction projects
- Planned events
- Camera video and images
- Incidents
- Weather information
Safety is always the first priority. Using your mobile device's built-in text-to-speech and GPS, a hands-free option allows the app to "read" a report to inform a traveler of what is ahead of them. Also, radar overlays can be added to the map giving travelers an expectation of what weather may be between them and their destination.
Customizing your app
Each customer has different needs. Customizations include:
- Twitter and social media feeds
- Integrated, personalized "push" notifications
- Trip planner (Virginia's "Reach the Beach" and Georgia's "Trip Calculator")
- Transit information
Integrated Corridor Management (ICM)
Smartphone applications developed as part of Integrated Corridor Management (ICM) projects. The technology provides an actionable service to the region's commuters, complementing existing 511 information in a new, timely and easy to understand manner.
The information provided includes:
- Travel times on freeway segments
- Color-coded maps indicating nearby traffic conditions
- Congestion levels
- En-route commuters receive location-based advisories of upcoming travel disruptions such as construction zones and incidents.
- Access to roadway camera images
- Tolling information
Travel time and cost information on alternative routes for transit and driving enables travelers to select optimum route and mode based on real-time conditions.
Pre-trip functionality allow travelers to view transit maps, fares and arrival times for transit routes in the region.
ICM information allows travelers to select their departure time and route based on predicted conditions.
Visit the San Diego 511 ICM for Android or iOS at the links below:
Smart Work Zones
The iPeMS® Work Zone application provides a turnkey, state-of-the-art construction work zone monitoring and analytics capabilities by creating a temporary virtual traffic management center for a specific project. Iteris solutions for smart work zones combine cloud-based software, field deployable Bluetooth units, mobile devices, and interfaces to dynamic message signs. Smart work zones increase traveler satisfaction and safety while reducing the costs of managing a work zone.
Specific Services
- Performance measurement
- Traveler information
- Virtual guidance


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Meet Dr. Raymond Thal
Dr. Raymond Thal is a board certified, fellowship-trained orthopedic surgeon in Reston, Virginia who specializes in the treatment of disorders of the knee, elbow and shoulder. He is a leader in the field of arthroscopic surgery of the shoulder, knee, and elbow and has authored numerous scientific articles and chapters.
Dr. Thal has been awarded more than 15 United States patents and 16 international patents for devices and instruments he has invented for arthroscopic procedures through his research efforts. He has received several invites from the international medical community to demonstrate and discuss these innovations and has been a guest speaker and surgeon throughout the United States, Europe, Asia, and Australia.
Dr. Raymond Thal is part of the team that makes up Town Center Orthopaedic Associates, P.C.
Watch Dr. Thal's Video Below:
Directions to our primary location in
How Dr. Raymond Thal Can Help You
- Orthopedic Surgeon
- Arthritis
- Elbow Disorders
- Elbow Injuries
- Elbow Pain
- Knee Injuries
- Knee Pain
- Rotator Cuff Injuries
- Shoulder Disorders
- Shoulder Injuries
- Shoulder Pain
- Arthritis Management
- Arthroscopic Surgery of the Elbow
- Arthroscopic Surgery of the Knee
- Arthroscopic Surgery of the Shoulder
- Knee Ligament Reconstruction
- Minimally Invasive Surgery
- Rotator Cuff Repair
What Patients are Saying About Dr. Raymond Thal
Just a note to say thank you so much for fixing my shoulder! I am back to full strength with golf, and more importantly, I can sleep! Now, tennis, is another story...might be back one day for my knees!— M.W.
J and I would like to thank you again for the successful surgery on our son's shoulder. He played the infield on a summer adult league and then 2nd base in the fall. This spring came along and he worked his way back out of center field! Thanks again.— J. and C. Family
It has been a year since I left your care. The knee that you repaired is doing great. I now run 5 miles 3 times a week, and 10+ miles once a week. I have no pain. I never believed I'd be able to run like this again. So, thank you for your care, your dedication to your work, and the healing your brought to me.— J.
Thank you again for all your help. I surely would not be doing what I honestly love to do if it wasn't for you. You have been a prominent figure in my life and words can not begin to explain how grateful I am.— R.A.
Thanks for making me feel comfortable. Your confidence in a successful outcome helped me through this operation. I am about as pain free as it gets.— T.F.
Accolades for Dr. Raymond Thal
Professional Distinctions
- Fellowship, Sports Medicine and Arthroscopy - Orthopaedic Research of Virginia, Richmond, VA
- Voted a "Top Doctor" by Washingtonian Magazine, 2005 - present
Professional Affiliations
- American Orthopaedic Society for Sports Medicine
- International Society of Arthroscopy, Knee Surgery and Orthpaedic Sports Medicine
Board Certifications
- Board Certified, American Board of Orthopedic Surgery
- Subspecialty Certification in Orthopaedic Sports Medicine
Office Information for Dr. Raymond Thal
Primary Location:
1860 Town Center Drive
Suite 300
Reston, VA 20190
Monday: 08:00 - 05:00
Tuesday: 08:00 - 05:00
Wednesday: 08:00 - 05:00
Thursday: 08:00 - 05:00
Friday: 08:00 - 04:00
Insurance Accepted
- Aetna
- Anthem PPO
- Blue Cross/Blue Shield
- Carefirst PPO
- Cigna
- MDIPA & Optimum Choice
- Medicare
- Tricare Standard
- United Healthcare
- Worker's Compensation
Additional Locations
6201 Centreville Road
Suite 600
Centreville, VA 20121
Driving directions
Profile last updated: August 30, 2016. This information was supplied by or on behalf of the provider.


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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Checkbox, how to control the horizontal spacingAsked by natupote on June 30, 2016 at 08:13 AM
how to control the horizontal spacing?
JotForm Support
I'm checking your form and I see that you have two forms that are currently being updated. I also noticed that you seem to be experimenting on different check boxes and radio buttons, you are also using some widgets for that.
The most effective way to redesign the spacing of the check boxes is by using the Form Designer Tool. You can click the check box and adjust the input width of it, that should also automatically adjust the spacing of your items:
Another way is to use custom CSS code, that should allow you to specifically design each elements on your form, but that means you will need to be familiar with CSS coding.
If you need more help, let us know the specific form and the specific field, but you will need to finalize or at least make the form almost complete for us to give you a custom CSS code.
I hope that helps.
thanks for help!


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"Total hip replacement"
Posted by John Williams
A highly professional surgical team at the top of their game supported by skilled and empathetic nursing and physiotherapy staff. Catering and reception staff are excellent. An example of how a small hospitall should be operated. A great success personally for "Choose and Book"
Do you have a similar story to tell? Tell your story & make a difference ››
Response from Renacres Hospital nearly 2 years ago
I am pleased to hear that we are your hospital of choice. Thank you! Thank you so much for your lovely comments. I am pleased you had a wonderful experience with our team.


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Attention passengers! This is your captain speaking. Your favourite comparison clinician is back for another sizzling session of monetary Q&A.
It's peak holiday season, when people in their droves will be heading to the airports for a bit of sun, sea and maybe even a bit of sangria – well, it'd be rude not to.
So, sit back, grab yourself a little aperitif from the minibar and relax with this month's round up.
Kindest Doctor Moore-Money,
I've been married to my doting hubby for many happy years now. We've done the lot, but over a few cheeky bottles of vino rosso the other night, he mentioned that he was up for trying something a little more adventurous…
"Oh really?" I giggled. "What did you have in mind?"
His eyes narrowed and he shot me a rakish look. "Well," he purred. "How about we try a spot of spelunking sometime soon?"
I didn't know what to say – our tastes have always been so… vanilla. I had no idea he'd been harbouring all these secret desires. He even mentioned that he'd be up for the two of us sharing a white water experience some time soon.
But in fact, now he's raised the idea, I'm raring to go. I could do with a few tips beforehand though – could you come up with the goods?
Bev, Lincoln
Dr Moore-Money says…
It's only natural that some couples like to spice things up with some adventurous antics after a few years.
But there are some big issues to take into account… namely, protection.
An adventure sports holiday potentially carries much greater risk than an average beach break, so getting your insurance in order is a must.
If you have an annual travel insurance policy – particularly if it's packaged as part of a deal from your current account or credit card provider – you may want to peruse the small print to see if you're covered for such activities.
If you aren't, then you may want to consider how much adding this sort of cover will cost, or think taking out a policy specifically for adventurous pursuits like riding, cycle touring or indeed rafting.
However, once that's in order, you should be good to go, secure in the knowledge that if anything goes awry, you'll be covered.
But in all likelihood, the worst thing you're likely to encounter is a face full of spume!
Dearest Dr Moore-Money,
Myself and the boys from the lawn bowls club recently embarked on a 'lads' holiday' to Magaluf.
It was as high-spirited as you might expect, but I'm afraid I've come back with something nasty which I hadn't bargained for – an angry, red overdraft.
On the third night, we crossed paths with a gaggle of fine fillies from Wigan. They had a mighty thirst, and were matching us drink for drink – and then some.
The banter was getting fairly close to the knuckle, and as the raki flowed, so did the Euros from my bank account.
As I awoke in the morning, nursing a dry mouth and a jumbo-sized hangover, I attempted to put together the pieces of what had gone on the night before.
I briefly recalled Bozza, Scoops and the Length (my accomplices from the team, since you ask) being strongly cautioned by the local Policia after some tops-off antics in the street.
The girls, as I remembered, also involved in an unedifying incident involving an inflatable sheep with a group of men from Swansea.
Then it hit me. Why hadn't I been more careful?
I'd been far too free and easy when it came to inserting my debit card, and now I'm terrified I've accumulated all sort of long-lingering nastiness on my credit file just because I fancied splashing out on those lasses to impress them.
Any tips on how I can set my mind at ease?
Geoff, Tiverton
Dr Moore-Money says…
You do seem to have gotten yourself into a bit of a pickle here, Geoff.
As the good times roll, it's very easy to forget about the sting that comes with playing fast and loose with your bank card abroad.
Flexing your card in a shop – or in your case, licensed establishment – will usually have a non-sterling transaction fee. Withdrawing cash will also be more expensive.
To mitigate these issues in future, a holiday credit card could come in handy.
These might have benefits like free use abroad, or even saucy bonuses. More on those later.
However, there are matters to consider, such as annual fees and whether they outweigh the savings, and whether the exchange rate offered by your travel credit card is as good as it could be.
Another good way of reducing your spend is changing money before you go.
Of course, with sterling taking something of a pounding over recent weeks, you won't be getting quite as much foreign dosh as you would have done a few months ago. But you can still shop around for the best rate, and I wholeheartedly suggest that you do so.
Dear Doctor-Moore Money,
My husband and I are thinking about joining the 'Mile High Club', but are a little worried about the risks involved.
We're frequent flyers, and often fantasise about maximising our pleasure by building up juicy rewards points to bag ourselves more trips abroad.
How might we go about it?
Maureen, Leominster
Dr Moore Money says,
Hi Maria. Firstly, I think you might be confusing 'the Mile High Club' with Air miles, or Avios, as the scheme is now known – it's an easy gaffe, but one people often make.
Building up points for an exotic trip abroad on spending you'd be doing anyway on your credit card is certainly a mouth-watering prospect, and one which many big spenders like to take advantage of.
But as always, there are various important caveats to take into account with any credit card which is rewards based.
To extract maximum value, you need to be spending a lot on your card, which means using it for every day spending, not just big-ticket items.
Because rewards-based cards can have high APRs attached to them, you'll also have to be paying off the balance in full every month in order for the rewards to make financial sense.
You might also want to consider whether you might get more bang for your buck (ahem) with a cashback credit card.
These are very versatile, as rather than having to splash your rewards all on flights, you can earn cold, hard cash.
This way, you can spend the spoils of your regular shopping on whatever you want.
Join us for another informative session with the doc next month. Until then, send us your problems


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LOS ANGELES (AP) Dwayne "The Rock" Johnson has split with his wife of 10 years. The 35-year-old pro wrestler-turned-action star announced his separation from wife Dany Garcia Johnson in a statement to People magazine on Friday.
"We've been fortunate enough to spend the last 17 years together as a couple and look forward to spending the rest our lives together as best friends and business partners," the statement said. "We will continue to advance and manage our business interests, our philanthropic efforts and most importantly the raising of our child together."
Garcia Johnson is the CEO of a wealth management firm. The pair have a 5-year-old daughter, Simone Alexander.
Johnson has appeared in the films Walking Tall,The Mummy Returns and The Scorpion King. He recently finished shooting a movie based on the television show Get Smart that stars Steve Carell.
Copyright 2007 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
Conversation guidelines: USA TODAY welcomes your thoughts, stories and information related to this article. Please stay on topic and be respectful of others. Keep the conversation appropriate for interested readers across the map.


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New 2017 Ram 1500 Rebel
- MAKE:Ram
- BODY TYPE:Truck
- ENGINE:Regular Unleaded V-8 5.7 L/345
- TRANS:Automatic
- LOCATION:Antioch
- EXT COLOR:Granite Crystal Metallic Clearcoat
- INT COLOR:N7x9
- MPG:15 city / 21 hwy
*Please note: we update our inventory daily. Please check with dealer to confirm vehicle availability.
Sellers Comments
Function has never had a more impressive form. The exterior of the Ram 1500 is re-engineered to look and perform better. With active aerodynamics and an upgraded front fascia, it's both beautiful and powerful. Available RamBox Cargo Management System offers lockable, lighted, drainable storage - perfect for storing tools, gear, and beverages. With new fabrics and leather trim choices for seats the interior of the Ram 1500 feels as good as it looks. From the displays to the stitching, it's the most sophisticated Ram Truck experience yet. With the Uconnect Access System, your truck can do double duty as a mobile office or entertainment center. The available 8.4-inch touchscreen offers available features like interior climate control and 3-D terrain maps. Bluetooth wireless connectivity, voice command, and steering wheel-mounted buttons mean you're always in complete control. Whether it's the legendary power of a 5.7L HEMI V8, the potent and efficient 3.6L Pentastar V6 with Best-in-Class fuel economy, or the 3.0L EcoDiesel V6 with exceptional torque and best fuel economy of any full-size pickup, there is an engine configuration right for you. Ram 1500 is the only light-duty truck in the U.S. with an available eight-speed transmission offering superior acceleration in low gears. Pair that with a 4-corner air suspension that keeps heavy loads level and you'll keep your payload on the right road. Dynamic front crumple zones and side-impact door beams help absorb impact forces from every angle. Ram 1500 side occupant protection system includes side-curtain airbags and side-impact door beams.
- TRANSMISSION: 8-SPEED AUTOMATIC (8HP70) -inc: 17 Aluminum Spare Wheel
- RAM 1500 BLACK REBEL GROUP -inc: Black Powder-Coated Front Bumper Wheels: 17 x 8 Matte Black Aluminum
- QUICK ORDER PACKAGE 26W REBEL -inc: Engine: 5.7L V8 HEMI MDS VVT Transmission: 8-Speed Automatic (8HP70) Dual Rear Exhaust w/Bright Tips Flat Black 5.7L Hemi Badge Flat Black Ram 1500 Badge Ram Glove Box Badge
- RADIO: UCONNECT 3C NAV W/8.4 DISPLAY -inc: SIRIUSXM Travel Link 1 Year Sirius Travel Link Service 5-Year SIRIUSXM Travel Link Service 5-Year SIRIUSXM Traffic Service 1 Yr Trial (Registration Required) HD Radio SIRIUSXM Satellite Radio No satellite coverage in Hawaii or Alaska GPS Navigation Uconnect Access 1 Year Trial SIRIUSXM Traffic 1 Year Sirius Real Time Traffic Service
- ENGINE: 5.7L V8 HEMI MDS VVT -inc: Electronically Controlled Throttle Hemi Badge Next Generation Engine Controller Heavy Duty Engine Cooling Heavy Duty Transmission Oil Cooler Engine Oil Heat Exchanger
- LUXURY GROUP -inc: LED Bed Lighting Overhead Console w/Garage Dr Opener Universal Garage Door Opener Sun Visors w/Illuminated Vanity Mirrors Exterior Mirrors Courtesy Lamps Glove Box Lamp Underhood Lamp Auto Dim Exterior Mirrors Exterior Mirrors w/Supplemental Signals Rear Dome w/On/Off Switch Lamp Power Heated Fold-Away Mirrors Rear View Auto Dim Mirror Power-Folding Mirrors
- Four Wheel Drive
- Tow Hitch
- Air Suspension
- Power Steering
- 4-Wheel Disc Brakes
- Brake Assist
- Aluminum Wheels
- Tires - Front All-Terrain
- Tires - Rear All-Terrain
- Conventional Spare Tire
- Tow Hooks
- Heated Mirrors
- Power Mirror(s)
- Intermittent Wipers
- Variable Speed Intermittent Wipers
- Privacy Glass
- Power Door Locks
- Automatic Headlights
- Fog Lamps
- AM/FM Stereo
- Satellite Radio
- Bluetooth Connection
- Steering Wheel Audio Controls
- MP3 Player
- Auxiliary Audio Input
- Power Driver Seat
- Power Passenger Seat
- Driver Adjustable Lumbar
- Passenger Adjustable Lumbar
- Pass-Through Rear Seat
- Rear Bench Seat
- Adjustable Steering Wheel
- Trip Computer
- Power Windows
- Leather Steering Wheel
- Heated Steering Wheel
- Keyless Entry
- Cruise Control
- Climate Control
- Multi-Zone A/C
- Cloth Seats
- Vinyl Seats
- Bucket Seats
- Passenger Vanity Mirror
- Floor Mats
- Remote Engine Start
- Telematics
- WiFi Hotspot
- Smart Device Integration
- Heated Front Seat(s)
- Engine Immobilizer
- Security System
- Traction Control
- Stability Control
- Front Side Air Bag
- Rear Parking Aid
- Tire Pressure Monitor
- Driver Air Bag
- Passenger Air Bag
- Passenger Air Bag Sensor
- Front Head Air Bag
- Rear Head Air Bag
- Child Safety Locks
- Back-Up Camera
- No information on specs available.
Vehicle Location
Antioch Chrysler Jeep Dodge Ram
1810 Auto Center Dr.
Antioch , California 94509


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After years of denials, Boston Cardinal Bernard Law will give a court-ordered deposition today about his role in allowing a pedophile priest to prey on Boston's children for more than 30 years.
Since Law is the most senior U.S. cardinal, his deposition could be seen as a low point for the Catholic Church — a rare intrusion of secular forces into its affairs. In the past decade, only one other cardinal has been hauled into court for questioning about sexual-abuse coverups.
But it is also an essential opportunity for cleansing. By protecting abusive priests, the church hierarchy and Law have destroyed their own credibility. Only a court can now provide convincing evidence that all information about priestly child abuse has been pried free.
That's how the current scandal was exposed. A civil suit against the Boston Archdiocese revealed Law's involvement, blowing the issue onto front pages. Eighty-six victims of convicted child molester John Geoghan allege that Law let the priest remain active long after his crimes were known.
Similar suits might easily have saved many children. Other dioceses have paid abuse victims at least $500 million, but most came in secret settlements that prevented the information from becoming public.
The motive, church officials say, was to spare the victims anguish while shielding perpetrators from the consequences of acts that the church then viewed as more sinful than criminal.
Regardless of the intentions, though, the secrecy compounded the abuse.
In case after case, pedophile priests were transferred to other parishes where they had the chance to abuse again.
In St. Louis, for example, victims who signed a secret settlement in a 1995 lawsuit found out this year that the alleged molester remained an active parish priest. The priest was removed in March, weeks after one of the abuse victims broke the agreement and went public.
Such hidden settlements are standard practice for businesses and other large institutions. In scores of lawsuits since 1991, for instance, Firestone demanded secrecy for internal documents and sworn statements. Had those documents not been hidden, the recall of 6 million faulty tires might have come sooner than 2000, averting dozens of deaths and injuries. But for a church to use the tactic in ways that endangered children is far more offensive.
Ironically, Law's deposition — which will become public at trial — comes because the Boston archdiocese abandoned a settlement with Geoghan's victims. Now when Law takes an oath he'll have to explain why he transferred abusive priests, hiding the risks they posed to children.
Only when all questions are answered openly can healing begin.


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Is It Headlines? By Emily, Michael and Tyler Defining Journalism OR A picture of society? We learn quickly from this week's readings that there are essentially a few "camps" which attempt to this question In One Corner: Craft In the other: Art Mechanisms? Journalism is the informed development of a craft which aims to serve the public through an interpretive explanation of the here and now. As history shapes and changes, the way the "here and now" is conveyed, likewise changes (and hence journalism is malleable and changing). Towards our own definition: Towards a definition of Journalism
What is journalism supposed to do?
Where is journalism going? Presentation What is journalism? Stuart Adam defines journalism as: Journalism as an Art Form "Journalism is a form of expression that is an invention of a creation, a product of the imagination" "The World is born in our imaginations, nature has no objective existence, the experience of nature is the work of the Imagination" Imagination with a Capital "I" And Journalism is a medium through which we express our imaginations If Journalism is a comment on the "here and now" taking place in the present public sphere THEN Putting these two thoughts together we understand that journalism expresses our cultural identity Adam's opinion:
Journalism is an art form, involving the "application of values we use to judge things and those values are reflected in the selection of subjects and in the judgements conferred by journalists on how well the world they reveal is working." Journalism is Art Rational Individualism: the world is made up of individuals and their rational calculations
Sociological: impulses which make individuals act
Individual but not rational (think Freud)
Cultural: human acts can be understood in light of conscious motives and unconscious motives The Principle of Meaning Human behaviour influences our story-telling Practitioners can be broken
up into two groups: Journalists and Their Style Styles of Journalism: 1) Invent within invention
2)Bureaucrats reproduce the invention without invention the bulletin
the proclamation
the announcement News on news judgement
Reporting or evidentiary method
Linguistic technique
Narrative technique
Method of interpretation or meaning The Principles of Design P. J. O'Rourke: American political satirist, journalist, writer, and author. Journalism as a Craft A necessity? "If you were a working class or lower-middle-class kid -- even as late as [in] my era growing up in the 1950s -- and you liked to read and you didn't like to get up real early in the morning and lift heavy things, you basically had two choices. You could become a newspaper reporter or you could become a priest."
-O'Rourke, June 2010 "But you always knew that what you were doing was a trade, like brick-laying. And like brick-laying it's a very difficult trade at times. It takes skill. It takes experience. But your job was simply to be the public rubberneck." Craft influenced by human behavior
human behavior influencing the craft? Adam VS. O'Rourke If there are two sides to the argument, then what is the right way to teach the study of journalism? The Education of Journalism Moving back to Adam's original question and critique of Journalism education, it is evident that he feels journalism as an academic field belongs in the humanities What is Journalism supposed to do? Nicholas Berthelot Lemann is dean and Henry R. Luce professor at the Columbia University Graduate School of Journalism in New York City.
Lemann is from New Orleans and he graduated from Harvard University in 1976, but has never attended a school of journalism. He is a journalist, editor, and author of several books on 20th century United States history Pushing the conversation a bit further we ask, can journalism be taught in a classroom? Brad and Angelina Cultural vs. Individual Imagination Mathrubhumi.com "Shit Happens" Vice President Joe Biden Image credit: The Daily Caller - Michael Schudson, Why Democracies Need an Unlovable Press "Journalists are supposed to be institutionalized outsiders even though they have in fact become institutionalized insiders." Marketplace of Ideas A cover from the National Review A cover from The Nation A Marketplace for Ideas A cover from Reason Magazine A cover from The Atlantic Photo originally published in the Washington Post Media as a Watchdog Image credit: Toronto Star Image credit to Jimmy Jeong of the Edmonton Journa. The First Amendment "rests on the assumption that the widest possible dissemination of information from diverse and antagonistic sources is essential to the welfare of the public, that a free press is a condition of a free society." Associated Press v. United States (1945) Justice Hugo Black Democracy needs "unfettered interchange of ideas for the bringing about of political and social changes desired by the people." Roth v. United States (1957) Justice William Brennan "…the press is free to do battle against secrecy and deception in government." "On the Press," Hasting's Law Journal Justice Potter Stewart The Sponsorship Scandal Where is Journalism Going? Newspaper Crisis
Siles and Boczkowski (2011) A) The "Death of the Newspaper" and the Rise of the Online Media Prominent factors behind decline:
Technological Wider set of tools for dissemination of content
-Deuze (2003)
Functional definition of online news-media
Hypertextuality Pros 'Death of Newspapers' Resources perspective
Effects on investigation and analysis
Re-prioritization in the newsroom Cons "Online journalism, particularly online journalism that is not sponsored by major print and television media, has so far shown little capacity for, or interest in, making the kind of large investments in investigation and analysis that make conventional media, especially newspapers, invaluable for democracy" (Shudson 17) Vos, Craft, and Ashley (2011)
"New Media, Old Practices"
"Transparency is privileged over objectivity, connection over detachment"
Mediaite, WorldNetDaily, BigJournalism
Stability of journalistic practices B) Journalism 'turned-in-on-itself' 'Watchblogs' and media criticism "[Our sites] will continue to fight the mainstream media – New York Times, Washington Post, LA Times, CBS, ABC, MSNBC, and CNN – who have repeatedly, and under the guise of objectivity and political neutrality, promoted a blatantly left-of-centre, pro-Democratic party agenda." (Andrew Breitbart, creator of BigJournalism.com in 2009) "He has no respect for actual journalism, seeking to turn the field into a province defined solely by partisanship and politically potent smears. Any reporter who cares about their craft should recognize that at his core, Breitbart would be all too happy to see them out of a job."
(John V. Santore, writing for Media Matters in 2009)
Returning to Stuart's disconnect between journalistic education and practice
MacDonald (2006)
"Teaching journalists to save the profession"
Echoing Shudson: reliance on official and institutional sources
Journalistic identities: professional or critical? Reform: media newsrooms or media classrooms? C) Journalism education Addressing the 'Professional Crisis' CONCLUSION 1. Does the emergence of 'new media', both in terms of the blogosphere and mainstream online news-media, represent a step closer to Stuart's ideal of Journalism-as-Imagination? Or does it further erode journalistic practice?
2. Do you believe that 'media-watchdog' sites effectively hold mainstream news organizations to account? Or are they simply another partisan tool that degrades objectivity?
3. Do you agree that the best solution to the 'professional crisis' currently afflicting journalism lies in education? Or does it have to come from the top-down (i.e. from within established news organizations)? Is there a need for 'professional certification' in journalism? Questions
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Journalism and Society
Defining Journalism
Emily Murthaon 17 September 2012
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text: Home \ TFCA Bookstore \ TFCA Authors G The Fabric of Faithfulness: Weaving Together Belief and Behavior (Revised & Expanded) By Garber, Steve How do you help Christian students, during one of the most eventful and intense periods of their lives, learn to connect what they believe with how they live. Drawing on a wide range of disciplines, Steven Garber vigorously engages this question. Get Up Off Your Knees: Preaching the U2 Catalog By Garber. Steve A thoughtful and provocative collection of sermons by a group of preachers from across the international church spectrum who have been moved to theological reflection on the art and work of U2. Revelations of a Single Woman: Loving the Life I Didn't Expect By Gilliam, Connally Through this collection of thoughtful, honest, and humorous memoirs, Gilliam delves into what it means to be single in a world that caters to couples and what it's like being the one who lives out moral values that her peers think died in the 1960s. Science But Not Scientists: How Everything Began: Chance or Creation By Grose, Vernon By means of example rather than hypothetical supposition, this book focuses on the problem of scientific literacy. American Hour By Guinness, Os An internationally known writer and speaker on religion and public life brilliantly analyzes the causes of our current moral malaise. Guinness examines how perilously close we have come to losing the shared beliefs, traditions and ideals that have helped shape America and sets forth a compelling view of a new role for religion. Character Counts: Leadership Qualities in Washington, Wilberforce, Lincoln, Solzhenitsyn By Guinness, Os Inspiring stories of four public figures who displayed strength of character in the face of adversity, enabling them to change the world. God in the Dark By Guinness, Os Author examines doubt from every angle and major perspective. Os Guinness pays special attention to the two basic questions "Why, O Lord?" and "How long, O Lord?" He tears away the layers of misunderstanding about doubt to reveal not only its dangers but its value. Prophetic Untimeliness: A Challenge to the Idol of Relevance By Guinness, Os Gives practical, constructive solutions for living with integrity in the midst of contemporary pressures. Guinness explores what it means to be both faithful and relevant, and how to be truly relevant without being trivial or trendy. The Call: Finding and Fulfilling the Central Purpose of Your Life By Guinness. Os The classic work on the central purpose of life is now offered with a full workbook included Unspeakable: Facing Up to the Challenge of Evil By Guinness, Os A leading public intellectual confronts America's inability to understand--let alone effectively respond to--evil, providing both a language and a strategy for a way forward. Mary: A Catholic-Evangelical Debate By Gustafson, David


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Patrick M. Maund is a San Francisco businessman who has produced significant scholarship on the works of L. Frank Baum. Perhaps most notably, he wrote a series of Bibliographica Baumiana articles, published in The Baum Bugle through the 1990s, that provide bibliographic analysis of the first editions of Baum's works. He also provided the annotations for The International Wizard of Oz Club's second edition of Robert Pattrick's Unexplored Territory in Oz (1979).
Maund is also a longtime activist in Oz fandom.


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Rishi Kapoor, who always manages to smile and sometimes even chuckle on people's face through his funny tweets, has pulled off a winner this time.
Recently, at an awards show, Parineeti Chopra bagged an award for losing weight. Rishi Kapoor, took to Twitter and posted this hilarious tweet saying,
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Parineeti Chopra, had recently shed weight and posted multiple pictures on social media with her newly toned body.
Parineeti Chopra, who stunned fans with the hashtag #builtthatway was the talk of the town during December 2015.
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"I couldn't believe it when they recently gave an award to Parineeti Chopra for losing weight. Can someone please give me an award for gaining weight?" says Rishi Kapoor.
Parineeti Chopra, shed all those extra kilos and became an inspiration to hit the gym and work out for a fit body.
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Rishi Kapoor, says that award shows these days have lost their true magic and reduced the credibility of the awards. A lot of people have expressed the same concern saying award shows have become just a fun event, and has lost its real purpose.
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Rishi Kapoor says, "I never did believe in popular awards. It's gotten much worse now. I use award trophies as doorstops in my home. What they have done to film awards is truly tragic. They've reduced them to trivia. Shameless. Agar sabko khush karna hai toh phir award ka matlab kya hua?"
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Also Read: 10 HOT PICS! Lisa Haydon's Photoshoot With Cosmopolitian Magazine India!