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Peripheral arterial disease (PAD) is a common disease with increasing prevalence, presenting with impaired walking ability affecting patient's quality of life. PAD epidemiology is known, however, mechanisms underlying functional muscle impairment remain unclear. Using a mouse PAD model, aim of this study was to assess muscle adaptive responses during early (1 week) and late (5 weeks) disease stages. Unilateral hindlimb ischemia was induced in ApoE−/− mice by iliac artery ligation. Ischemic limb perfusion and oxygenation (Laser Doppler imaging, transcutaneous oxygen pressure assessments) significantly decreased during early and late stage compared to pre‐ischemia, however, values were significantly higher during late versus early phase. Number of arterioles and arteriogenesis‐linked gene expression increased at later stage. Walking ability, evaluated by forced and voluntary walking tests, remained significantly decreased both at early and late phase without any significant improvement. Muscle glucose uptake ([18F]fluorodeoxyglucose positron emission tomography) significantly increased during early ischemia decreasing at later stage. Gene expression analysis showed significant shift in muscle M1/M2 macrophages and Th1/Th2 T cells balance toward pro‐inflammatory phenotype during early ischemia; later, inflammatory state returned to neutrality. Muscular M1/M2 shift inhibition by a statin prevented impaired walking ability in early ischemia. High‐energy phosphate metabolism remained unchanged (31‐Phosphorus magnetic resonance spectroscopy). Results show that rapid transient muscular inflammation contributes to impaired walking capacity while increased glucose uptake may be a compensatory mechanisms preserving immediate limb viability during early ischemia in a mouse PAD model. With time, increased ischemic limb perfusion and oxygenation assure muscle viability although not sufficiently to improve walking impairment. Subsequent decreased muscle glucose uptake may partly contribute to chronic walking impairment. Early inflammation inhibition and/or late muscle glucose impairment prevention are promising strategies for PAD management.
- M1 and M2 macrophages
- MR spectroscopy
- peripheral arterial disease
- Th1 and Th2 cells
- tomography
Peripheral arterial disease (PAD) is a common disorder mainly due to atherosclerosis characterized by stenosis and/or obstruction of lower limbs arteries leading to decreased muscle perfusion and oxygenation. PAD represents a major public health issue. Its prevalence is ~12% in the adult population, increasing to 20% above 70 years (Hirsch et al. 2006; Norgren et al. 2007; Olin et al. 2010). Symptomatic PAD patients suffer symptoms of intermittent claudication (IC), defined as fatigue, discomfort, or pain occurring in limb muscles during effort, due to exercise‐induced ischemia, with rapid relief at rest (Hirsch et al. 2006). As a result, patients with PAD and IC are physically impaired and have a markedly reduced quality of life (Hirsch et al. 2006; Norgren et al. 2007; Olin et al. 2010). Moreover, PAD is associated with a significant increase in cardiovascular (CV) morbidity (myocardial infarction and stroke) and mortality (CV and all cause) (Hirsch et al. 2006; Norgren et al. 2007; Olin et al. 2010). PAD management includes strict CV risk factors control, and patient encouragement to regular walking exercise. If needed, revascularization procedures are proposed to avoid lower limb amputation. Unfortunately, no specific treatment for PAD is yet available.
Due to the complexity and multifactorial origins of PAD, as well as the differences in muscular adaptive responses, precise PAD pathophysiological mechanisms are still largely unknown. Although blood flow limitation to active muscle is of critical importance, little is known about factors independent of blood flow and intrinsic to skeletal muscle that may also contribute to disease process and functional limitations in PAD patients.
More than 90% of cases of PAD are secondary to atherosclerosis, which is now recognized as a chronic inflammatory disease. Atherosclerotic plaques contain abundant immune cells, mainly macrophages and CD4+ T cells, that orchestrate many of the inflammatory processes occurring throughout atherogenesis (Hansson and Hermansson 2011; Ketelhuth and Hansson 2011). These cells can polarize toward different phenotypes (pro‐inflammatory or anti‐inflammatory) according to various stimuli present in their surrounding microenvironment. Thus, CD4+ T‐cell subtype Th1 (pro‐inflammatory cells) and CD4+ T‐cell subtype Th2 (anti‐inflammatory cells) exist in plaques, each having a distinct function influencing lesion's fate, that is, development of rupture‐prone unstable versus stable plaque phenotype (Hansson and Hermansson 2011; Ketelhuth and Hansson 2011). Likewise, macrophages can polarize into two different subsets: classically pro‐inflammatory M1 macrophages, driven by Th1 cytokines, or alternatively anti‐inflammatory M2 macrophages, driven by Th2 cytokines. Recent evidence indicates that macrophage polarization balance is a crucial element in determining plaque outcome (Hoeksema et al. 2012). Besides their role in atherosclerosis, macrophages and CD4+ T cells have been implicated in ischemia‐induced neovascularization through the synthesis of local angiogenic/arteriogenic factors (Silvestre et al. 2008). However, although emerging evidence shows a role for CD4+ T cells and macrophage phenotype switch in atherosclerosis, no study has addressed this significance in PAD.
Few studies have reported abnormal skeletal muscle metabolism in patients with PAD and IC, including impaired skeletal muscle glucose uptake (Pipinos et al. 2007, 2008; Anderson et al. 2009; Pande et al. 2011), however, this potential mechanistic explanation has not been studied in early and late phases of PAD.
Using a mouse model of peripheral ischemia with impaired walking ability, aim of present study was to assess skeletal muscle adaptive responses during early and late stages of PAD focusing on glucose and high‐energy phosphates metabolism, and M1/M2 macrophages and Th1/Th2 cells polarization.
Mouse model of PAD and IC
Unilateral hindlimb ischemia was induced in 14–16‐week old male hypercholesterolemic and atherosclerotic C57BL/6J Apolipoprotein E knock‐out (ApoE−/−) mice (Charles River Laboratories, L'Arbresle Cedex, France) by right common iliac artery ligation. Briefly, mice were anesthetized using isoflurane inhalation (1–2% in O2) and placed on a heated pad during surgery. Hindlimbs and inferior abdominal area were shaved. Through a small abdominal incision, right common iliac artery was exposed and ligated with 7–0 silk suture just above the internal–external iliac artery bifurcation. Iliac vein and nerve were preserved. Abdominal incision was then sutured with a resorbable 5–0 silk suture. Sham‐operated contralateral nonischemic hindlimb served as control. One week prior to surgery, mice were treated with Dafalgan (200 mg/kg) via the drinking water for 14 days. In addition, mice were administered Temgesic (0.01 mg/kg, s.c.) following surgery.
Mice were fed regular rodent chow, and accessed water ad libitum throughout the study. Animal experiments were performed according to the Swiss Federal guidelines (Ethical Principles and Guidelines for Experiments on Animals). The protocol was approved by the local Institutional Animal Committee (Service Vétérinaire Cantonal, Lausanne, Switzerland). All efforts were made to minimize animal suffering during the experiments.
In vivo transcutaneous oxygen pressure measurement
Before ischemia, 1 week, and 5 weeks postischemia, skin oxygenation in ischemic hindlimb was determined by measuring transcutaneous oxygen pressure (TcPO2) using a TcPO2‐monitoring system equipped with a Clark electrode (TCM30; Radiometer, Copenhagen, Denmark). TcPO2 measurements are routinely used in vascular clinical practice as a measure of ischemia severity in lower extremities of PAD patients. Reproducibility and accuracy of TcPO2 measurements in mice were tested in preliminary experiments in control nonischemic mice. Measurements were performed in anesthetized mice placed in the supine position on a heated pad to maintain body temperature at 37 ± 1°C. Prior to each measurement, electrode calibration was performed according to manufacturer's instructions. After calibration, the electrode was connected to a ring filled with contact solution, to avoid oxygen air interference, and fixed to the skin just above the knee. After a 15 min period of stabilization, TcPO2 (expressed in mmHg) was continuously recorded during 15 min. Values measured at 15 min were used for analysis.
In vivo laser Doppler perfusion imaging
Skin perfusion of both ischemic and contralateral nonischemic hindlimbs was evaluated using a Laser Doppler Imager (Moor Instruments, Axminster, U.K.) in anesthetized mice placed in a prone position on a heating pad. Before ischemia, 1 week, and 5 weeks postischemia, five consecutive plantar foot images were recorded at 30‐sec intervals, and averaged. Perfusion status was calculated on the basis of colored histogram pixels within the region of interest (ROI) using Moor LDI Image Review software. Tissue perfusion in ischemic hindlimb was expressed as a percentage of that measured in contralateral nonischemic hindlimb. This calculation allows minimizing biases due to variables such as ambient light and even minimal temperature variations.
Total walking distance assessment
Total 24‐h walking distance (24hTWD) was assessed at three time points: before ischemia, 1 week, and 5 weeks postischemia. Mice were housed in individual cages containing a 12‐cm diameter wheel and were free to run during 24 h. The wheel was connected to a counter recording number of revolutions allowing 24hTWD (kilometers) calculation for each animal.
Maximal walking distance and time assessment
Maximal walking distance (MWD) and maximal walking time (MWT) were determined before ischemia, 1 week, and 5 weeks postischemia using a forced treadmill (Columbus Instruments, Columbus, OH). Mice were subjected to an incremental speed protocol, starting at a speed of 9 m/min for 3 min with an increase of 2 m/min every 3 min until speed reached 19 m/min (0% slope). Mice were encouraged to run as long as possible with the use of an electric grid located at the back of the treadmill (1.5 mA, 3 Hz). The test was stopped when mice were exhausted (remained on the shock grid for five continuous seconds). MWD (kilometers) and time (minutes) were then calculated for each animal.
Clinical evaluation of ischemia and limb function
At 1 and 5 weeks postischemia, mice were observed and scored according to an ischemia grade scale (0 = normal, 1 = foot discoloration, and 2 = tissue necrosis). Limb function was also assessed using a gait abnormality grade scale (0 = normal, 1 = limping, 2 = dragging of foot).
In vivo [18F]fluorodeoxyglucose PET imaging and glucose metabolism of hindlimb muscle
At 1 and 5 weeks postischemia, noninvasive [18F]fluorodeoxyglucose (18FDG) positron emission tomography (PET) hindlimbs imaging was performed using an avalanche photodiode microPET scanner (LabPET4; Gamma Medica, Sherbrooke, Canada) (Seyer et al. 2013). Mice were anesthetized with a mixture of 1.5% isoflurane in 100% O2 (0.9 L/mL, 2.5 bars) and tail vein catheterized into. Animals were prone positioned with extended legs. Fifty‐minute list mode acquisitions were acquired with field of view (FOV) containing both ischemic and contralateral nonischemic hindlimbs. i.v. injection of 18FDG (≈50 MBq) through tail vein catheter was initiated within the first 10 sec of PET scan, followed by 100–500 μL of saline chase solution. Number of detected single events/s was used to evaluate and control intravenous 18FDG delivery. During the entire scanning period, mice were maintained under isoflurane anesthesia using a face mask. Temperature and breathing rate were continuously monitored. An energy window of 250–650 keV and a coincidence timing window of 22.2 nanoseconds were used. For image reconstruction, storage of coincidence events, recorded in list mode files during the PET scan, were binned according to their line of response, as previously described (Selivanov et al. 2000). Voxel size measured 0.5 × 0.5 × 1.2 mm, giving a typical resolution of 1.2 mm at the center of FOV. For spatial histogramming, scans of 50 min duration were reconstructed in three blocks (15, 15 and 20 min, respectively) using a FOV of 46 mm, a span field of 31 and a "maximum likelihood expectation maximum" (MLEM) from 20 to 60 iterations, intermediate images were saved every five iterations. After correcting for different count‐rates of each line of response and for quantitative 18FDG calibration, images of accumulated intracellular 18FDG‐6P at steady state were quantitatively expressed using standardized uptake value (SUV) (mean ROI activity [kBq/cm3])/(injected dose [kBq]/body weight [g]). Images were corrected for nonuniformity of the scanner response, dead time count losses, and physical decay from time of injection. No correction was applied for attenuation and partial‐volume effects. Images were analyzed with PMOD 3.2 software (PMOD Technologies, Zurich, Switzerland). ROIs were manually drawn by optical reading of well‐delineated hindlimb muscle. Glucose uptake in ischemic hindlimb muscle was expressed as percentage of that in contralateral nonischemic hindlimb.
Inter‐hindlimb variability in individual mice was assessed in three control nonischemic animals. Results demonstrated <2% variability in 18FDG uptake between right and left hindlimb.
In vivo 31‐phosphorus magnetic resonance spectroscopy
Mice were measured on a 9.4 T Varian VNMRS spectrometer (Varian, Palo Alto, CA) in supine position 1 and 5 weeks postischemia. Animal anesthesia, body temperature, and breathing rate were continuously monitored through the measurement. A home‐built 18 mm‐diameter dual 1H quadrature/10 mm‐diameter 31P single‐loop surface radiofrequency coil was used and positioned over mice hindlimbs. Thereafter, hindlimbs were fixed in order to prevent any movement leading to signal deterioration. T2‐weighted turbo‐spin‐echo images were obtained in the axial plane of ischemic and nonischemic quadriceps muscles using a FOV 30 × 30 mm and 1 mm slice thickness. For spectroscopy, volume of interest (VOIs) of about 60 mm3 was chosen. Static field homogeneity in selected VOI was adjusted by an echo‐planar‐imaging version of FASTMAP (fast, automatic shimming technique by mapping along projections) using the 1H signal of water (Gruetter and Tkac 2000). Spectroscopic localization was achieved by outer volume saturation, that is, by applying slice selective inversion in the upper horizontal plane and saturation pulses in all planes around the selected VOI (Mlynarik et al. 2006). Overall, 160 transients were collected with a repetition time of 4 sec. Total measurement time for imaging and 31P spectroscopy was about 1 h. Peak intensities of inorganic phosphate (Pi), phosphocreatine (PCr), and adenosine triphosphate (γ‐ATP) were obtained by fitting to a Lorentzian function using AMARES (Vanhamme et al. 1997) from the jMrui software (http://sermn02.uab.cat/mrui7). Ratios of PCr to γ‐ATP and PCr to Pi were calculated from the respective peaks intensities. PCr/γ‐ATP and PCr/Pi in ischemic hindlimb were expressed as percentage of PCr/γ‐ATP and PCr/Pi in nonischemic contralateral hindlimb, respectively. Preliminary data showed similar PCr to γ‐ATP and PCr to Pi ratios between right and left hindlimb in control nonischemic mice.
Muscle histology and immunohistochemistry analysis
On week 5 postischemia, ischemic quadriceps and gastrocnemius skeletal muscles were isolated and fixed with 10% buffered formalin. Quadriceps and gastrocnemius muscles were also harvested from two independent groups of mice sacrificed at pre‐ischemia (control nonischemic mice), and 1 week postischemia. After fixation, specimens were further embedded in paraffin, and tissue sections (5 μm thick) prepared. Transverse sections were hematoxylin and eosin stained. Pictures were acquired with a high‐sensitivity color camera (Leica DC300F Camera, Wetzler, Germany). Muscle fiber size (μm2) was determined using morphometric analysis (Qwin software, Leica). For each sample, a minimum of 50 muscle fiber sizes were quantified, and results averaged.
For arteriogenesis evaluation, muscle sections were immunostained with a mouse monoclonal α‐SM actin antibody, followed by a secondary biotinylated anti‐mouse antibody. Antibodies were revealed with a peroxidase‐linked avidin‐biotin detection system (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) as previously described (Mazzolai et al. 2004). The number of arterioles in each section was counted in a blinded fashion in five randomly selected fields using the Qwin software. Arteriolar density, number of arterioles per muscle fiber, was then calculated.
Real‐time reverse transcription‐polymerase chain reaction
Total RNA was isolated from ischemic and nonischemic quadriceps and gastrocnemius muscles, at both 1 and 5 weeks postischemia, using Trizol reagent (Invitrogen, Switzerland) followed by the RNeasy Cleanup Kit (Qiagen, Switzerland). RNA concentration and purity were spectrophotometrically estimated by calculating the A260/A280 ratio. cDNA was then synthesized by reverse transcription using the iScriptT™ cDNA Synthesis Kit from Bio‐Rad (Reinach, Switzerland). Quantitative Real time PCR was performed on IQ™‐Cycler (Bio‐rad, Switzerland) using iQ™ SYBR® Green Supermix (Bio‐Rad, Switzerland) according to manufacturer's protocols. The following primers were used: Hypoxia‐inducible factor‐1α (HIF‐1α): sens 5′‐TCAAGTCAGCAACGTGGAAG‐3′, and antisense 5′‐ TATCGAGGCTGTGTCGACTG‐3′; Angiopoietin‐2 (ANG2): sens 5′‐GCATGTGGTCCTTCCAACTT‐3′, and antisens 5′‐ TGGTGTCTC TCAGTGCCTTG‐3′; CD11c: sens 5′‐ACACAGTGTGCTCCAGTATGA‐3′, and antisense 5′‐GCCCAGGGATATGTTCACAGC; CD206: sense 5′‐CATGGATGTTGATGGCTACTGGAG‐3′, and antisense 5′‐GTCTGTTCTGACTCTGGACACTTG‐3′; Interferon‐gamma (IFN‐γ): sense 5′‐ TGAGACAATGAACGCTACACACTG‐3′, and antisense 5′‐TTCCACATCTATGCCACTTGAG‐3′; Interleukin‐4 (IL‐4): sense 5′‐TCAACCCCCAGCTAGTTGTC‐3′, and antisense: 5′‐TGTTCTTCGTTGCTGTGAGG‐3′; and 36B4: sense 5′‐ATGGGTACAAGCGCGTCCTG‐3′, and antisense 5′GCCTTGACCTTTTCAGTAAG‐3′. All samples were run in duplicates. Post PCR melting curves were analyzed to ensure primer specificity. Data were analyzed using the comparative threshold cycles (CT) method (Livak and Schmittgen 2001). Briefly, all results were normalized for the housekeeping 36B4 gene. mRNA expression of genes from ischemic muscles was expressed as fold change in those from non ischemic contralateral muscles.
Statin treatment
ApoE−/− mice were administrated oral atorvastatin (20 mg/kg per day in drinking water, kindly provided by Pfizer) (Wang et al. 2011) 1 day before ischemia until 1 week postischemia. Nontreated mice were used as controls. 24hTWD was assessed in mice before and after atorvastatin treatment using the voluntary walking test while gene expression analysis for CD11c and CD206 was performed in ischemic and nonischemic quadriceps at the end of the treatment according to methods described above.
Statistical analysis
All data are expressed as mean ± SD. Statistical significance was evaluated using one‐way analysis of variance (ANOVA) or repeated measures ANOVA followed by the Tukey post hoc analysis for multiple comparisons. For comparison between two groups, statistical significance was determined by the paired or unpaired t‐test. Differences in ischemia and limb function scores were evaluated by the Fisher exact test. A value of P < 0.05 was considered to be statistically significant.
Mouse model of PAD
Following artery ligation, perfusion of ischemic hindlimbs, assessed by laser Doppler, significantly decreased by 47% 1 week postintervention (early stage of ischemia) compared to the pre‐ischemic situation (P < 0.0001; Fig. 1A). Perfusion remained significantly low (−26%) also at later phase of ischemia (5 weeks postartery ligation; P < 0.0001 vs. pre‐ischemia) although values resulted higher than those observed in the earlier ischemic phase (P < 0.001; Fig. 1A). Similarly, tissue oxygenation of ischemic hindlimbs, assessed by TcPO2, significantly decreased 1 week postischemia compared to the pre‐ischemic situation (P < 0.001; Fig. 1B). This decrease remained significant at 5 weeks (P < 0.05) although values were increased compared to the 1 week levels (P < 0.05; Fig. 1B).
Number of ischemic muscles arterioles was also evaluated using α‐SM actin immunostaining. Consistent with laser Doppler imaging and TcPO2 results, number of arterioles significantly increased, after 5 weeks, in ischemic quadriceps muscle (P < 0.01 vs. 1 week; Fig. 1C). At similar time point, number of arterioles increased also in gastrocnemius muscle although not significantly (P = 0.08; Fig. 1C).
Along the same line, mRNA expression of pro‐angiogenic factor HIF‐1α was significantly upregulated both in ischemic quadriceps (2.2‐fold) and gastrocnemius (1.5‐fold) muscles already at 1 week postischemia (P < 0.05 vs. respective nonischemic muscle; Table 1). ANG2 expression (an arteriogenic factor) was significantly upregulated in ischemic quadriceps muscle at 5 weeks postischemia (1.3‐fold, P < 0.001 vs. nonischemic one), and in ischemic gastrocnemius muscles both at 1 and 5 weeks postischemia (2.1‐fold and 1.7‐fold, respectively, P < 0.05 vs. nonischemic muscles) (Table 1).
Walking abilities of mice at early and late stages of limb ischemia
Walking ability of mice was evaluated at early (1 week postartery ligation) and late (5 weeks postartery ligation) stages of ischemia using voluntary and forced walking tests. 24hTWD significantly decreased by 50% during early stages of ischemia compared to the pre‐ischemic phase (P < 0.01; Fig. 2A). Walking impairment significantly persisted at 5 weeks (P < 0.05 vs. pre‐ischemia; Fig. 2A) and up to 14 weeks postischemia (data not shown). Similarly, both MWD and MWT significantly decreased by 85% (P < 0.01 vs. pre‐ischemia), and 80% (P < 0.001 vs. pre‐ischemia), respectively during early ischemic phase (Fig. 2B and C). These decreases remained significant also after 5 weeks (P < 0.05 vs. pre‐ischemia; Fig. 2B and C). Interestingly, no significant improvement in walking ability was observed between early and late phases of ischemia (Fig. 2A–C).
Clinical observation of mice revealed impaired limb function both at early and late stages of ischemia, characterized by limping and foot dragging (Fig. 3A). No discoloration and/or necrosis was observed. As expected, histological analysis showed muscle fiber atrophy in ischemic quadriceps and gastrocnemius muscles (Fig. 3B).
In vivo resting muscle glucose metabolism in early and late stages of limb ischemia
Noninvasive [18F]fluorodeoxyglucose PET imaging was used to quantify glucose uptake in ischemic hindlimb muscles. Representative 18FDG PET images of phosphorylated 18FDG levels at steady state are shown in Figure 4A. During early phase, glucose uptake significantly increased in ischemic hindlimb muscle compared to the nonischemic one (P < 0.01; Fig. 4A and B). On the contrary, at later stages of ischemia, ischemic muscle glucose uptake significantly decreased not only compared to the nonischemic muscle (P < 0.05) but also compared to the early stage within the ischemic muscle (P < 0.001) (Fig. 4A and B).
In vivo resting muscles energetic state in early and late stages of limb ischemia
Muscle energy state was assessed by 31P‐MRS. Peaks of high‐energy phosphate metabolites (PCr, γ‐ATP), and Pi were, respectively, identified in spectra measured from ischemic and nonischemic contralateral hindlimb muscle. As shown in Figure 5A and B, PCr/γ‐ATP and PCr/Pi ratios were not different between ischemic and nonischemic hindlimb muscles either at early or at later stages of ischemia. Similarly, no change in integral intensity of the ATP peaks (in terms of signal‐to‐noise ratio) was observed in ischemic hindlimbs.
Muscle macrophage phenotype in early and late stages of limb ischemia
Macrophage phenotype was characterized by examining expression of specific pro‐inflammatory M1 (CD11c) and anti‐inflammatory M2 (CD206) macrophage markers using real‐time PCR. At early stage of ischemia, CD11c mRNA expression was significantly upregulated both in ischemic quadriceps (10.5‐fold, P < 0.01), and gastrocnemius muscles (8.1‐fold, P < 0.05) (Fig. 6A). At later stage, CD11c mRNA expression remained significantly upregulated exclusively in ischemic quadriceps muscle (2.2‐fold, P < 0.05) although values were significantly lower than those observed at 1 week (P < 0.001; Fig. 6A). During early phase of ischemia, CD206 mRNA expression was significantly upregulated (2.1‐fold) in ischemic quadriceps muscle only (P < 0.01; Fig. 6B). Based on these results, calculation of CD11c to CD206 ratio (index of M1/M2 macrophage balance) resulted significantly higher both in quadriceps (4.9‐fold, P < 0.05), and gastrocnemius (5.4‐fold, P < 0.01) ischemic muscles, compared to nonischemic ones, during early stage of limb ischemia. This increase was no longer significant at later stage (Fig. 6C).
Phenotype of muscle T cells in early and late stages of limb ischemia
Phenotype of T cells (pro‐inflammatory Th1 cells vs. anti‐inflammatory Th2 cells) in hindlimb muscles was also evaluated. As shown in Figure 8A, IFN‐γ mRNA expression (pro‐inflammatory Th1 marker), was significantly upregulated in early ischemic phase both in quadriceps (8.0‐fold, P < 0.05 vs. nonischemic one) and gastrocnemius (3.8‐fold, P < 0.05 vs. nonischemic one) muscles. However, this upregulation was no longer present in later ischemic stage (Fig. 7A). IL‐4 mRNA expression (anti‐inflammatory Th2 marker) was not significantly modulated by ischemia, both at early and late phases (Fig. 7B). As a consequence, IFN‐γ/IL‐4 ratio (index of Th1/Th2 cell balance) resulted significantly higher in ischemic quadriceps (7.3‐fold), and gastrocnemius (3.8‐fold) muscles during early phase of ischemia (P < 0.05 vs. respective nonischemic muscles; Fig. 7C).
Effect of muscle inflammation inhibition on walking ability of mice in early stage of limb ischemia
Markers of M1/M2 macrophage balance were determined in quadriceps muscle 1 week postischemia in mice treated with or without atorvastatin. As shown in Figure 8A, while the ratio of CD11c to CD206 was significantly higher in ischemic quadriceps muscle than in nonischemic one in control nontreated mice (P < 0.05), no significant difference was observed in atorvastatin‐treated mice. Interestingly, atorvastatin treatment prevented the significant decrease in 24hTWD observed in control mice (P < 0.05) (Fig. 8B).
Results from this study show that in a mouse model of PAD, different muscular adaptive mechanisms take place in response to early and late stages of ischemia. During early phase, muscle glucose uptake raises significantly while decreasing at later phases. Local inflammatory reactions take place with significant macrophage and polarization of T cells toward a pro‐inflammatory phenotype during early phase. This inflammatory imbalance is, however, restored at later phases.
It is known that abnormal ischemic limb hemodynamic status (reduced limb oxygenation and perfusion) does not completely explain functional limitations experienced by patients with PAD (Szuba et al. 2006; Pipinos et al. 2007; Anderson et al. 2009). Our mouse model closely reproduces this human process. Indeed, walking capacity remains profoundly impaired even though limb perfusion and oxygenation tends to significantly increase with time (though remaining inferior to the pre‐ischemic situation). Along the same line, muscle arteriolar density tends to increase. This corroborates the hypothesis that additional intrinsic ischemic muscle factors, independent of hemodynamic ones, are likely to play a role in PAD pathophysiology.
Plasma‐derived glucose importantly contributes to muscle energetic fueling. PET scan, using 18FDG glucose tracer, is a well‐established approach to measure in vivo skeletal muscle glycolytic activity (Kelley et al. 2001; Gondoh et al. 2009). It allowed us to demonstrate, for the first time in a mouse model of PAD, a significantly increased glucose uptake in resting ischemic mouse muscles during early phase of ischemia. Modulation of glucose uptake during early ischemia was also documented in a porcine model of myocardial infarction (Lautamaki et al. 2009). In this study, 18FDG PET also revealed increased glucose uptake in the hypoperfused infarcted area early after myocardial infarction. Contrary to the early phase situation, during the later phase of ischemia, we observed a significantly decreased glucose uptake. This result is in accordance with a recent study showing decreased calf muscle glucose uptake in chronic PAD patients with IC compared to healthy control subjects (Pande et al. 2011). Interestingly, this metabolic abnormality characterizing later phases of peripheral ischemia in our model relates to impaired walking ability, suggesting that decreased muscle glucose uptake may predict exercise limitation. Taken together, these findings suggest that transient initial increase in muscle glucose uptake allows maintaining basal muscle viability despite significantly reduced limb perfusion but that at later stages failure to maintain a sustained muscle glucose metabolism impairs ameliorating walking performances despite increased limb perfusion. Future investigations are needed to determine molecular mechanisms responsible for glucose uptake modulation occurring in peripheral ischemia.
Systemic inflammation has been shown to play a role in the pathophysiology of PAD. Indeed, previous works showed a strong relationship between elevated serum or plasma levels of various inflammatory markers and PAD claudication severity (McDermott et al. 2008; Brevetti et al. 2010). The implication of inflammation at the local level (i.e., muscle) remains, however, poorly investigated. Previous works have demonstrated presence of M1 and M2‐polarized macrophages as well as CD4 Th1 and Th2 cells in atherosclerotic plaques. Additionally, growing evidence strongly suggests that modulation of the M1/M2 and/or the Th1/Th2 balance affects pathogenesis, evolution, and complications of atherosclerosis (Mantovani et al. 2009; Ketelhuth and Hansson 2011; Hoeksema et al. 2012). Although macrophage and T‐cell balance plays a major role in atherosclerosis, few experimental data are available as yet to substantiate their role in PAD. In the present work, phenotypic analysis of muscle infiltrating M1, M2 macrophages and Th1, Th2 cells revealed that early ischemia is accompanied by macrophage and T‐cell balance shift toward a pro‐inflammatory state. This proinflammatory phenotypic switch returns to neutral state at later stages of ischemia. Therefore, pro‐inflammatory cells may be critical players in the initial phase of ischemic events, and may contribute to impaired walking capacity. To test this hypothesis, mice were treated with a statin to selectively inhibit muscular inflammation, especially pro‐inflammatory M1 activation state. The rational for the use of statin as a therapeutic agent to inhibit macrophage polarization has been demonstrated recently (Li et al. 2013; van der Meij et al. 2013). Results show that M1/M2 shift inhibition at early stage of limb ischemia prevented impaired mice walking ability, thereby demonstrating that pro‐inflammatory muscular state plays a critical role in PAD‐related impaired walking ability in our mouse model. 18FDG used in PET imaging is uptaken by macrophages (Joshi et al. 2011). Thus, PET scan results showing increased muscle glucose uptake in early ischemic phase may also reflect increased number of infiltrating pro‐inflammatory macrophages. Rapid local muscle inflammation in response to ischemia may, therefore, be a transient deleterious mechanism contributing to walking ability impairment. With time, compensatory increased limb perfusion will provide sufficient basal state muscle nutriment though not enough to guarantee appropriate energy fueling during functional activity (i.e., walking).
To gain insights into skeletal muscle energy metabolism, in response to early and late stages of ischemia, index of energetic state (PCr/γ‐ATP and PCr/Pi ratios) was measured using 31P‐MRS. 31P‐MRS is at present the only available technique permitting noninvasive in vivo measurement of major phosphorylated compounds involved in muscle energy metabolism: that is, ATP, and PCr (Boesch 2007). ATP is a substrate for all energy‐consuming cellular reactions, as its hydrolysis provides free energy (Ten Hove and Neubauer 2007). PCr acts as an energy buffer and serves as an energy transport molecule in the creatine kinase/PCr energy shuttle (Ten Hove and Neubauer 2007). No differences in resting ischemic muscle energy state were observed in our mouse model, both in early and late phases of limb ischemia, relatively to nonischemic contralateral limb. On the other hand, Boring et al. (2013) recently showed a drop of PCr and ATP levels 1 week after femoral artery ligation and excision in C57BL/6 wild‐type mice, compared to those before femoral ligation. In our 31P‐MRS experiments, a drop in the PCr/γ‐ATP concentration after iliac artery ligation and a significant increase in PCr/γ‐ATP and PCr/Pi ratios between early and late ischemic stages were observed not only in the ischemic hindlimb but also in the contralateral nonischemic one used as internal control for each mouse (data not shown). This phenomenon may be due to a systemic response following unilateral arterial occlusion, as previously reported (Yan et al. 2009), and may have interfered with local metabolite changes in ischemic limb in this study. However, clinical studies also reported no significant differences in resting muscle metabolism between PAD patients and control subjects (Pedersen et al. 2009). Similar concentrations of high‐energy phosphate metabolites have also been reported under resting conditions in patients with type 2 diabetes and hypercholesterolemia, two majors CV risk factors for PAD, as compared to corresponding control healthy subjects (Laaksonen et al. 1996; Scheuermann‐Freestone et al. 2003). Taken together, these data indicate that energy reserve is unchanged in resting skeletal muscle compared to nonischemic muscle at any stage of peripheral ischemia.
In conclusion, results presented herein provide novel insights into the intrinsic adaptive mechanisms taking place in early and late phases of muscle ischemia. These ischemia‐associated changes in skeletal muscles may contribute to PAD‐related walking ability impairment.
Clinical perspective
PAD is a common disorder limiting patient's walking ability. To date, no pharmacological therapy for PAD exists. Only a better understanding of biological mechanisms underlying PAD may help develop new therapeutic strategies aiming at improving patient function and quality of life. This study indicates that both pro‐inflammatory T cells and macrophages are implicated in early ischemia whereas late ischemia is associated with impaired muscle glucose uptake in a mouse model of PAD. Hence, treatment aiming at preventing modulation of muscle macrophage and T cells toward a pro‐inflammatory phenotype in early disease stage and/or impaired muscle glucose uptake during late disease stage might improve clinical symptoms of patients with PAD.
We thank Pascal Laurant (University of Avignon, Avignon, France) for lending us the rodent treadmill.
Conflict of Interest
None declared.
Funding Information
This study was in part funded by the CIBM of the UNIL, UNIGE, HUG, CHUV and EPFL and the Leenaards and Jeantet Foundations.
- Manuscript Received: January 15, 2014.
- Manuscript Accepted: January 20, 2014.
- © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.


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The Phoenix Suns are not only a resilient bunch, but an optimistic group of players, too.
First they bounced back from a 2-0 deficit in the best-of-seven Western Conference finals to even the series then quickly shifted their focus from a surprising loss Thursday to forcing a possible Game 7.
The Suns lost Game 5 on Thursday on a last-second shot by Ron Artest that gave the Lakers a 3-2 edge in the series, but they weren't despondent or even discouraged by the sudden turn of events in the waning moments of the game.
Phoenix, which trailed by as many as 18 points in the third quarter, caught the Lakers down the stretch and tied the game on Jason Richardson's 3-pointer with 3.5 seconds left.
"We just fought back," Channing Frye said. "We just started believing in each other and just kind of started playing our style of basketball, just picking up the pace. The guys were playing extremely unselfish, attacking the rim and being aggressive."
On the next possession, though, Kobe Bryant took a pass from Lamar Odom, but his jumper fell short of the basket and Ron Artest took the rebound and put it back for the game-winner. The Suns, stunned by Artest's shot, walked dazed off the court.
Their shock didn't last long, though.
"We got back into the locker room (and) we already started shifting our focus to Game 6," Suns guard Steve Nash said. "If we continue to encourage each other, continue to draw the best out of each other, you know, I think we can have a lot of positive energy back home in Game 6. Our fans will be terrific and we have a lot of belief (in ourselves)."
Nash has seen his share of remarkable game-winners and this was just one more.
"We just got to be really fortunate it didn't happen in a deciding game," Nash said. "So at the end of it, we go home and got a chance to hold serve. And like I said, we have a great opportunity after that to come back here for a Game 7 and avenge this one."
Said Amar'e Stoudemire: "We got to swallow this one …. We had a chance to win. We didn't quite pull it out. We got to get back to Phoenix, try to get that win, tie the series back up. See if we can do that."

subset: commoncrawl


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The Web is full of entertaining videos, but I usually don't see them unless someone sends me a link.
The problem is that those interesting videos are hidden like Easter eggs around the Web. You have funny user-generated content and mash-ups on YouTube, original comedy on FunnyOrDie and the Onion News Network, "Saturday Night Live" shorts on Hulu, and other videos scattered elsewhere.
What this situation is crying out for is a respected aggregator, a TV Guide for the Web. I have yet to find a Web site that fits the bill. I would have thought TV Guide or EW.com would have stepped up to do it, but no. Sites are either pushing their own content or are cluttered with a lot of crap videos.
Just for grins, I've started a separate Web site on Google's Blogger to embed some of my favorite videos and provide links to others. The site is called One Stop Video. For now, it's just an experiment.
A word of caution, however, some of these videos are rated R for language and content. I'll post ratings for each video.


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Today I'll be sifting through the extensive responses to our Best China Books survey, which was a very short questionnaire tied to the very short reading list in our very short new book This Is China. The respondents gave us *lots* more suggestions, as well comments on and criticisms of the books we selected, which I'll be incorporating in a new and longer survey where we'll start breaking the list down by level of familiarity with China and also by subject and study goals. We'll keep a current list posted online, too – I may do this at Librarything, to make it easier to find books.
In the meantime, you can see how respondents rated the books we listed at Best China Books. The top rating for history was a well-known standard: Fairbank, John King, & Goldman, Merle. (1998). China: A New History. More generally, Jonathan Spence's books are leading the lists. Across the board, he and Peter Hessler got top marks, but many other friends and colleagues aren't far behind. For business readers, Tim Clissold's Mr. China holds the top spot. In fiction, Lu Xun's The True Story of Ah Q is in the lead for fiction. It's set in the revolutionary China of 1911.
Only one respondent who reminded me of the people who write most of the "answers" on Yahoo and other online Q&A sites, with comments like, "I don't know much about this but in my opinion…." or "Just guessing but maybe it's…."
The survey is still open, so do join in if you haven't already by clicking here.


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This afternoon at the San Diego Convention Center, the beautiful cast of True Blood took the stage, delighting thousands of fans who had waited in line for literally hours. Before they were introduced, however, we were treated to a short clip of what we can expect from the rest of the season. Fans, you do not want to miss this. For those of you who have been thinking that this season has been a bit too slow for your liking, strap yourselves in and hold on. Eric/Sookie fans squealed – a lot. Bill fans roared their pleasure. Sookie amazes with a fantastic display of her power. Well, you can see for yourself in the clip we posted here.
First on the stage was the lovely Kristin Bauer Van Straten. Pam is a fan favorite and they let her know it with a loud welcome. Next up, Alexander Skarsgard. I still do not have my hearing back. The applause was tremendous, and yet Skarsgard maintained a cool and almost humble demeanor. Kevin Alejandro was next followed by Nelsan Ellis. The crowd roared for Lafayette almost as much as for Skarsgard, and Ellis takes it all in stride. Ryan Kwanten came out sporting a bit of a new look, and the crowd loves it – and him! Deborah Ann Woll was introduced next, and Jessica is another Truebie favorite. Rutina Wesley brought on more cheers, because what's not to love about Tara? Stephen Moyer and Anna Paquin finish off the cast with Alan Ball last, but certainly not least, in my book.
Moderator Tim Stack begins asking his questions and he only asks a few so that he can open up the floor to the fans. Nice touch, Tim! Anna Paquin talks about Sookie being a bit divided. "There's a part of her that's sweet and a part of her that has really dirty vampire sex. Possibly with two vampires. And maybe werewolf sex!"
Nelsan Ellis reveals that Lafayette doesn't really want to do the supernatural thing, especially with witches.
Deborah Ann Woll talks about the final scene in episode 4.04, when Jessica gives Jason some of her blood to heal him. "Giving Jason blood has after effects, and that's what's going to be interesting."
Alan Ball said that we will find out about the creepy doll and whether or not Arlene's baby is evil. He asked the crowd what we thought about the baby, and most believe he's evil. One fan shouted out "KILL THE BABY!" That shocked even Alan, who replied, "I knew True Blood fans were sick, but not that sick!" It was all in fun!
Regarding the famous and highly anticipated "shower scene", the only thing Alan would say was, "There will be a moment on the show when Sookie and Eric are in the shower. It will be weirder and dirtier."
Alexander told us his favorite scene so far this season: Eric's swim in the sunlight. Deborah's was when Jessica was throwing eggs at Hoyt. She said they had a lot of fun with that. Alan said his fave was Jason's gang rape, and Ryan said, "I'll take it!" Rutina's favorite scene was the cage fight. She secretly loves the UFC.
In talking about some points for the rest of the season. Kevin Alejandro was asked if Jesus was dark, and he says, "There's a little darkness in all of us, but we'll have to wait and find out what that is."
Kristin Bauer delighted fans when she talked about filming the scene where her face starts to peel off from Marnie's curse. After applying all the makeup and goop made out of tapioca pudding and coffee grounds, they told her to 'find the hole and push up.' She loves it when the show can make us squeamish.
The floor was opened up to fan's questions, and there were some really great ones. Alan was asked if Eric would remember his time with Sookie after he regains his memories. He would not say because he didn't want to give that spoiler away. He was also asked if we would get to see when Eric made Pam his child. Alan said yes, in season five.
When asked if there was any hope for Sookie and Bill, Anna said, "It's complicated." She also answered a fan's question about the lack of Sookie's telepathy this season. "It's still there, but we don't always see it."
Nelsan was asked if Lafayette weren't dating Jesus, who would he choose, Eric, Bill or Jason? Ellis says Jesus is pretty happy with his current squeeze, but he's do all four!
The next fan didn't really have a question. She just wanted to yell, "What were you thinking? You are all going to hell!" Who could be so bold? Only Janina Gavankar (Luna), who stood in line patiently waiting her turn to ask her fan Q.
An interesting question came from a fan who wanted to know how Eric's memory loss would effect his part in vampire relations and politics, when he eventually is healed. Alan says, "The vampire community is under siege this season, so they all band together to fight a common enemy."
Are Sam and Tara over? Alan says, of course, nothing is ever over on True Blood.
Anna explained to one fan who wanted to know where she got her inspiration to play Sookie without all the powers. "Even though these characters are supernatural, they are grounded in human reality. What Sookie goes through is real."
Stephen adds, "It all comes down to the writing. Unless the writing is good, you can't do it. We've got an unbelievable group surrounding Alan."
Alan talked about some supernaturals that we have yet to see. "Spirits without bodies. Brujo-based creatures. More research to come."
Alexander wants Eric to fly more. He enjoys that and the flashbacks to his Viking past.
Alan was asked about the differences between "Six Feet Under" and "True Blood". "True Blood is never depressing. The supernatural nature makes it fun to come to work."
What if Pam lost her memory & Eric had to take care of her? Kristin says, "I'd like to think he'd do anything to save Pam." Skarsgard adds, "He'd do anything for her. We saw last season that he'd die for his progeny."
The inevitable book vs. show question was answered by Alan. "One, the books are all Sookie's story. We can't do that. Two, if we stuck to books, there'd be no surprises. Though we stray a bit from the books, we try to stay very true to the spirit of them."
Fan: will we every get some waitresses at Merlotte's with some staying power for the guys to look at? I don't know where this guy has been for three seasons, but he missed Sookie and Arlene and Jessica. Alan answers though. "She (Sookie) takes her clothes off all the time! Just wait for this season." Alan says we'll meet more waitresses this season, but how long they stay he won't say.
The cast were asked about their auditioning process for True Blood. Nelsan remembers thrusting his crotch slightly at Alan during his audition. A tip to remember, Alan says. Kevin had to do a chemistry read with Nelsan as did other actors. 'I won.' Deborah remembers rolling around on the floor during her audition. 'I guess it worked.' Rutina almost wrecked her car when she got the call that she landed the role. Stephen's story had me teary-eyed. He was burgled the same day he put his audition on tape. Computer was stolen. His son immediately ran into his room and checked for his teddy. He got the call to live audition the next morning. Met Anna the next day. 'It's been alright.' Anna stalked Alan until he said yes.
And that's about it! We've got hundreds of photos loaded in our gallery, with more on the way, so be sure to head over there with a cup of your favorite refreshment and browse!


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For true relaxation Maldivian style, a tranquil environment and amazing 24 hour diving, Ellaidhoo Maldives by Cinnamon ticks all the boxes. There's no denying the main attraction on this small island is the incredible house reef, and the PADI certified dive school organises lessons and excursions so you can discover the spectacular underwater scenery both day and night.
Your journey starts here
Experience one of the best diving reefs in the world, home to a wide range of marine life including sharks and manta rays. A school of 100 giant jack fish even come by the hotel decks each evening to be fed by the hotel staff. Fabulous diving, a relaxing spa and excursions and activities ensure that there's plenty to suit everyone's interests.
In North Ari Atoll approximately 1 hour 30 mins by speedboat (included in the price of your holiday). To upgrade to a seaplane transfer which takes approximately 20 minutes, please call us.
All inclusive in a Standard room with private facilities.
There's no need to shop around. Find a cheaper like-for-like tour from another company, and we'll not only match their price but also give you a £100 discount when you book with us.
They have in-depth knowledge of the places you'll visit and will be on hand every step of the way with commentary entirely in English. 96% of customers rate them 'excellent' or 'good'.
We understand things change, often beyond your control. If you need to change your holiday plans, we offer a flexible deposit up until your balance is due.


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Charles Apellaniz
I am a published comic book artist and storyboard artist but have been looking to do voices for a loooooooonnnnnnngggggg time. i do voices everyday when I draw comics to get the feel for the characters i am drawing and i love it and my wife a kids catch me all the time. so even though i am new you can see that i will have fun. i also did plays at church when i was either the main character or supporting and had to not only change my voice but my look as well.
English (North American), English
North American
Middle Aged
Special Skills
Audiobooks, Cartoons, Educational, Internet, Jingles, Music, Podcasting, Radio, Telephone, Television, Videogames
I have none as far as professional voice work, but as a I said i love to do voices for the characters i draw. please try me out if anything.
associates in graphic and web design
headset with mic
Computer & Software
laptop with CS5 creative suite
Payment Terms
Hire Charles as a voice actor. Payment is made through Voices.com SurePay, an escrow service that guarantees your work is done to your satisfaction. Send payment securely by Visa, Mastercard or PayPal through Voices.com then download your files from the website.
Watch a video to see how to make a payment
Next Steps
To contact Charles Apellaniz about your project, request a quote today. We'll walk you through the process step-by-step. It's fast and easy.
This talent has not uploaded any demos yet.
Hire Charles Apellaniz as a voice actor. Working with Charles Apellaniz is fast and easy. We'll walk you through the process step-by-step.
Request a quote for your project today.


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Range Reports Record Level Production in 2Q
Range announced that its production for the second quarter 2010 reached a record level. Production for the second quarter averaged 472 Mmcfe per day, representing a 9% increase over the prior-year period. Adjusting for the sale of the Ohio properties, which closed at the end of March 2010, the second quarter production growth rate would have been 13%.
Second quarter 2010 production materially exceeded the Company's previous guidance of 450 to 455 Mmcfe per day. Due to the better than expected performance, Range was able to achieve its 30th consecutive quarter of sequential production growth. Driving the record level of production was higher production from all of the Company's divisions. In particular, production from the Marcellus Shale division saw the largest increase due to continued outstanding drilling results.
The Company also announced that preliminary second quarter 2010 oil and gas price realizations (including the impact of derivative settlements) averaged $5.07 per mcfe. This represents an 18% decrease from the prior-year period and a 9% decrease versus first quarter 2010. For the second half of 2010, 77% of anticipated natural gas production is hedged at an average floor price of $5.54 per mcf and for 2011 51% is hedged at a $5.73 floor.
Second quarter drilling expenditures totaled approximately $240 million, funding the drilling of 87 (65.6 net) wells. A 97% success rate was achieved. For the first six months of the year, 159 (123.5 net) wells were drilled. At June 30, 53 (38.6 net) wells were in various stages of completion or waiting on pipeline connection. To date, Range has drilled 146 horizontal Marcellus wells, of which 29 are awaiting completion and four are awaiting pipeline hook up.
Commenting on the announcement, John Pinkerton, Range's Chairman and CEO, said, "Our plan indicated that it would be mid third quarter, before we would be able to overcome the loss of production from the Ohio property sale. Fully overcoming the loss in the second quarter is extraordinary performance by our operating team. While all our divisions delivered, the Marcellus Shale is leading the way. Our 2010 year-end exit rate target for the Marcellus Shale of 180 to 200 Mcfe per day, net, is now well within our sights. In addition to rising production, our emphasis on a low-cost structure is continuing to pay dividends, as second quarter unit costs once again declined. With our large inventory of high-return projects, low cost structure, attractive hedge position and strong financial position, we are exceedingly well positioned to continue to drive up per share value in the quarters ahead."
- Range Recalls 'Incredibly Creative Solution' for Marcellus/Utica 'Problem' (Apr 26)
- Oil, Gas CEOs Optimistic about Future of E&P in North America (Mar 10)
- PwC: Oil Price Stability Drives Uptick in M&A Deals (Jul 28)


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- Virginia native Kelly Millspaugh Thompson's company Stylish Patina is where vintage meets modern in home furnishings & decor. She believes that all spaces should have modern function and a vintage soul. ... read moreFrequently Asked Questions
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It is learnt that since the time, Amitabh Bachchan had said that Vidya Balan reminded him of his favourite actress Waheeda Rehman, the actress just can"t stop blushing.
The senior Bachchan had said in a recent interview that Vidya 'has the grace and beauty of Waheeda Rehman. He added that Vidya"s Indianness reminded him of the star with whom he worked in his earlier films like Trishul, Kabhie Kabhie and Adalat.
A pal of Vidya"s reveals to us that Vidya is extremely delighted by the superstar"s compliments. "What more she could ask for. She is deeply humbled and at the same time dumbfounded." The pal tells us.
Vidya has stated in this regard, "It was very kind of him (Amitabh Bachchan) to say something like that. Waheedaji was known for her beauty and grace and being compared to her is like receiving a great award. Amitji has been generous with his compliments. I"m speechless."
She further adds, "I got the opportunity to work with him and Abhishek and it has been a great experience. I sincerely wish I get more roles like this in the future."


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In my listening to others to myself as well I have come to recognize one most common desire by nearly all of us. That most frequent want is: to add, to improve, to mitigate or eliminate something within our lives, more succinctly nearly all of us wish to make a positive change in our life by either increasing and or decreasing a condition. Unfortunately, however, our choices of where to focus our energies and which approach to implement with our desire to attain our success often run from misguided, to nonexistent, to unrealistic, to ineffective, to unsustainable and more often than not, are doomed to fail from the onset, or worst of all we end up succeeding but realize we have achieved one important outcome and have irretrievably lost two more precious parts of our life in the process with our unbalanced approach.
In his legendary bookthe Art of War Sun Tzu states If you know the enemy and know yourself, you need not fear the results of a hundred battles. Likewise another quote from Sun Tzu: Strategy without tactics is the slowest route to victory. Tactics without strategy is the noise before defeat. Perhaps this great ancient Chinese military generals philosophy can also apply nicely to each of us with our quests to achieve, to contribute, and gain harmony with our lives and relationships. First and foremost perhaps we must come to know ourselves and be courageous and candid with both our gifts and deficiencies. To truly know our enemy must we not fully know ourselves? Whether our challenges are health/fitness, relationships, financial, serenity or causes we desire to champion; our foundation for moving forward must rest on the understanding of who we are and how we have more often than not stood in our own way. I have met the enemy, and I see them clearly each morning in my own mirror.
So what War do you wish to achieve victory with my friends? Is it your health? Is it with relationships? Is it financial? Is it a cause for Humanity? And once you have decided upon the ONE important focus for yourself, do you see, do you have the clarity to discern that you are not a victim, but rather the master of your fate?
Seek out mentors, ponder extensively, educate yourself with research. Write out your vision outcome and then, and only then, plan the tactics, the specific activities that will progress your war forward with incremental victorious battles. Be wise with what you do, as well as with what you dont do, and victory, success and feeling a sense of completeness and peace will be yours. As Shakespeare writes in The Merry Wives of Windsor the world will be your oyster to open with your sword of wise strategy swung in the direction dictated by effectively planned tactics.
No greater tragedy can occur with our lives than to Win Our War and with this success come to realize that we had wasted our time, resources and energy in fighting the WRONG War. Choose your Wars wisely my friends, choose them most wisely, for there is but one, and only one Today, one lifetime for each and every one of us. Then move on to pick the battles you will fight in support of your WAR; do so with courage, wisdom, enthusiasm, determination and yes, perspiration as well.
Michael Starr is the owner of Executive Coaching Services. He can be reached at www.executivecoachingservices.net or by calling 501-908-2298.


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It's about socialism. Didn't know that either. Compare the actions of Raskolnikov with those of the communist revolutionaries (Lenin, et al). A system of government that uses violence as a legitimator, with the good intention of spreading wealth, can succumb to happenstance (shit can happen) and the whole thing can go wrong. Those in any way involved in the violence and killing inherit some heavy karma.
Excellent psychologial novel. The question in this murder mystery is not "who" but "why".
Crime and Punishment
by Feodor Dostoevsky
Posted by Thomas Beek at 10/12/2005 07:44:00 PM
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Yissel Ayala
Experienced Fashion Designer For Hire
With both a BFA in women's sportswear and over five years of retail experience, I'm ready to provide a fresh perspective and diverse skill set to increase your sales via a creative role in a corporate setting. I live for research and development, and I thrive in a fast-paced environment where every day brings new challenges and surprises.
Specialties: ENTHUSIASM, r&d, CAD flats, public speaking, presentation, self-starting, strong color sense
- Extensive knowledge of fashion industry and design trends
- Proficiency in Adobe Creative Suite, Microsoft Word, Excel
- Hand illustration and computer-aided sketching skills
- 4+ years in retail in a managerial role
DC Comics/Vertigo Designer Oct. 2016
Contributed designs to Gail Simone's, "Clean Room," series, used on main character
Geek Girl Brunch (GGB) Co-founder May 2014 - Present
Ensure consistent brand design and voice by co-managing over 80 international chapters
Research geek culture trends to develop themes for GGB objectives and brand
Collaborate with artists and partners to develop visual content for events and campaigns
Curate and procure goods and services from vendors and marketing companies through cold emailing, networking
and following through with warm and qualified leads. This includes coordinating shipments of promotional goods from
Warner Bros., securing a year-long sponsorship from Forbidden Planet, acquiring free catering for our NYCC afterparty
Present at and host various GGB events and comic book conventions
Valiant Entertainment Design & Sales Intern May 2015 - Oct. 2015
Hand illustrated comic pages for editorial and promotional purposes
Conceptualized, designed, and photographed original content for social media
Researched graphic design trends for marketing, forecasts, and creative inspiration
Created and maintained several ongoing newsletter, sales, and image databases
Grossed over 2k in one weekend by hand-selling at a convention
Dr. Martens AirWair USA Senior Keyholder Oct. 2010 - Oct. 2014
Earned the store over 13% of its annual sales via display and excellent customer service
Designed, sourced, shopped, and budgeted for in-store displays
Michael Kors Inc. Design Intern – Special Markets Feb. 2010 - May 2010
Supported all areas of design including: hand drawing and garment specs, CAD renderings of original denim embellishments, updating pieces on PDM, assembling boards for line meetings, managing linesheets, choosing and filing swatches and denim samples, trim sourcing, sewing
Contributed original designs to the Holiday and Spring 2011 MICHAEL Michael Kors collections
Maintained the showroom and set up merchandise presentations for client meetings
Intricate Concepts/ Fourfront Clothing Graphic Design Assistant Feb. 2008 - Aug. 2008
Landed the company it's greatest exposure to date-- 3 million people-- by negotiating a deal to have our product repeatedly featured on a hit MTV Show.
Contributed a featured design to client's brand used in marketing and packaging
Produced full color CAD renderings of garments
Assisted in the design of t-shirt graphics and branding for various clients
Museum @ FIT Photography Assistant Aug. 2004 - May 2005
Organized photographs for documentation, publicity, and promotion
Handled museum pieces from Costume & Accessories departments for shoots
Fashion Institute of Technology, State University of New York Bachelor of Fine Arts - Women's Sportswear May 2010
Politecnico Di Milano, Milan, Italy Knitwear Design Focus Fall 2008
H. S. of Fashion Industries, New York, NY Fashion Endorsed Diploma May 2004
Hand Sketching, Adobe Photoshop, Illustrator, In Design, Draping, Flat Pattern Making, Microsoft Office, CAD Design, Garment Construction, Concept and Line Development, PDM, Spanish, Flat Sketching, Spec'ing
Volunteer StyleCareers Fashion Career Fair NY Fall 2015, 2014, 2011, 2010
Volunteer Food Server Bowery Mission Winter 2015
Featured Guest Artist HarperCollins Publishing Spring 2014
Counselor/ Design Teacher Camp Move IT Summer 2010
Volunteer Mercedes Benz Fashion Week F/W 2009, S/S 2008, F/W 2007
NJ Comic Expo 2016 Anime Fan Fest 2016 NJ Comic Expo 2015
Ladies of the Con Pokemon 20th Anniversary Panel DC Comics Dialogues
#MakeComics: Art of Lettering #MakeComics: Designing Spectacular Covers
#MakeComics: Digital Comics
TL;DR : Where I've contributed
Geek Girl Brunch, DC Comics/Vertigo, Valiant Entertainment, Dr. Martens AirWair USA, Michael Kors Inc. , Intricate Concepts/ Fourfront Clothing, Museum @ FIT,
StyleCareers Fashion Career Fair NY, Bowery Mission, HarperCollins Publishing, Mercedes Benz Fashion Week,
among others...
It serves to prove that the fashion world is full of geeks and people inspired by them and their subcultural interests-be it tech, sci-fi, comics, Steampunk, anime, etc… Designers teach us that what we often consider to be the most geeky — a term that hasn't always been viewed in the most flattering light — can too serve as a driving force behind a bevy of beautiful, wondrous things, that are seen by the general (read: fashionable) population as commercially viable, praiseworthy, and sought after.
Leave it to the fashion industry to elevate geek culture to haute heights.
Geeks are no longer the underdogs, or the outcasts. We need not be afraid of fashion because, as this blog will prove, we are clearly the ones creating it.
Here's to bridging the gap between geek and chic.


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William Paterson University student job postings
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Find information about William Paterson University student job postings, and registering for elective seminars. Which college degrees give you the best chances of finding employment after graduation? You can start working in a high-paying business field with just an associates, or a bachelors degree. To begin, browse accredited business degree programs and certificate courses online. Concentrating on William Paterson University student job postings, for example, you may gain an advantage in corporate recruiting being done on-campus. Check with the workforce development department for course registration deadlines, and career training program requirements. Please use the form above to request admissions info for William Paterson University.
As the economy improves, many firms are beginning to hire. Companies are in need of candidates that can fill a particular job position, so set up an introductory meeting to ask questions about the firm's history, and current business opportunities. Review your prior work experiences, internships, volunteer experience, interests and hobbies. For each item on your list, identify the skills and knowledge you developed, because many skills are transferable to the job you're applying for.
Before a first interview, analyze the open position that you are applying for. Review the job description carefully, and highlight both the employment qualifications and job responsibilities. At the same time, learn as much as you can about the company's mission, corporate services, product lists, and main competitors. Start your research on the company's website, then do an article search on Google, and attend career information sessions and job fairs that are held on campus. Beyond major requirements within the workforce development department, you may consider Copper Mountain College GED course as a complementary elective to your primary course of study.
Human Resources Analyst
UC San Diego
Under direction of the HR Manager, provide independent analysis and recommendations on issues pertaining to human resource administration that are often..
21 hours ago Administrator II
Los Angeles Superior Court
The structured interview will consist of customer service, decision making, fact finding and data analysis, financial management, integrity/honesty..
4 days ago Summer Undergrad Intern
Bond Street
Bond Street is transforming small business lending with technology, data and design. Inspired by our mission to help small businesses realize their dreams.
2 days ago Police Officer 1-Intermittent
State of Ohio
Checks identity of unknown persons on grounds & nature of their business; The mission of the Ohio Department of Mental Health and Addiction Services (OhioMHAS)..
4 hours ago Credit Analyst
West Town Bank & Trust
West Town Bank & Trust is currently seeking a Credit Analyst . A Bachelor's Degree in Finance or Accounting or equivalent relevant experience..
2 hours ago Account Specialist
This is a high-activity, high-volume presales role focused on maximizing the number of well-qualified new business opportunities..
5 hours ago


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Hi Associate,
We have an Urgent opening .Please see the Job Description Below and let me know if you have any consultant available. *Job Title : Senior ETL Developer and Data Modeler* *Location : Newark, NJ* *Duration: 6+ months* *Rate: $45/hr c2c* *Need visa copy and photo id during submission* *Job Title Senior ETL Developer and Data Modeler* *Technical/Functional Skills * - Senior ETL (Informatica, MS SSIS is a plus) developer and Data Modeler with at least 8 years of IT experience (atleast 4 years of onsite) with great communication and technical knowledge. - Need to know supporting technologies like Unix Scripting etc - Should be able to independently work with clients and Business to discuss, analyze and gather requirements - Should be able to follow process, provide process and technical improvements - Should be able to lead, work with and guide offshore and should set-up , improve process and quality. *Generic Managerial Skills * Should be able to schedule and drive meetings with Business for gathering requirements *Thanks * *Wasim AhmedUnited Software Group Inc.. 565 Metro Place South. Suite # 110 Dublin, OH 43017 Phone: 614-588-8477Fax: 1-866-764-1148 * *wasimah...@usgrpinc.com* <wasimah...@usgrpinc.com> *www.usgrpinc.com* <http://www.usgrpinc.com/> To unsubscribe from this mailing, please reply to this email with the word UNSUBSCRIBE <techsupp...@usgrpinc.com,pan...@usgrpinc.com?subject=UNSUBSCRIBE%E2%80%9D.>in the subject line, or click here. *_______________________________________________________________________________* *USG is an Equal Opportunity Employer. Qualified applicants will receive consideration for employment without regard to race, color, religion, sex, age, disability, military status, national origin or any other characteristic protected under federal, state, or applicable local law.* -- You received this message because you are subscribed to the Google Groups "Resumes" group. To unsubscribe from this group and stop receiving emails from it, send an email to resumes+unsubscr...@googlegroups.com. To post to this group, send email to firstname.lastname@example.com. Visit this group at https://groups.google.com/group/resumes. For more options, visit https://groups.google.com/d/optout.


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Thank you so much for stopping by to check out this week's Amusing Challenge! 🙂 With Valentine's Day just around the corner, we'd love to see what Valentines you are creating. Throughout the month I have different sorts of challenges…"use it", themes, sketch, etc. I will have a Weekly Winner and at the end of the month, those 4-5 winners will go into a prize drawing for one lucky Grand Prize winner. February's Prize Winner will receive sn@p cardstock birthday sticker sheets. This is perfect for project life type scrapbooking or cardmaking!
Carol had a wonderful idea of creating a cascade of hearts using the A Muse Studio heart trio die set. She also used polka dot ribbon from amuse studio. Love the layers of red and richness of this card.
Tricia (of 1PhatChic) showed us her playful side…I always love to include a little treat with my Valentines and this Tic Tac package is adorable! Tricia used Mad for Plaid and My Punny Valentine in this yummy Shell and French Roast cardstock combination. All supplies are from amuse studio.
I love the googly eyes! Even the inside of her Valentine treat is finished off perfectly with this little band. Sweet!!
Cyndi did some paper piecing with these little ladybugs from the A Muse Studio stamp set, My Punny Valentine. It's a colorful and fun idea – especially if you don't feel like pulling out markers or pencils. She also used Cherry Baker's Cording and the 6×6 paper pack called Everything's Rosy…which are also from A Muse Studio.
I went super CAS with my Valentine. I used a few A Muse Studio stamp sets: Say it With Style, Brushstrokes and All for Love. I also used Blush ink, Blush Splash and Blush Baker's Cording as well as Kraft and Vellum cardstock from A Muse Studio. I was inspired by a card I saw on Pinterest and set about creating my own version with supplies from amuse studio and what I had on hand.
PS. Don't forget that you can still play along with the current Amusing Challenge going on until midnight, Sunday night. It's a "flocking" use it challenge and you can enter it here!
My Punny Valentine is part of the A Muse Studio January Collection and the LAST day to order from that is Feb 14th…which will be before the next Amusing Challenge. Don't miss out on these stamps, heart doilies, sequin mix and fantastic die sets.
I hope this inspired you to make some Valentines as well! It's super easy to play along with Amusing Challenges, and just as a reminder: 2 entry limit and no digis please!
■Link your creation to your paper crafts blog, gallery (SCS or Flickr) before midnight of the following Sunday.
■Use any product but please follow the challenge criteria.
■Double your chances to win by using A Muse Studio products (just leave me a reminder in the comments please)


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Baku. 5 November. REPORT.AZ/ Ministry of Education and Science of Russia approved the form of a certificate that migrants will receive after passing comprehensive examination on history, law, and Russian language. Report informs citing ITAR-TASS, the corresponding order has been prepared by the Ministry.
From January 1, 2015 the certificate will become mandatory for the legal employment of migrants, obtaining a temporary residence permit or permanent residence.
To obtain a work permit a person will need to correctly answer at least 30% questions of every test. Exam modules contain 20 tasks.