text:
While ryanodine receptor 1 (RyR1) critically contributes to skeletal muscle contraction abilities by mediating Ca2+ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK) senses contraction-induced energetic stress by phosphorylation at Thr172. Phosphorylation of RyR1 at serine2843 (pRyR1Ser2843) results in leaky RyR1 channels and impaired Ca2+homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca2+-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise.
Determine the effects and early time-course of resistance exercise on pRyR1Ser2843 and pAMPKThr172 in type I and II myofibers.
7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg) performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser2843 and pAMPKThr172 levels were determined by western blot and semi-quantitative immunohistochemistry techniques.
While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser2843 increased 15 min and peaked 30 min (p<0.01) post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser2843 levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05). pAMPKThr172 also increased 15 to 30 min post exercise (p<0.01) in type I and II myofibers and in whole skeletal muscle.
Resistance exercise induces acutely increased pRyR1Ser2843 and concomitantly pAMPKThr172 levels for up to 30 min in resistance exercised myofibers. This provides a time-course by which pRyR1Ser2843 can mechanistically impact Ca2+handling properties and consequently induce reduced myofiber contractility beyond immediate fatiguing mechanisms.
Citation: Gehlert S, Bungartz G, Willkomm L, Korkmaz Y, Pfannkuche K, Schiffer T, et al. (2012) Intense Resistance Exercise Induces Early and Transient Increases in Ryanodine Receptor 1 Phosphorylation in Human Skeletal Muscle. PLoS ONE 7(11): e49326. https://doi.org/10.1371/journal.pone.0049326
Editor: Petras Dzeja, Mayo Clinic, United States of America
Received: June 10, 2012; Accepted: October 10, 2012; Published: November 16, 2012
Copyright: © 2012 Gehlert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This investigation was funded by the Federal Institute of Sports Science (BISp) IIA1-507 070103/09-10. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Human skeletal muscle tissue represents the vital organ for human locomotion. As such, it exerts a high plasticity to adapt to various modes of exercise [1]. For example, intense contractions of myofibers (as occurs during exercise) initially result in gradually decreased skeletal muscle contraction properties, including acutely reduced myofiber contractility, which occurs already after few seconds of high intense exercise [2], [3]. More importantly, intense exercise also induces sustained alterations in skeletal muscle contractility that last up to several min post exercise [4], [5]. This phenomenon is described as the slow recovery curve of myocellular fatigue [6] in which a sustained increase in relaxation time between periodic contractions of myofibers can be observed [7]. The molecular mechanisms contributing to these phenomena are still a matter of debate [8]. However, calcium (Ca2+) plays a fundamental role in the onset of skeletal muscle fatigue [9], [10], [11], [12] because its oscillation in myofibrillar areas links sarcolemma depolarization to muscle contraction, which is also known as excitation-contraction (EC) coupling [13]. The ryanodine receptor 1 (RyR1) channel complex is mainly involved in facilitating the accurate release of Ca2+from sarcoplasmic compartments into myofibrillar areas and triggers the important calcium oscillation that is required for continued myofiber contractions. RyR1 has been observed to be posttranslationally modified at several residues, predominantly by phosphorylation at serine2843 (pRyR1Ser2843) [13]. This modification contributes critically towards skeletal muscle contraction impairment upon exercise as it destabilizes the closed state of RyR1, thereby increasing the ratio of open channels [13], [14], which, consequently, facilitate uncontrolled Ca2+release and inaccurate distribution of Ca2+ions in myofiber compartments [14], [15]. Bellinger and co-workers determined that chronic exercise in a mouse model results in increased phosphorylation of RyR1 on Ser2843 and, consequently, impaired skeletal muscle contractility [14]. Additionally, total RyR1 levels in human skeletal muscle have been found to decrease in response to bed rest and increase due to long term exercise [16]. However, to date, no data are available concerning the acute phosphorylation level of RyR1 in response to exercise. Increased RyR1 phosphorylation is likely to occur in response to acute and severe resistance exercise as this exercise mode requires a high rate of Ca2+-oscillation and induces stress within myofibers. RyR1 phosphorylation might have an impact on Ca2+handling structures, transiently increased Ca2+levels and also on kinases relevant for mediating its own phosphorylation. Thus, an early increase in RyR1 phosphorylation after exercise could potentially provide a subtle cellular indicator for impaired Ca2+handling properties in a sensitive recovery period after exercise without affecting general myofiber contractility. Therefore, we hypothesized that increased phosphorylation of RyR1 at Ser2843 will be observed in an early and transient time-course in response to high intense resistance exercise in human skeletal muscle. ATP-dependent ion channels and myosin ATPase enzymes exert high rates of ATP turnover and, thus, energetic stress [17] in contracting myofibers, which consequently induces increased phosphorylation of AMP-activated protein kinase (AMPK). Because this emphasizes a contraction-induced effect, we further postulated that exercise-induced phosphorylation of RyR1 is accompanied by elevated pAMPKThr172 levels, indirectly indicating that increased pRyR1Ser2843 results from myofiber contractions.
Type I and type II myofibers show distinct mechanic and metabolic properties and offer different recruitment patterns during exercise. A distinct recruitment of type I and II myofibers in response to exercise [18], [19], [20] may induce a different load on these myofibers. This may consequently lead to different magnitudes of stress on downstream effectors which mediate the molecular bases for cellular signaling [21] and EC coupling [13]. Thus, we hypothesized that type I and II myofibers would present with differences in pRyR1Ser2843 and pAMPKThr172 levels in response to exercise, as would individual fibers within each myofiber population due to unequal recruitment of fibers in exercising skeletal muscle.
A model of maximum eccentric leg extensions on human subjects was used to apply high intense muscular workload. Here, we show that pRyR1Ser2843 and pAMPKThr172 levels increase significantly in an early and transient manner in both type I and type II myofibers 15 to 30 min post exercise. These data are highly significant as they describe the fiber type-specific response of RyR1 phosphorylation in response to acute exercise bouts and beyond.
Materials and Methods
7 healthy active, but not exceptionally resistance- or endurance-trained, male volunteers (age: 23±2 years, height: 185±7 cm, and weight: 82±5 kg) participated in the present study. All subjects were informed orally and in writing of the study's purpose and the possible risks involved before providing written informed consent to participate. The study was approved by the Ethic Committee of the German Sport University Cologne in compliance with the Declaration of Helsinki. Dates and times of exercise and muscle biopsies were documented for each subject. Subjects were assigned an anonymous ID during the study.
Standardization of Diet and Activity before Testing
Subjects were instructed to refrain from vigorous physical activity two days prior to baseline biopsies as well as prior the main stimulus. After fasting overnight, the subjects were advised to drink one standardized energy drink (Fresubin® protein energy drink, Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany; containing 20 g protein, 24.8 g carbohydrate 13.4 g fat, and providing 1260 kJ) two hours prior the training intervention to carry out training in the fed state. Subjects were allowed to drink water ad libitum.
Experimental Trials
On the day of the main trial, subjects reported to the laboratory at 07:45 AM. Prior to the resistance exercise bouts, a 5 min warm-up with a workload of 1 W*kg–1 on a cycle ergometer was carried out. A combined warm-up and main exercise program was carried out subsequently on an isokinetic leg extension machine. The program was automatically operated by the control unit of the isokinetic device. Range of motion, angle velocity, as well as resting time between exercise sets were equalized for each subject. The first phase featured 3 sets of 4 submaximal (30% of maximum force) concentric and eccentric repetitions and 90 sec resting intervals between the sets. The angular velocity was determined by 65°*sec–1 movement speed in concentric and eccentric phases. After a 3 min resting phase, the main stimulus was applied featuring 3 sets of 8 maximal eccentric repetitions with a 3 min rest between the sets. The angular velocity during the eccentric contraction phase was carried out at 25°*sec–1. The leg was passively returned to the extended knee position over the concentric phase to the starting position with an angular velocity of 75°*sec–1. All subjects were verbally encouraged during the exercise session to perform all 24 contractions with maximum force. The exercise regimen induced approximately 70 sec of maximum eccentric force on skeletal muscle.
After the exercise was completed, subjects walked a short distance (≈ 250 m) to the local outpatient clinic of the German Sport University Cologne. After a local anesthesia was applied to the vastus lateralis at its incision, muscle biopsies were collected from the exercised leg.
Muscle Biopsies
To prevent potential injury due to damaged muscle tissue under maximum eccentric contractions, baseline biopsies of the vastus lateralis muscle were taken at rest 5 days prior the exercise intervention. On the day of the experimental trial, biopsies were collected 15 min, 30 min and 60 min post exercise. The 3 biopsies were taken 2 cm distal from each other starting 2 cm distal of the resting biopsy. Muscle biopsies were obtained from the middle region of the vastus lateralis between the spina iliaca anterior superior and the lateral part of the patella. Biopsies were gathered 2.5 cm below the fascia using the percutaneous needle biopsy technique [22].
Tissue Processing
Muscle samples were freed from blood and non-muscle material, embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, Netherlands), frozen in liquid nitrogen-cooled isopentane and stored at −80°C. For immunohistochemical analyses, 7 µm cross sections of all biopsy time points were mounted in triplicates on Polysine® slides (VWR International GmbH, Darmstadt, Germany), aligned carefully for cross-sectional analysis, air-dried and stored at −80°C until further analysis.
Immunohistochemical Staining and Analysis of pRYR1 Ser2843 and pAMPKThr172
Immunohistochemical analysis was conducted as described previously [23]. Cryo-sections were brought to room temperature and subsequently incubated for 10 min at 37°C. Sections were fixed for 5 min in −20°C pre-cooled acetone. Sections were blocked in TBS containing 5% BSA for 1 hour at room temperature. Slides were then incubated overnight at 4°C with monoclonal primary phospho-specific antibodies. Antibodies recognizing phosphorylated RyR1Ser2843, (Abcam, Cambrigde, UK) [14], [24], total RyR1 (Abcam, Cambridge, UK), phosphorylated AMPKThr172 (Cell Signaling, Beverly MA, USA), total AMPK (Cell Signaling, Beverly, MA, USA) and A4.840 (Developmental Studies Hybridoma Bank, Iowa City, USA), which is raised against adult human slow myosin heavy chain type 1 (MyHC1), were used. Antibodies were diluted 1∶200 (rabbit pRyR1Ser2843 and total RyR1), 1∶100 (rabbit pAMPKThr172 and total AMPK) or 1∶300 (mouse A4.840) in TBS containing 0.8% BSA. Slides were rinsed 5 times for 5 min each with TBS and then incubated for 1 hour with appropriate polyclonal biotinylated secondary antibodies (DakoCytomation, Glostrup, Denmark) diluted 1∶400 in TBS. Negative controls were processed in the same way as the experimental cross-sections; however, primary antibody incubations were substituted by incubation with 0.8% TBS. Thereafter, slides were incubated for 1 hour with Streptavidin biotinylated Horseradish Peroxidase complex (Amersham Biosciences, Uppsala, Sweden) diluted 1∶400 in TBS. After rinsing, staining was carried out using a 3,3`-diaminobenzidine (DAB) solution (0.09 M phosphate buffer (pH 7.4), 2.2 mM DAB, 7.03 mM ammonium chloride, 0.93 mM nickel sulfate, 10.44 mM ß-D-glucose and 0.000024 mM glucose oxidase). After dehydration, the stained sections were embedded in Entellan (Merck, Darmstadt, Germany) and supplied with a coverslip. Muscle cross sections from all biopsy time points of 1 subject were stained within a single batch using the same antibody dilution and development time to minimize variability in staining efficiency. Duplicate mounted serial sections of each time point were stained on a single slide for MyHC1 with the monoclonal A4.840 and polyclonal pRyR1Ser2843 antibodies as well as for pRyR1Ser2843 and pAMPKThr172. The intensity of myofibrillar staining for pRyR1Ser2843 and pAMPKThr172 was determined separately for type I and type II myofibers. Additionally, myofiber type-specific levels of total RyR1 and total AMPK were determined separately for each time point. pRyR1Ser2843 and pAMPKThr172 localized specifically to type I and type II myofibers as did total RyR1 and total AMPK.
Staining of pRyR1Ser2843 and pAMPKThr172 was additionally carried out in identical myofibers to determine the impact of exercise on both parameters in single myofibers. All slides were examined with a Zeiss Axiophot 200 light microscope coupled to a Sony 3CCD Color Video Camera. The background intensity was equalized for each slide and subject prior to image capturing. Differences in staining intensity between time points were determined as the difference between background intensity and measured sarcoplasmic intensity. Digitally captured images (200x magnification) were analyzed to determine the fiber type. To determine the fiber type-specific phosphorylation pattern in the sarcoplasmic compartment of myofibers, pAMPKThr172- and pRyR1Ser2843-stained cross sections were compared to the consecutive area, showing the corresponding type I and II myofiber distributions. The specific staining intensity was quantified for each myofiber by selection of the sarcoplasmic region of the myofiber and its subsequent assessment by optical densitometry using the software ImageJ® (National Institutes of Health, USA). Intracellular pRyR1Ser2843 and pAMPKThr172 content was set as the mean staining intensity. 70±6 myofibers per biopsy time point and subject were analyzed for fiber type-specific pRyR1Ser2843 and pAMPKThr172 signals.
Tissue was prepared as described under "Immunohistochemical Analysis". The following primary antibodies were used: mouse α-actinin (Sigma-Aldrich, Steinheim, Germany; dilution 1∶250) and rabbit pRyR1Ser2843 (Abcam, Cambridge, UK; dilution 1∶100). The following secondary fluorescent antibodies were used: goat anti-mouse Alexa488 (Invitrogen, Karlsruhe, Germany; dilution 1∶500) and goat anti-rabbit Alexa555 (Invitrogen, Karlsruhe, Germany; dilution 1∶500). Pictures were taken using a Zeiss confocal laser scanning microscope equipped with a Plan-Neofluar 40x/1.3 Oil DIC objective (LSM 510Meta, Zeiss, Jena, Germany). Alexa488 was exited by an Argon laser and detected using the filter set BP505-530. Alexa555 was exited by a Neon laser and detected using the filter set BP565-615.
Western Blotting
20 mg of muscle tissue was homogenized in ice-cold lysis buffer (Cell Signaling, Boston, U.S.A.) using a commercially available micro-dismembrator (Braun, Melsungen, Germany) and stored at –80°C for further analysis. The protein content of each homogenate was determined by the Lowry test kit (BioRad, Munich, Germany). 30 µg of homogenate protein was thawed on ice, suspended in buffer (0.5 M Tris-HCl, 10% glycerol, 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol, and 0.05% bromophenolblue), and heated at 95°C for 5 min. Tissue proteins were separated on a 6% acrylamide self-casted minigel using a gel casting system (PerfectBlue Vertical Double Gel Systems, Twin S, PeqLab, Erlangen, Germany) in Tris-glycine electrophoresis buffer (0.25 M Tris-HCl, 1.92 M glycine, 1% [w/v] SDS). After electrophoretic separation, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad, Munich, Germany) by semidry blotting (65 min at 12 V, 155 mA, and 3 W) using a semidry blotting system (PerfectBlue Semidry Electro Blotter, Sedec S, PeqLab, Erlangen, Germany). The membranes were blocked in 5% low-fat milk for 75 min. Afterwards, membranes were washed with Tris-buffered saline containing Tween (150 mM NaCl, 10 mM Tris-HCl, 0.05% Tween 20, pH 7.5). Membranes were incubated overnight at 4°C with rabbit polyclonal total AMPK and phosphorylated AMPKThr172 antibodies (Cell Signaling, Beverly, MA, USA; dilution 1∶1000) total RyR1 and RyR1Ser2843 antibodies (Abcam, Cambridge, UK; dilution 1∶500 total RyR1 and 1∶750 pRyR1 Ser2843). After membrane stripping using commercially available stripping buffer (ThermoScientific, Rockford, U.S.A.), membranes were re-incubated overnight at 4°C with mouse monoclonal anti myosin heavy chain-1 (MyHC1) antibody (A4.951 dilution 1∶2000, DSHB, University of Iowa, U.S.A.). Total RyR1 western blot analysis was performed on separate gels and membranes. Respective secondary antibodies (goat anti-rabbit and goat anti-mouse, dilutions in TBST for both 1∶2000) were purchased from Thermo Scientific (Rockford, U.S.A.) and membranes were incubated for 1 hour at room temperature. Proteins were detected by an enhanced chemo- luminescence assay (ECL Kit, Amersham-Life Science, Buckinghamshire, UK) exposed to X-ray film (Kodak X-OMAT Engineering, Eastman Kodak Co., Rochester, NY). Total RyR1 and MyHC1 served as internal loading controls and pRyR1Ser2843 signals were normalized to total RyR1.
All data are expressed as means ± standard error of means (S.E.M.). A multifactorial ANOVA for repeated measures with the factors 'time' and 'fiber type' was applied to determine the differences in the phosphorylation pattern of sarcoplasmic RyR1 content over time. In the case of a significant difference between factors, a Bonferroni post-hoc test was applied to locate the differences in fiber type dependent analysis. Tukeys post-hoc analysis was conducted for the western blotting analysis. Statistical significance was set at p<0.05. Linear regressions between variables were assigned using Pearson's product moment correlation index.
Immunohistochemistry and Localization of RyR1
To demonstrate the pRyR1Ser2843 staining specificity, immunofluorescence and DAB staining was performed, which further verified localization of pRyR1Ser2843 in junctional sarcoplasmic areas. α-actinin (Figure 1A) and pRyR1Ser2843 (Figure 1B) were stained in representative longitudinal cross sections of skeletal muscles. As demonstrated, pRyR1Ser2843 was located in cross striated areas in representative longitudinal skeletal muscle sections of immunofluorescence stained (Figure 1C) and DAB stained (Figure 1E) myofibers. Immunofluorescence staining demonstrates Z-bands (α-actinin) located in the proximal vicinity of junctional sarcoplasmic reticuli (pRyR1Ser2843) surrounding the t-tubuli which are located in the transitional zone of A- and I-bands. The negative control (Figure 1D) demonstrates that the obtained pRyR1Ser2843 signal was specific.
(A) Representative picture of longitudinal arranged myofibers displaying α-actinin staining (Alexa488) and (B) pRyR1Ser2843 staining (Alexa555) (C) Merged picture demonstrates localization of Z-bands (α-actinin, Alexa488) in close vicinity of junctional sarcoplasmic reticuli (pRyR1Ser2843, Alexa555). (D) Myofiber cross-section displaying an unstained negative control without primary antibody (α-actinin and pRyR1Ser2843) incubation. (E) Immunohistochemical DAB staining of pRyR1Ser2843 in longitudinal skeletal muscle sections (63x magnification). Arrows indicate pRyR1Ser2843 localization in cross striated areas.
Determination of Total RyR1 and Total AMPK in Myofibers
Salanova and co-workers [16] demonstrated that type I myofibers have less total RyR1 than do type II myofibers in human soleus muscle. To determine whether the human vastus lateralis muscle shows a comparable pattern of total RyR1 abundance and whether acute and severe resistance exercise induces changes in total RyR1 levels over the investigated time-course, we studied the levels of total RyR1 by means of immunohistochemistry. As shown in Figure 2D, the abundance of total RyR1 was significantly different between type I and type II myofibers; however, the amount of total RyR1 in the myofiber types did not change over the time-course (Figure 2G). Importantly, type II myofibers were found to have significantly more total RyR1 than type I myofibers (Figure 2G).
Illustration of DAB stained myofiber cross-sections (20x magnification) showing (A) total AMPK, (B) pAMPKThr172 and (C) corresponding MyHC1 staining displaying type I myofibers (black) and type II myofibers (unstained). No fiber type-specific differences in total AMPK were observed; Illustration of DAB stained consecutive myofiber cross-sections showing (D) total RyR1, (E) pRyR1Ser2843 and (F) corresponding MyHC1 staining displaying type I myofibers (black) and type II myofibers (unstained). As displayed in (D, F) total RyR1 density was considerably lower in type I compared to type II myofibers. (G) Assessment of total RyR1 density in type I and II myofibers at baseline, 15 min, 30 min and 60 min post resistance exercise. Type II myofibers displayed significantly higher RyR1 densities than type I myofibers (p<0.01). Total RyR1 did not change over the time-course in type I and II myofibers. *headed bars display significant differences between myofibers types (p<0.01).
It was recently demonstrated [25] that total AMPK levels do not differ between type I and type II myofibers in human skeletal muscle. Furthermore, the authors did not find any changes in total AMPK levels after exercise [25]. In line with these observations, we did not find any changes in the total amount of AMPK between type I and type II myofibers (Figure 2A), nor did we find any influence of acute resistance exercise on total AMPK levels up to 60 min after exercise (data not shown).
Resistance Exercise-induced Phosphorylation of pRyR1 Ser2843
The differential investigation of pRyR1Ser2843 abundance within type I and type II myofibers was carried out by staining consecutive cross-sections for pRyR1Ser2843 and type I myosin heavy chain (MyHC1). Figure 3A displays pRyR1Ser2843 immunohistochemical staining before exercise and its consecutive MyHC1 stained cross-section showing type I and II myofibers (Figure 3B). Figure 3C shows an increased abundance of pRyR1Ser2843 in stained cross-sections at 30 min post exercise. Figure 3D displays its consecutive cross-section stained for MyHC1. Figure 3E shows the phosphorylation levels of pRyR1Ser2843 in type I and type II myofibers over the time-course from baseline conditions up to 60 min post exercise in relation to total RyR1 levels. At baseline conditions, no fiber type-specific differences in RyR1 phosphorylation levels were detected. As early as 15 min post exercise, significant increases (p<0.01) in pRyR1Ser2843 staining intensity in both fiber types were detected. The RyR1 phosphorylation level in type I and II fibers remained significantly increased (p<0.01) up to 30 min after resistance exercise. pRyR1Ser2843 decreased significantly (p<0.01) 60 min after exercise in comparison to the 15 and 30 min time-points, which showed peak phosphorylation in both myofiber types. pRyR1Ser2843 phosphorylation levels returned to near-baseline levels in type II myofibers, but remained significantly elevated in type I myofibers (p<0.05) at 60 min post resistance exercise.
(A) pRyR1Ser2843-stained myofiber cross-section at baseline (PRE) (20x magnification). (B) Consecutive myofiber cross-section specifically stained for type I MyHC1 (A4.840) assigning type I (dark) and type II (unstained) myofibers. (C) pRyR1Ser2843-stained myofiber cross-section in type I and type II myofibers 30 min post exercise. (D) Consecutive myofiber cross-section of figure 3C displaying type I (dark) and type II (unstained) myofibers (E) Densitometry analysis of pRyR1Ser172 staining in type I and II myofibers cross-sections at baseline as well as 15, 30 and 60 min after resistance exercise. Significantly (p<0.01) increased levels 15 and 30 min post exercise compared to baseline levels in type II (§) and up to 60 min in type I (#) myofibers. (F) Western blotting analysis of pRyR1Ser2843 (n = 7). pRyR1Ser2843 (band size is observed at 270 kDa according to [14]) was analyzed PRE, 15, 30 and 60 min following exercise in comparison to total RyR1 levels. Myosin heavy chain (MyHC1, 220 kDa) served as an additional internal loading control. (§) pRyR1Ser2843 significantly different towards baseline levels at 15 min (p<0.05) and 30 min (p<0.01) after resistance exercise.
To further confirm the findings that pRyR1Ser2843 levels are significantly increased at 15 and 30 min post exercise, as determined by quantitative immunohistochemistry, western blots were conducted using whole muscle lysates (Figure 3F). The RyR1 complex has a molecular weight of about 565 kDa [13]. However, it was demonstrated that a band around 270 kDa obtained by western blot analysis is specific for skeletal muscle RyR1 [14], [26]. Total RyR1 (270 kDa) and MyHC1 (220 kDa) served as internal loading controls. Representative examples in Figure 3F show that both total RyR1 and MyHC1 levels remain unchanged within 60 min post resistance exercise. Compared to baseline levels, pRyRSer2843 levels increased considerably at 15 min after resistance exercise (p<0.05), peaking at 30 min post resistance exercise (p<0.01) and returning towards baseline levels at 60 min after exercise.
Resistance Exercise Induced Phosphorylation of pAMPKThr172
All subjects completed 3 sets of 8 repetitions of maximum voluntary eccentric knee extensions. pAMPKThr172, which has been described to show increased activity upon single bouts of resistance exercise [27], was used as a marker for contraction-induced energetic stress. As shown for pRyR1Ser2843, fiber type-specific pAMPKThr172 levels were determined in consecutive cross-sections of myofibers stained for pAMPKThr172 and MyHC I at baseline (Figure 4A and B) and 30 min post resistance exercise (Figure 4C and D). pAMPKThr172 staining intensities are depicted in relation to total AMPK levels. Fiber type-specific phosphorylation of AMPKThr172 (Figure 4E) showed significantly increased levels 15 min (p<0.01) post exercise, peaking after 30 min (p<0.01) in both myofiber types. By 60 min post exercise, pAMPKThr172 dropped down to baseline levels. Increased pAMPKThr172 levels were confirmed by western blotting in muscle homogenates at both 15 and 30 min post exercise (p<0.05, respectively) (Figure 4F) and confirmed to decrease to baseline levels within 60 min post resistance exercise. These data demonstrate the impact of the applied exercise regime on skeletal muscle cellular stress.
(A) pAMPKThr172-stained myofiber cross-section at baseline (PRE) (20x magnification). (B) Consecutive myofiber cross-section specifically stained for type I MyHC1 (A4.840) assigning type I (dark) and type II (unstained) myofibers. (C) pAMPKThr172-stained myofiber cross-section in type I and type II myofibers 30 min post exercise. (D) Consecutive myofiber cross-section of figure 3 C. (E) Densitometry analysis of pAMPKThr172 staining in type I and II myofibers at baseline as well as 15, 30 and 60 min post resistance exercise. Significantly (p<0.01) increased levels 15 to 30 min post exercise compared to baseline levels in type I (#) and II (§) myofibers. (F) Western blotting analysis (n = 7) of pAMPKThr172 to the respective time points showing significantly increased pAMPKThr172 levels 30 min post exercise (p<0.01) (§).
pRyRSer2843 and pAMPKThr172 Levels in Identical Myofibers
We investigated by immunohistochemical techniques the phosphorylation pattern of pAMPKThr172 (Figure 5A) and pRyR1Ser2843 (Figure 5B) at baseline and 30 min post exercise (Figure 5C and D) in the same myofibers of exercised subjects. The investigation was carried out using multiple consecutive cross sections stained for pAMPKThr172 and pRyR1Ser2843 from biopsies obtained at baseline and 30 min post exercise. Figure 5E depicts the data points obtained from one representative subject showing the combined staining of pAMPKThr172 and pRyR1Ser2843 before and 30 min post resistance exercise. As depicted in Figure 5E, control and exercised myofibers presented a broad range of different intensities of both AMPK and RyR1 phosphorylation within myofibers. It is clear, however, that pRyR1Ser2843 and pAMPKThr172 levels were increased simultaneously in the same myofibers as a consequence of resistance exercise. Hence, the majority of myofibers shifted within 30 min post exercise towards an increased amount of pRyR1Ser2843 (Figure 3E) and also pAMPKThr172 (Figure 4E) phosphorylation compared to baseline levels. Together, these data demonstrate that RyR1 is phosphorylated in the same fibers as AMPK and, therefore, strongly suggest that RyR1 phosphorylation is a direct consequence of resistance exercise.
(A) pAMPKThr172-stained myofiber cross-section at baseline (PRE) (20x magnification). (B) Consecutive myofiber cross-section specifically stained for pRyR1Ser2843 at baseline (C) pAMPKThr172-stained myofiber cross-section 30 min post exercise showing increased staining intensities compared to baseline condition. (D) Consecutive myofiber cross-section of figure 4C showing increased staining intensities compared to baseline. (E) Distribution of combined phosphorylation levels of pAMPKThr172 and pRyR1Ser2843 within a population of single myofibers of one representative subject at baseline and 30 min post exercise. Each data point reflects the values of pAMPKThr172 and pRyR1Ser2843 within a single myofiber to the specified time point. The scatter plot reveals a heterogeneous distribution of combined phosphorylation levels of AMPK and RyR1 within single myofibers at baseline (○). 30 min post exercise the majority of myofibers offered significantly, however gradually increased pAMPKThr172 and pRyR1ASer2843 phosphorylation levels (•).
Ca2+represents an essential ion in skeletal muscle as it primarily mediates the basis of electromechanical coupling in myofibers by its rapid and thorough oscillation between sarcoplasmic and myofibrillar compartments. However, severely contracting skeletal muscle inevitably fatigues in response to an intense workload [4], which leads to gradually reduced contraction properties of myofibers, lasting from seconds up to several minutes after cessation of exercise. This fatigue reduces the ability to perform repeated exercise bouts within a tight time-course [28]. Several cellular factors contribute to this event, including the regeneration of phosphates [29], neuronal factors [30] and Ca2+homeostasis [8].
The present study investigated acute effects of eccentric resistance exercise on phosphorylation levels of the skeletal muscle Ca2+release channel RyR1. Phosphorylation of RyR1 has been shown to induce "leaky" RyR1 channels [14], impaired myofibrillar Ca2+handling and, consequently, reduced contraction performance in chronically loaded mouse skeletal muscle. However, the previous data reveal a lack of knowledge regarding the time-course of RyR1 regulation in response to acute high intense exercise and, in particular, concerning whether different myofiber types are affected to different magnitudes. A primary novel finding of this study is that basal RyR1 phosphorylation levels can be rapidly and transiently increased up to 30 min in human myofibers as a response to 70 sec of maximum eccentric contraction and muscular tension.
We hypothesized that the impact of the applied exercise setup would not affect pAMPKThr172 and pRyR1Ser2843 levels to an equal extent in the proportion of type I and II myofibers as skeletal muscle constitutes a heterogeneous tissue in which type I and II myofibers are not consistently recruited during exercise [19]. This fact might affect distinct RyR1 and AMPK phosphorylation levels [21] within both populations. pRyR1Ser2843 was found to increase significantly in whole skeletal muscle, including both myofiber types. However, relatively higher increases in type I myofibers were observed, which remained elevated up to 60 min post exercise.
To obtain indirect clues for contraction-induced RyR1 phosphorylation, in parallel with the pRyR1Ser2843 studies, we investigated phosphorylation levels of AMPKThr172, which occurs in response to exercise in skeletal muscle [25]. Its phosphorylation is caused by energetic stress due to increased ATP turnover at ATP-dependent ion channels and myosin ATPase enzymes during electromechanical coupling [17] in myofibers. Here, we determined whether, parallel to elevated pRyR1Ser2843 levels, significant increases in pAMPKThr172 could be seen in type I and II myofibers up to 30 min post resistance. As we indeed did observe parallel increases in phosphorylation, we conclude that the observed raise in pRyR1Ser2843 levels occurred as a response to myofiber contraction. Despite a heterogeneous range of co-regulation of pAMPKThr172 and pRyR1Ser2843 within the investigated myofiber population (Figure 5E), we surmise that, 30 min after exercise, the parallel increase of both parameters within identical myofibers (Figure 5A–D) reflects a contraction-induced event. We speculate that the observed heterogeneity is due to distinct activity levels of motoneurons at rest and during exercise [19]. Indeed, AMPK and RyR1 phosphorylation can be triggered in response to exercise-induced stress [14], [25]; but it remains unclear whether they directly relate to each other at the molecular level. However, due to the observed paralleled changes in pAMPKThr172 and pRyR1Ser2843 in the current study, the co-regulation of both molecules, especially those lying in spatial vicinity to each other might be expected. Thus, it will be of future interest to determine whether exercise-induced regulation of AMPK and RyR1 in skeletal muscle myofibers is particularly occurring locally, in mechanically and energetically stressed environment.
Increased AMPK phosphorylation has recently been shown to occur in both myofiber types up to 120 min following endurance exercise [25]. However, the metabolic and mechanical impact of 70 sec of maximum eccentric contractions differs considerably from extended endurance exercise [31], which might have contributed to the shorter time-course of increased pAMPKThr172 in the present study. Because AMPK phosphorylation reflects a direct response to contraction-induced energetic turnover [27], this strongly suggests a broad contraction involvement of both type II an especially, type I myofibers in the present study. The observed increase in pRyR1Ser2843 takes place in a time-course corresponding to that of AMPKThr172 phosphorylation and shows a relatively higher increase in type I compared to type II myofibers up to 60 min post exercise. Our results emphasize the overall higher impact of the applied exercise regimen on type I myofibers. However, besides the observed changes in total RyR1 levels in response to unloading or chronic overload [16], no data are available concerning fiber type-specific responses of RyR1 phosphorylation in response to acute exercise. Our data provide evidence that the observed responses of transiently elevated RyR1 phosphorylation levels within myofibers reflect the impact of strenuous exercise on RyR1 channels within myofibers. Importantly, this can reveal differences in stress-induced regulation of structures involved in Ca2+handling in functionally distinct myofiber types. However, molecular differences concerning RyR1 regulation within human myofibers may exist, which to date are not known. This might include a different density of kinases within type I and II myofibers that contribute to RyR1 phosphorylation, e.g CAMKII and PKA, as well as responsible phosphatases that mediate its de-phosphorylation [13]; this would affect the time-course of increased RyR1 phosphorylation in myofibers regardless of any load dependent properties of myofiber types. Thereby, it might be speculated and should be considered in future studies that the observed pRyR1Ser2843 elevations are likely be driven by acute activations of CaMKII at Thr286 and more importantly by the formation of the catalytic subunit of protein kinase A (PKAc), as both kinases are activated upon acute exercise stimulations [32], [33].
Our data, combined with the described impact of RyR1 phosphorylation on Ca2+handling properties by Bellinger and co-workers [14], suggest that RyR1 phosphorylation is increased in response to intense resistance exercise, contributing to a sustained reduction in contraction properties up to 30 min after exercise. This time-course corresponds to a slow recovery period after intense myofiber contractions in which a reduction in myofiber contractility and relaxation ability has been observed [6], [7]. This phenomenon is decidedly one of the most important effects of RyR1 phosphorylation [14], although its phosphorylation has not been linked to the post exercise recovery period after fatiguing muscle contractions.
This is the first study showing the kinetics of increased pRyR1Ser2843 after resistance exercise in humans. Our data adds important knowledge regarding the time-course of RyR1 phosphorylation and, importantly, to a time-dependent and partly distinct response in subpopulations of type I and type II myofibers. However, as the difference in RyR1 phosphorylation between fiber types was found to be significant only at the 60 min post exercise time-point, further subpopulation exercise-based studies are needed. Replication of these findings is important as knowing that distinct myofibers may differ in their susceptibility to RyR1 phosphorylation in response to exercise. This what can have considerable impact on the behavior of competing athletes.
The current study does not link the impact of RyR1 phosphorylation within myofibers to decreased myofiber contractility, as multiple biopsies made the investigation of contraction abilities impossible to the investigated time points. However, the current results do extend the known mechanism of RyR1 phosphorylation. Until now, RyR1 phosphorylation has been solely investigated under chronic loading at an early and transient time period after exhausting exercise in human skeletal muscle. Combined approaches, such as EMG derivations, strength testing and multiple biopsies, are needed to investigate the extent to which increased RyR1 phosphorylation alters the contraction abilities of myofibers in general and following repeated training bouts within short recovery periods after exercise. Knowing this time-course would crucially affect a broad spectrum of training behaviors. It will be important to determine whether RyR1 modification can be altered by changes in physical activity between single bouts of exercise. Besides affecting myofiber contractility, pRyR1Ser2843-mediated changes in sarcoplasmatic Ca2+levels may also offer an important function in exercise-induced Ca2+signaling, as elevated sarcoplasmic Ca2+levels may trigger enhanced Ca2+binding to its targets, including NFAT, Calcineurin and CaMKIV [34]. These factors influence the modulation of myofiber plasticity, e.g. activity-induced myofiber shifting [35] or mitochondrial biogenesis [36], and thus emphasize their importance for exercising skeletal muscle. As each exercise bout induces a transient increase in intracellular Ca2+levels, it can be speculated that RyR1Ser2843 phosphorylation might also contribute indirectly to early Ca2+-induced signaling and, thus, be important for skeletal muscle micro-adaptations.
The present study indicates that 70 sec of maximum eccentric exercise induces transiently increased phosphorylation levels of pRyR12843, up to 30 min post exercise, in human type I and II skeletal myofibers. These results provide a sensitized time period in which pRyR1Ser2843 can exert a sustained impact on impaired calcium handling properties. This may affect skeletal muscle contraction performance of critically preloaded myofibers in sports beyond immediate fatiguing mechanisms. pRyR1Ser2843 may further provide a marker to detect subtle disturbances in skeletal muscle myofiber performance.
The authors are grateful to Kathryn D. Rogers for critically reading the manuscript. The authors thank Katharina Hermanns (Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine) for expert technical assistance.
Author Contributions
Conceived and designed the experiments: SG FS. Performed the experiments: SG LW TS FS. Analyzed the data: SG KP FS. Contributed reagents/materials/analysis tools: YK. Wrote the paper: SG GB WB FS.
- 1. Berchtold MW, Brinkmeier H, Muntener M (2000) Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. Physiol Rev 80: 1215–1265.
- 2. Bigland-Ritchie B, Jones DA, Woods JJ (1979) Excitation frequency and muscle fatigue: electrical responses during human voluntary and stimulated contractions. Exp Neurol 64: 414–427.
- 3. Choi SJ, Widrick JJ (2009) Combined effects of fatigue and eccentric damage on muscle power. J Appl Physiol 107: 1156–1164.
- 4. Bigland-Ritchie B, Johansson R, Lippold OC, Smith S, Woods JJ (1983) Changes in motoneurone firing rates during sustained maximal voluntary contractions. J Physiol 340: 335–346.
- 5.
Bigland-Ritchie B, Cafarelli E, Vollestad NK (1986) Fatigue of submaximal static contractions. Acta Physiol Scand Suppl 556: 137–148.
- 6. Edwards RH, Hill DK, Jones DA, Merton PA (1977) Fatigue of long duration in human skeletal muscle after exercise. J Physiol 272: 769–778.
- 7. Westerblad H, Lannergren J, Allen DG (1997) Slowed relaxation in fatigued skeletal muscle fibers of Xenopus and Mouse. Contribution of [Ca2+]i and cross-bridges. J Gen Physiol 109: 385–399.
- 8. Allen DG, Lamb GD, Westerblad H (2008) Skeletal muscle fatigue: cellular mechanisms. Physiol Rev 88: 287–332.
- 9. Plaugher G, Hendricks AH (1991) The inter- and intraexaminer reliability of the Gonstead pelvic marking system. J Manipulative Physiol Ther 14: 503–508.
- 10. Westerblad H, Allen DG (1991) Changes of myoplasmic calcium concentration during fatigue in single mouse muscle fibers. J Gen Physiol 98: 615–635.
- 11. Bruton JD, Lannergren J, Westerblad H (1998) Mechanisms underlying the slow recovery of force after fatigue: importance of intracellular calcium. Acta Physiol Scand 162: 285–293.
- 12. Allen DG, Lamb GD, Westerblad H (2008) Impaired calcium release during fatigue. J Appl Physiol 104: 296–305.
- 13. Bellinger AM, Mongillo M, Marks AR (2008) Stressed out: the skeletal muscle ryanodine receptor as a target of stress. J Clin Invest 118: 445–453.
- 14. Bellinger AM, Reiken S, Dura M, Murphy PW, Deng SX, et al. (2008) Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity. Proc Natl Acad Sci U S A 105: 2198–2202.
- 15. Betzenhauser MJ, Marks AR (2010) Ryanodine receptor channelopathies. Pflugers Arch 460: 467–480.
- 16. Salanova M, Schiffl G, Rittweger J, Felsenberg D, Blottner D (2008) Ryanodine receptor type-1 (RyR1) expression and protein S-nitrosylation pattern in human soleus myofibres following bed rest and exercise countermeasure. Histochem Cell Biol 130: 105–118.
- 17. Hardie DG (2011) Sensing of energy and nutrients by AMP-activated protein kinase. Am J Clin Nutr 93: 891S–6.
- 18. Sahlin K, Soderlund K, Tonkonogi M, Hirakoba K (1997) Phosphocreatine content in single fibers of human muscle after sustained submaximal exercise. Am J Physiol 273: C172–C178.
- 19. Beltman JG, de Haan A, Haan H, Gerrits HL, van Mechelen W, et al. (2004) Metabolically assessed muscle fibre recruitment in brief isometric contractions at different intensities. Eur J Appl Physiol 92: 485–492.
- 20. Altenburg TM, Degens H, van Mechelen W, Sargeant AJ, de Haan A (2007) Recruitment of single muscle fibers during submaximal cycling exercise. J Appl Physiol 103: 1752–1756.
- 21. Godin R, Ascah A, Daussin FN (2010) Intensity-dependent activation of intracellular signalling pathways in skeletal muscle: role of fibre type recruitment during exercise. J Physiol 588: 4073–4074.
- 22. Bergstrom J (1975) Percutaneous needle biopsy of skeletal muscle in physiological and clinical research. Scand J Clin Lab Invest 35: 609–616.
- 23. Gehlert S, Theis C, Weber S, Schiffer T, Hellmich M, et al. (2010) Exercise-induced decline in the density of LYVE-1-positive lymphatic vessels in human skeletal muscle. Lymphat Res Biol 8: 165–173.
- 24. Munch G, Bolck B, Sugaru A, Brixius K, Bloch W, et al. (2001) Increased expression of isoform 1 of the sarcoplasmic reticulum Ca(2+)-release channel in failing human heart. Circulation 103: 2739–2744.
- 25. Lee-Young RS, Canny BJ, Myers DE, McConell GK (2009) AMPK activation is fiber type specific in human skeletal muscle: effects of exercise and short-term exercise training. J Appl Physiol 107: 283–289.
- 26. Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, et al. (2009) Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med 15: 325–330.
- 27. Dreyer HC, Fujita S, Cadenas JG, Chinkes DL, Volpi E, et al. (2006) Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. J Physiol 576: 613–624.
- 28. Girard O, Mendez-Villanueva A, Bishop D (2011) Repeated-sprint ability - part I: factors contributing to fatigue. Sports Med 41: 673–694.
- 29. Baker JS, McCormick MC, Robergs RA (2010) Interaction among Skeletal Muscle Metabolic Energy Systems during Intense Exercise. J Nutr Metab 2010: 905612.
- 30. Bigland-Ritchie B, Woods JJ (1984) Changes in muscle contractile properties and neural control during human muscular fatigue. Muscle Nerve 7: 691–699.
- 31. Atherton PJ, Babraj J, Smith K, Singh J, Rennie MJ, et al. (2005) Selective activation of AMPK-PGC-1alpha or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation. FASEB J 19: 786–788.
- 32. Rose AJ, Kiens B, Richter EA (2006) Ca2+-calmodulin-dependent protein kinase expression and signalling in skeletal muscle during exercise. J Physiol 574: 889–903.
- 33.
Chasiotis D (1983) The regulation of glycogen phosphorylase and glycogen breakdown in human skeletal muscle. Acta Physiol Scand Suppl 518: 1–68.
- 34. Chin ER (2004) The role of calcium and calcium/calmodulin-dependent kinases in skeletal muscle plasticity and mitochondrial biogenesis. Proc Nutr Soc 63: 279–286.
- 35. Serrano AL, Murgia M, Pallafacchina G, Calabria E, Coniglio P, et al. (2001) Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth. Proc Natl Acad Sci U S A 98: 13108–13113.
- 36. Wu H, Kanatous SB, Thurmond FA, Gallardo T, Isotani E, et al. (2002) Regulation of mitochondrial biogenesis in skeletal muscle by CaMK. Science 296: 349–352.


text:
Sunshine Griffith faces down her destiny.
The Sunshine Girl trilogy concludes as the title character returns from training and prepares to battle the deadly superdemon, Dubu. Surrounded by her biological, luiseach parents, Aidan and Helena, her boyfriend, Nolan, her adoptive, human mom, Kat, her childhood, human best friend, Ashley, and a fellow luiseach, Lucio—all evidently white with the exception of Latino Lucio—Sunshine is filled in on prophecies, hidden histories, and last-minute skill sets. The promising series stumbled in its second installment by wedging Sunshine into a poor "chosen one" narrative. But now Sunshine is back on her home turf and ready to slay some demons. The author embraces the campy, gothic roots that inspired the series, offering sordid romantic histories, a mysterious luiseach council, and eleventh-hour betrayals. Sunshine is admirably strong, observant, and self-reliant. She may have lots of backup, but at the end of the day she is perfectly capable of taking care of herself. The only storyline that's a whiff is Sunshine's relationship with Nolan. There's very little heat to this romance. Sunshine's relationship with Aidan and Helena is much more interesting, crackling with conflict and character. The spooky set pieces remain excellent, and the author's handle on action has improved over the three books. The first installment remains the best, but this is a fine way to end Sunshine's story.
An excellent end to an uneven but enjoyable series. (Paranormal suspense. 12-16)

subset: commoncrawl


text:
Automatically playing similar songs. To disable, switch Autoplay to 'OFF' under Settings
Get Notified about the latest hits and trends, so that you are always on top of the latest in music when it comes to your friends.
Add to queue
drop here
This will remove all the songs from your queue. Are you sure you want to continue?
Released by T-Series May 2007 | 6 Tracks
By Bhai Satvinder Singh Ji, Bhai Harvinder Singh Ji
Tu Thakuro Baragro - Satvinder Singh,Bhai Harvinder Singh Ji
added to MyPlaylist


text:
DNS Administration
Administering your DNS settings is a key requirement for any good domain management utility. The control panel that comes with all of your Speednames domains provides the ability to configure all DNS elements, delivering complete flexibility for you to manage your names.
Your control panel allows you to create, edit and remove an astounding number of DNS records, including:
A records (or web records)
Allow you to point your domain to an external server via an IP address. It is usually used for sub domains
Allow you to point your domain to an external server via an IP address. It is usually used for sub domains
(eg: forum.example.com A 184.108.40.206 / www.example.com A 220.127.116.11 / example.com(root) A 126.96.36.199)
MX records (or email records)
Give you the opportunity to point your domains to multiple mail servers. Two MX records is the minimum required to be able to send and receive emails and can be prioritised.
(eg: example.com MX 10 mail.example.com / example.com MX 20 mail2.example.com)
CNAME (alias records)
Allow your domain to be an alias of another, which could be useful if several service are running from the same IP address but on different port.
(eg: help.example.com CNAME forum.example.com)
TXT (for SPF records for instance)
This type of records is mainly used to increase the security on your domain. SPF records for example is used to prevent other users to use your domain to send their emails from.
(eg: example.com TXT "v=spf1 a mx -all")
Name Servers (up to 12)
These servers are the one telling your domain where every part of it should go. All the records explained above need the name servers to relay the information to the worldwide web. We provide our name servers by default, but you can change them to your own at anytime and can add up to twelve.
(eg: example.com NS ns1.speednames.com / example.com NS ns2.speednames.com)


text:
Updo hairstyles include the dressed-up pony, the basic ballerina bun, the teased topknot, the side-pony, the side bun and the mini bouffant. The Gibson tuck, the low loop and the knotted bun and the French twist are other popular updos.
The dressed-up pony is similar to a basic ponytail updo, but with some lengths of hair left out of the ponytail. A basic ballerina bun is a bun at the top of the head, and the teased top-knot is a slightly messier version of the ballerina bun. For the side bun and side pony, the hair is tied at the side of the head. Cosmopolitan.com and Babble.com offer tips and tutorials to create these updo styles.


text:
I logged the issue with the WCAG WG's public comment form and will follow up with the group to help get this changed. Thanks, AWK Andrew Kirkpatrick Group Product Manager, Accessibility Adobe Systems email@example.com http://twitter.com/awkawk http://blogs.adobe.com/accessibility -----Original Message----- From: Vivienne CONWAY [mailto:email@example.com] Sent: Monday, August 27, 2012 9:02 PM To: firstname.lastname@example.com list Subject: Testing flash techniques - aDesigner Hi Jan and group Kentarou Fukuda from IBM has kindly provided the following information about aDesigner as requested: Hi Vivienne, Thank you so much for letting us know about it! At first, I apologize for the confusion. I think it is caused by aDesigner's history. Here is a rough summary. --- 2004: aDesigner was published at IBM alphaWorks (Free but IBM proprietary.) 2007: aDesigner2 that supports Flash evaluation function was published at IBM alphaWorks. 2008: Eclipse ACTF aDesigner was published. (Open-sourced version of aDesigner2. ACTF aDesigner 0.5 is almost equivalent to aDesigner2) At the same time, aDesigner2 was graduated from IBM alphaWorks. Further developments and enhancements are performed at Eclipse ACTF project. --- As you know, aDesigner that supports Flash evaluation is now available from Eclipse.org, and its latest version is 1.0. So, if possible, would you please ask editors to replace "aDesigner2" with "aDesigner" (or "ACTF aDesigner") in the Flash techniques page? If needed, I can post it to w3c-wai-ig ML. BTW, I found that the link to aDesigner in "Resources" section was already updated and now it linked to ACTF's download page (http://www.eclipse.org/actf/downloads/tools/aDesigner/). Thank you for your support for it. Many thanks, Kentarou --- Kentarou Fukuda, Ph.D. IBM Research - Tokyo Tel: +81-3-5144-2852 E-mail: firstname.lastname@example.com I trust that this will clear up any confusion. Kind regards Vivienne This e-mail is confidential. If you are not the intended recipient you must not disclose or use the information contained within. If you have received it in error please return it to the sender via reply e-mail and delete any record of it from your system. The information contained within is not the opinion of Edith Cowan University in general and the University accepts no liability for the accuracy of the information provided. CRICOS IPC 00279BReceived on Tuesday, 28 August 2012 11:15:25 UTC
This archive was generated by hypermail 2.3.1 : Tuesday, 13 October 2015 16:21:45 UTC


text: Prolific actor Robert Peters (Ocean's Eleven, Catch Me If You Can) makes his feature directorial debut with this musical romantic comedy about an optimistic "happiness counselor" named Bob who falls head over heels in love with his cynical handler Greta. Bob has just penned a self-help book that sold a meager one copy in the entirety of the United States, yet the eternal optimist is quick to note that he's a big hit in Deutschland. Now, as bob straps on his guitar and boards a plan bound for Germany with his handler Greta, an intellectual realist who refuses to see the world though rose tinted glasses, these two polar opposites are about to discover that opposites really do attract.


text:
I remember lying on top of cracked pots on a cool yet sunny Baguio day. Just a few seconds ago I was at the top of a steep hill, about to attempt what my older brother, Joseph, just did with no difficulty: ride a bike down the hill. For some reason I've already forgotten, I was using Joshua's bike which had broken breaks (but we didn't know that). My dad instructed me, "You can do this David. Just ride it down, break if you feel like you're going too fast, that's it."
So I kicked off and started going down. Very quickly it was apparent that something was wrong. I couldn't break and the sight of a row of plants on large pots, a lamppost, and a drop in front of me just kept getting closer. The next thing I know, I'm squinting at the glare of a bright sun, my head hurts like crazy, I can feel sharp ceramic and thorns on my skin, and I'm saying "Lord" over and over.
Then I saw my dad run up, worried as hell, check me, and then I felt thorns plucking out of my jacket as he lifted me. "You're really a tough kid." my dad said. "I told you not to pedal. I told you to break." He continued. "I shouldn't have pedaled but I couldn't break" I explained.
When we got into the house my mom wiped the blood from the side of my head. "You're really like Rambo now" my dad told me, and I remember picturing a bloody Rambo from a movie I used to watch over and over. "Yeah". I replied proudly.
"Ok, rest a bit, let's fix the breaks, then let's go back out there."
And we did. And I rode down the hill with no problem. And my dad and brothers and I rode down that hill, and other hills, and other paths, and trails, and roads.
I remember that incident and I realize now what my dad did, he wanted me to learn the right lesson. The lesson wasn't that riding bikes downhill is dangerous. The lesson wasn't you're too small for steep hills. The lesson wasn't you're no good at biking. The lesson was: here's how you conquer things. Here's how you conquer cliffs. Here's how you face your fears. Here's how you get back up from failure.
It's similar to when, just the day after my brother Joseph nearly killed both of us in a total car wreck by driving too fast, my dad throws the keys to his car and says, "You drive for us". He wanted Joseph to learn how to be careful. He didn't want him to learn how to be afraid of driving.
Many times I find that different people in very similar contexts, living in the same county, working in the same offices, or sitting in the same classrooms, walk away with very very different lessons.
Most people go to class thinking: I need to pass. The smartest people I know are always thinking: How do I understand. The former will learn how to pass (even if it involves cheating). The latter will learn how to understand. Guess who'll become more successful?
Some people are stressed at work and learn: "My boss is an ass" or "I need a vacation" or "work sucks" or "the setup sucks". The most productive people I know, the ones who make things happen, are learning: "What can I learn from my boss?", "What can I improve?" or "How do I lead up?" or "How do I manage my time better so that I can be productive AND recharge?" or "How do I change my context to make me more efficient?" The earlier group will learn how to rationalize their mediocrity. The latter group will change their workplaces for the better.
Some people go through a breakup and learn: "I need to kiss dating goodbye" or "guys suck" or "girls are liars" or "everyone is a cheater" or "I'm not pretty" or "no one will ever want me". Others will go through one and learn "That's the last time I'm making looks my main priority" or "Next time I won't rush" or "it's time I really clearly state my values". One group will become bitter, fearful, and probably terrible future partners or never have a partner (which isn't so bad really). The other group will be wiser and better prepared for a new beginning.
Some people tried the entrepreneurial route, went bust, and learned: "It's risky", "There's no money", "It's hard", "I'm better off with a stable job". Others will learn "So that's what I should have done!", "I need to choose my partners better". Guess who will build that next great business?
A lot of religiously superstitious people will fail at something and chalk it up to it not being "God's will", and will miss the true learning opportunities. Maybe the lesson needed was stewardship and discipline. Maybe it's perseverance.
A lot of people will see get-rich-quick stories and think that's what they need to figure out instead of learning about timing, about character-building, about the reality of breaks.
Some people will see an evil person succeed, a violent man gain power, or a manipulative woman move ahead, they'll learn that the world is unfair, that good people finish last. They'll miss the lesson that we get what we run after, we get what we sacrifice for, but even more, they'll miss the opportunity to reflect that maybe money, and power and fame aren't the barometers of success, and they'll miss the chance to know their own personal values.
All of these examples are to say one thing: As you go through life don't learn the wrong lessons. The wrong lessons like I mentioned in my last article are lessons that teach us fear, teach us pride, teach us bitterness. They are the lessons that trap us under the guise of wisdom. They are the lessons that teach us to mistrust instead of learn who to trust. They are the lessons that harden us for the sake of security, that make us selfish for the sake of practicality, that teach us pride for the sake of self-esteem.
No one progresses by learning the wrong lessons. They will only make us more narrow minded. A narrow mind is a mind choked by the wrong lessons. It's so full that new ideas, new concepts, even the consideration of new thoughts, can't pass, so discovery is killed at inception.
Instead, learn the right lessons. Failure should teach you to improve. Heartbreak should show us that we can bounce back from anything, even more, heartbreaks should remind us of how broken the world is, and of how we desperately need God. Goliaths should remind us of the Davids that toppled them, and the faith that powered them. Darkness should activate us to be the lights, even better to daily live as a light, like a star automatically shining bright in a world of cold nights. If we foster a mind that learns the right lessons from anything, we will find that the wisdom we need to face the things we have to face, do the things we ought to do, and be the person we hope to be is not just available but all around us.


text:
Last week, after watching highly respected City Council candidates Doug "Shin-Kicker" Bruce and Ed "Not Really Counting On the Black or Jewish Vote" Bircham appear together to discuss their planned Reform Team takeover of our village government, I did what most of you did.
That's right, I curled up in the fetal position under my psychiatrist's couch and sucked my thumb for three days.
But we're not here to write about the angry and volatile Bruce-Bircham ticket (official campaign motto: "Tippecanoe and #@%* You, Too").
Instead, we'll visit the ol' mailbag to see what you, the loyal readers, are thinking. And just so you don't have any big expectations for this column, I'll tell you right now that none of the recent notes rival last year's "Dear Butthead" letter from reader "YS," who called me, among other things, a "lazy, wothless (shitbrained liberal" [sic] and closed by asking the ages-old philosophical question: "How's that bear bite you in the ass u dumbass liberal?"
By comparison to that eloquent dissertation, today's e-mails appear to have been written by people who did not stick a butter knife into an electrical outlet when they were little.
Take this actual note about the aforementioned candidate Doug, from reader Bill: "I am convinced Mr. Bruce's problem is that he needs to get laid. I have been asking my female friends to lie down and take one for the team, but so far have found no takers. Maybe with your larger circle of friends you could have greater success. Not fooling around here. This is serious."
Note to Bill: You make a good point. And I'll ask around. I'm sure there are people who consider being kicked in the leg a type of foreplay. But enough about the women of Nebraska.
Here's another interesting note that arrived as a letter to the editor: "Clearly, Rich Tosches must have slept through the unit on satire in journalism school."
To which I would say, in all seriousness: journalism school?
My No. 1 fan went on to demonstrate a Harvard-worthy knowledge of finance and business when he wrote: "It's a good thing the Indy is free, or they couldn't afford to print your waste of trees."
The note was signed: Rock Chasko, La Mesilla, N.M.
(I don't know if Rock is his actual name or just a nickname based on the most popular children's toy in New Mexico.)
Another note came after I announced my candidacy to become the next mayor of our village, via a column in which I confessed to having 11 toes (nine of them on my right foot) and some other stuff that I don't remember.
From reader Studmuffin: "You go from talking about your toes to some obscure Ted Haggard reference to running for mayor. Does anyone take anything seriously anymore?"
To which I'd say sure, some people do. I just find my group of people, called "idiots" or "loons," to be more fun. Oh, and by the way, Mr. Studmuffin, if your wife gave you that nickname, it's possible she's in a third group of people. They're called "sarcastic."
Reader "Ronnie1" reacted to a recent column about UFO Phil, a fellow who wants to build a flying saucer-refueling station atop our very own Pikes Peak (that old idea again), by saying there are millions of aliens whizzing across our skies in spaceships all the time, causing gamma-ray flashes.
"These Terrestrial Gamma-ray flashes," the reader informs me, "send beams of anti-matter arcing through the Earth's atmosphere in thunderstorms ... and are sometimes referred to as Blue Jets."
These so-called Blue Jets of light should not be confused with another natural phenomenon often demonstrated back in the 1970s by George, my college roommate. He called his scientific displays — which involved a cigarette lighter, a burrito and him lying on his back in his underwear with his legs over his head — Blue Darters.
Some notes contain medical information, too. Following a column about the Gazette, which used to be a newspaper, "gregR" offered this informative snippet: "My bird gets the squirts when I put the Geezette in the cage."
Which is, quite frankly, startling on two fronts. First, the guy has a bird that can read. And even more surprising, somewhere in our village there's a guy who still gets the Gazette.


text:
Folk erotic art from the battlefield is hardly unheard of — remember nose art on bombers? Here's a sample from an even more unlikely place to find beauty, namely, the latrines at Ali Al Salem Air Base, near Kuwait City:
In the best tradition of comic folk erotica, our latrine siren is ready and willing; the speech bubble appears to say "any hole".
From The Walrus.
Shorter URL for sharing: http://www.erosblog.com/?p=2234


text:
1/ Matching iPhone covers that my mother and I bought yesterday. We're so sickening like that.
2/ Starting a fresh, new diary for positive things and thoughts, and planning and ideas for posts.
3/ Outfit of the day. Sportsgirl neon jumper and black jeans, Topshop boots, Michael Kors watch, skull bangle from eBay, Diva choker and mixed metal Russian wedding ring (not worn as a wedding ring, mind you).
4/ Details


text:
A TOP United States official visiting Somalia recently was told this story: One of the country's many young gunmen, in search of food, shot a woman carrying a bag of rice. When he stepped forward to claim the booty, he discovered he had killed his own wife.
Apocryphal or not, the tale accurately describes the extent of Somali lawlessness and despair. Civil society no longer exists; only people with guns or armed protectors eat. The rule of law has been replaced by the rule of anarchy. The result: the worst humanitarian disaster in the world.
Final realization of this state of affairs is what seems to lie behind the US decision to offer troops for the protection of Somali food aid. Attempts to send relief through existing channels are just no longer working. Fighting between warlords is blocking aid flights from airports, keeping aid trucks off highways, and - worst of all - forcing aid ships to stay at sea.
"It's going to require some kind of stronger military action there to safeguard these relief supplies," White House spokesman Marlin Fitzwater said Nov. 27.
In crossing the line between simply sending food and C-130s to sending the Marines, the Bush administration has taken a big step. For one thing, officials are offering the largest deployment of US force under a United Nations flag since the Korean War. For another, they are ignoring the sovereignty of a nation, albeit a battered one, in the name of humanitarian concern.
Pressure for intervention in other disasters, such as the Balkans and Liberia, will likely increase. "This is a pretty dramatic change. I'm not sure it's entirely a good one," says Carol Lancaster, an Africa expert at the Georgetown University School of Foreign Service.
As of this writing, the UN had taken no final decision on the US offer to send up to a division's-worth of force, roughly 20,000 troops, as part of an international Somalia effort. Reports from the UN indicated that Secretary-General Boutros Boutros-Ghali was likely to approve some sort of expanded military effort.
In Somalia, both major warlords who control the capital of Mogadishu, Ali Mahdi Muham-mad and Gen. Muhammad Farah Aidid, have welcomed the US offer.
About 500 Pakistani soldiers are already in Somalia operating under the UN flag. But they have been largely ineffective, as uncooperative warlords have blocked their widespread deployment. The Security Council has also voted to send another 3,000 troops from various nations, but they have not yet arrived.
Many Africa experts and relief officials applauded the sudden US offer of armed help. It was seen as a welcome exception to the recent US tendency to focus most of its foreign-policy energy on developments and strife in Eastern Europe and Russia. Considering the natural ebbing of energy that characterizes a lame-duck presidency, it was also seen as something of a surprise.
"I can't honestly see how there can be any opposition to this," says Mohamed Kahlil, a former high official of the Sudanese government now in Washington as a fellow of the US Institute of Peace.
IT is possible to have a military deployment to Somalia that will be able to stabilize the situation, and not become an open-ended commitment, Mr. Kahlil says. In any case, the state of decay in Somalia now means that inevitably "the world is going to become more involved there," he says.
Some aid officials, however, actually are not happy about the prospective increase in US involvement. They feel that US troops could act as an affront and incitement, turning Somalis against all outside intervention, including relief distribution, and could endanger the safety of their in-country staff. A statement issued in London by the Save The Children organization claimed "imposing troops on Somalia as suggested by the United States would be a disaster."
Professor Lancaster says that while humanitarian concerns may have prompted the administration's offer of troops, the action appears to be a hasty one and leaves many questions: "What do you do if you start to take casualties? Or, what do you do if you succeed? Do you have a plan for an interim government?."
Reportedly, administration officials see a phased operation, with heavy US forces eventually giving way to several thousand non-US UN troops, and finally to locally hired guards able to protect well-established relief efforts.
Then there will be the inevitable comparisons of the US effort in Somalia to its reticence in other world disaster areas. It is easy to make a case that the Balkan situation is much harder. There, well-armed insurgents lurk in terrain that is inhospitable to fighting. But there are other nations where the situation is more similar to Somalia - such as battered Liberia or Zaire.


text:
Washington, DC Online American Studies Certificate Programs
If you are interested in enrolling in an online American studies certificate program, you can expect to take courses in the humanities, social sciences and the arts. They will give you the foundation to specialize in a specific area or a culture of your choosing, be it African American studies graduate certificate programs or another culture that has been significant to the growth of the United States.
Online graduate school certificate programs in American studies will give you the flexibility to learn anytime, anywhere and will also fit into your busy lifestyle. If you are just starting your search, why not look online for a suitable American studies certificate program or search the many online certificate degree programs that are available from American studies schools?
Sponsored Listings
- OnlineOnline
Minnesota State University - Mankato
Ethnic and Multicultural Studies
The Department of Ethnic Studies, an interdisciplinary program, is academically committed to promoting multicultural and ethnic knowledge, values, and skills both within and outside the US and to preparing our students for effective function across the...
- OnlineOnline
University of North Carolina at Greensboro
African American Studies Program
The UNCG African American Studies Post-Baccalaureate Certificate Program is available to currently enrolled graduate students...


text:
A Need For Environmental Friendliness
Waste disposal services have grown a prime necessity these days. Every home, office, hospital or public place has to deal with waste disposal companies every day. If you do not dispose the garbage on time, it may cause plenty of problems embracing serious diseases and severe infections. Waste disposal is nothing but the dumping of garbage for collection. A Waste Disposal company collects all the garbage and dump them in a garbage transfer zone for segregation. The garbage disposal companies recycle all the recyclable items and pour the rest into the landfills.
A lot of people do not even know where all these waste items go, after their disposal. All these trash materials normally go to a dumping zone. The junk clearance companies dispose the trash either over the ground or deep inside the earth.
Waste disposal companies, fortunately, offer the junk clearance services throughout the year. The dumping grounds collect the junks as long as there is no more space left in it. When the dumping ground reaches a saturation point, the rubbish removal companies have to look for a new waste removal zone. If there is no dumping spot in a city, you must send the junks to a different city or town.
A lot of companies offer waste disposal or trash clearance services these days. The companies mainly focus on different construction sites, homes, offices and retail outlets. So, soon as you hire a reliable garbage clearance company, their professionals will come to check the junks, sort them and dispose of them in a systematic and efficient way.
Some Commonly Prevailing Methods Of Waste Disposal
As far as effective waste management is concerned, the companies that belong to this industry have several options to pick from. These methodologies are all designed in such a way that they are highly effective and ensure robust result. Some of them are being discussed below.
Ocean dumping
The first method of effective waste removal is given the name of ocean dumping and it has several advantages and disadvantages. Let us review some of them.
Advantages of ocean dumping
- This process is extremely convenient for waste management
- It is inexpensive as there are no major factors that can involve expenditure
- It becomes a very remarkable source of nutrients, shelter and breeding
The disadvantages
- The oceans suffer a remarkable burden of dumping
- It facilitates the destruction of natural food resources
- This also causes the destruction or killing of plankton
- The threat of desalination is always prevailing with this method of waste removal
Listed below are the advantages of sanitary land filling method of rubbish removal
- The volume can be increased drastically with just a little addition of manpower and resources
- The filled land can be reused for any task, including community purposes
- The completed landfill areas can settle down and therefore pose a threat and also require maintenance
- This process can require proper planning, designing and operation when it comes to using it for waste removal
The advantages of incineration are –
- It requires minimum land
- It can be operated in any weather
- It produces stable odor-free residue
- The refuse volume is reduced by half
Listed below are some remarkable disadvantages.
- It is an expensive thing to build and operate for waste disposal
- There is a high energy requirement
- It requires skilled personnel and continuous maintenance
- It is unsightly – smell, waste, vermin
How You Could Help Bulk Waste Collection Adelaide Firms?
Irrespective of where you want, the fact remains the same that waste management comes alive only after rubbish removal is done. This becomes even more mandatory in domestic areas or homes, but the advantage is that there are several easier methods to get rid of the domestic garbage.
For this, it is important that you first reduce garbage generation as much as possible. Only then, you can think about implementing effective disposal ideas. The reason is that quite obviously, you'll be doing this work as DIY without any specialized tools, setup of skills. In this situation, you can think about attaining success only if smaller amounts of wastes are tried upon.
- According to bulk waste collection firms, a very influential step that you can take is reducing the amount of plastic and use cloth market bags.
- These bags are available in different sizes and designs at the markets and can be used for buying grocery or in several other tasks where you were earlier using plastic bags.
- This step, if made effective can reduce 60 percent of the problems that we are confronting currently about our Mother Nature or the environment.
- Another very similar step is buying the products with the least packaging. This way, you'll automatically reduce the amount of garbage that bulk waste collection Adelaide firm would collect from your home.
- Rather than going to supermarkets and buying packed vegetables, consider visiting your local market, where you can buy them fresh and open to store them in your bag.
- Another idea is reducing the amount of bottled, packed or canned products. This will also reduce the amount of garbage that you generate on a daily basis.
- A very brilliant step is of composting the garbage, as much as you can.
- This will fetch two benefits for you and the first would be an effective waste management.
- The second would be that you'll get high quality and completely organic compost to put in your lawn or garden.
Tips On Using Skip Bins Adelaide To Their Best
In several projects that we undertake, the biggest hassle associated is of garbage disposal. But this problem appears huge to only those who have no ideal till now about skip bins. This concept has emerged to help in garbage collection and disposal at such sites, industries, housing societies and even homes. By using them, users can save a huge amount of time, money and efforts that they would have wasted on disposal of garbage.
With several companies offering services related to skip bin hire Adelaide on the Internet, hiring skip bins Adelaide is no more a tricky task. All you have to do is visit the website, select the size that you want, fill in the details of the location where you want it and that's it. In some time, it would be delivered to you by the skip bin hire services offering firm.
A very notable feature of these firms is that they don't just supply empty skip bins Adelaide, but they also carry away those that have been filled. They are in collaboration with waste removal firms and sometimes extend their own services to add disposal and removal services.
How to be in profit while you hire skip bin
- By selecting the right size
The first thing that you do to ensure that you are in profit is selecting the appropriate size according to your needs. They are available in three most basic sizes and by selecting the right size; you can save a lot of money.
2. Elevating the hire skip bins Adelaide
As you place them in your premises, be sure to keep it a bit elevated from the ground (maybe 1 foot or so) to ensure that when it is lifted, it does not damage the ground underneath.
3. Sorting your rubbish
Another very robust step that you can take towards saving money is sorting your garbage and dropping it in the appropriate bin. This way, you'll be able to use the available space in a better manner and will also contribute directly towards our mother nature.
The Need of Proper Waste Management Adelaide
For cosmopolitan cities like Adelaide, London, New York, Mumbai and several others, there is an utmost necessity of proper waste management. The reason is the regularly deteriorating condition of our eco-system and according to environmentalists, this happens to be one of the most robust steps that we can take for damage control.
The problem is that people are not interested in this because according to them, dropping their garbage in skip bins is all that can be done. To some extent, this can be justified that management starts from ground level itself and this actually means managing the garbage at individual levels.
Types of wastes
There are basically two different kinds and the first is disposable, while the second is recyclable.
Those termed as disposable can be disposed by waste disposal Adelaide firms after use. This basically involves everything that is not threatening to the environment, i.e. green garbage.
On the other hand, there are those that can be recycled and this could contain different things that can be hazardous as well as non-hazardous. If not disposed properly, it can put a drastic impact on natural and our resources.
Residential waste management Adelaide
In the past few years, it had been observed that residential waste management has become a rather bigger problem than assumed. The reasons are several and the most prominent are the lack of proper waste removal Adelaide methodologies and production of more rubbish than what local councils can handle. The solution is also present there in the form of hiring a waste management Adelaide firm that has the techniques for proper removal as well as disposal.
Tips for Better Management of Bulk Waste Collection Adelaide
If you own a business that has a production unit as well, quite obviously, you'll be generating a lot of waste. Because you are generating it, surely you would be concerned about its proper disposal.
This is quite obvious because not only that is unsafe for us in a number of ways, but its disposal is time-consuming and costly too. In this post, we will review some important aspects that you need to know to ensure its effective and economic disposal.
Tip # 1
Examining the contents carefully
Perhaps the most important thing that you should do is examining the constituents that constitute that garbage. This is quite important because different items need different disposal and at different locations as well. For example – mattresses are to be sent to a mattress recycling Adelaide company and similarly, for furniture, you need to get in touch with a furniture recycling Adelaide firm.
However, if you have collaborated with Distribution 360, you need not find separate companies because as the most reputed bulk waste collection Adelaide firm, it offers all-inclusive services to its clients.
Tip # 2
Estimating the load
The second very crucial step that needs to be taken is estimating the load. You should be aware of the fact that waste is mainly measured in weight. This means that the heavier items will cost more whereas lighter items will be less expensive to dispose.
Tip # 3
Checking the access route to your wastes
If your garbage is tough to get to the spot, you'll have to inform this to your bulk waste collection Adelaide firm. Similarly, if you require extra labor, you'll have to inform your contractor in advance. To make job easier for the firm, you can bundle up or pack as much squander as possible. You can use tough bags and string for smaller materials. You need not pack or bundle larger items as they can be picked in the natural condition itself.
Making Waste Management Adelaide Easier With Skip Bins Adelaide
No matter where you are, if you are engaged in some kind of production, manufacturing, demolition or cleaning, obviously, you are generating some amount of waste. One of the best ways to manage this garbage is searching for a company that offers skip bins Adelaide. This point has its share of benefits and some of them are mentioned below.
They are accessible
Suppose you don't have a trailer or a vehicle to pull a trailer, these bins can be an ideal solution for you. They will straightly save you from traveling miles to the closest dumping or landfill zone to dump your waste. The company that you have collaborated with will supply them at your place and pick the one that is done.
The help in conserving the environment
Another benefit of going for the concept of skip bin hire Adelaide is that you play a very active role in safeguarding the nature from the ill-effects of different hazardous wastes. The companies that offer them under waste management Adelaide program send several items like scrap materials, timber, paper, plastic, metals, glass, e-waste, tires, mattresses and several other things for recycling. This way they play their part and help you play a similar role in making this earth a better place to live.
They are safer and healthier
The staff coming to deliver and picking up the used one is trained for offering high quality waste management Adelaide services. They actually have all the knowledge of how, when and what tools and equipments to use to complete their tasks safely.
They are reasonably priced
Another benefit is that availing skip bin hire services Adelaide for garbage collection is a much more affordable option than using DIY and deciding to dump your garbage on your own. Moreover, you can get the size that you need and get them delivered and picked right from your place.
Services Offered By Waste Disposal Adelaide Firms
Waste comes in a variety of shapes and sizes and this is why tackling it can become a really tricky task to perform through DIY. Often it has been observed that that garbage collection has resulted in a build-up of a pile of additional garbage. Therefore, it has become a necessity to avail the service of a commercially operating waste management Adelaide firm. The good thing about these firms is that they offer a really wide variety of services and perform this task of waste removal and collection in several ways. Some of them are mentioned below.
In this service, the experts from these firms will visit your place to clean all types of rubbish prevailing there. An estimate of home cleaning relies on several factors like location, volume and time. While searching for the firm, you can obtain 5-6 quotes to compare and then, select the best of them in terms of cost and services.
Hard rubbish collection
Removal of large and hard squander is one of the best reasons behind availing the services of these waste management Adelaide firms. In this segment, we can include names like –
- Lawn mowers
- Television
- Fridge
- Washing machine
- Dishwasher
- Scrap metal
- Furniture
- Electrical appliances
- Air conditioners
- White goods (white colored home appliances and goods)
Office waste removal
Getting rid of the office waste
Offices and commercially running arenas generally create large piles of garbage at very frequent intervals. Therefore, for the owners of such places, it becomes the best option as with that they can be assured about absolutely neat and clean office. Some of the most common materials that the experts from these firms pick are –
- Electronic recycling and waste collection
- Removing old furniture
- Disposing of rubbish after a move
Other related services include –
- Deceased Estate
- Garbage Collection
- Garden And Greenery garbage Collection
- Collecting Renovation Garbage
- Carpet Removal
- Furniture Removal
- Mattress Removal
Tips on Selecting the Best Rubbish Removal Adelaide Firm
Often it becomes a challenging thing to search for a company that can help you effectively in keeping your surroundings clean. This is because there are several points that are to be applied or followed while searching for them. These points will ensure that you come across the most reputed service provider working in your area. Let us review some of these points.
Reputation of the firm
Before you finalize, it is important to shortlist at least 5-6 and researches everything about them as this will let you get aware of their respective market reputation. The best way to shortlist the finest companies is by knowing about them through referrals. You can also rely upon the Internet to search them and their status can be determined well with the help of reviews and feedbacks that they have received from their past clients.
A very important point to take note of before finalizing a firm, you should get the price quote from at least 5-6 companies before inking the contract. You need to fetch the cost details and compare them to know the most affordable one. This will also let you know about the company that offers the maximum number of services in the least price.
Before inking the deal with a bulk waste collection Adelaide firm, it is important that you inquire about the "insurance" status of the shortlisted companies. It is important that the rubbish removal Adelaide firm has a minimum of third party liability protection set in place. This will be an added advantage for the people who want to avail their services.
Before taking the last step of fixing the deal, you need to make sure that the company has the most updated equipments, machinery and infrastructure for bulk waste collection Adelaide as well as furniture recycling. They should have trucks of different sizes to meet different requirements of their clients and also a well-suited fleet of vehicles to help you get rid of the waste in a quick manner.
Different Approaches of Medical Waste Management Adelaide
We, the people have been generating different kinds of wastes from different resources and one of the most concerning of them all is the medical garbage. The term medical garbage includes materials generated as a result of healthcare practices and healthcare institutions and hospitals are the biggest generators.
Ever since our consciousness has grown towards waste disposal Adelaide and the impacts of not doing this, this form of garbage has been a very major concern for all of us. There are different approaches used to reduce generation of this form of garbage as well as its disposal. Here are some very remarkable features of the methods and approaches used for medical waste disposal Adelaide.
The rule says that all kinds of medical wastes and squander generated by the hospitals should be treated before being disposed.
This includes bio-hazardous and sharp material like needles, blades, knives, syringes that are generated because of the following.
- Diagnosis
- Treatment
- Production or testing
- Immunization
The third rule of rubbish removal Adelaide firms says that medical wastes should not be confused with –
- Medical solid elements like paper towels, cotton, bandages as in most cases; they are not bio-hazardous.
- The household wastes
- The excretes generated from livestock management on farms
- Those garbage generated in the procedure of food processing that does not include blood, infectious substances or different forms of blood products
The fourth rule says that for medical waste disposal Adelaide, there are a few rules to be followed without any laziness. These rules include –
- Steam sterilization
- Microwave technology
- Incineration
- Generators


text:
Paparazzi, photogs, shutterbugs, no matter what you call them, lately they seem to be pestering celebrities more than ever. So what's the matter with them you may ask? Are these celebrity photographers or nuisances to people who want to just live their life?
After the death of princess Diana was blamed on paparazzi, the whole world was in uproar. Angry at their behaviour, of hounding celebrity's every move. As the years have passed, it seems as if everything has blown over and the paparazzi are now more than ever out to get "that shot".
But who should be blamed, the paparazzi, the tabloids, or consumers? The reality of the matter is that when you become a celebrity, you are celebrated. Fans obsess about whatever it is that made you famous, whether that be reality TV, music, movies, or "being famous, for being famous". Regardless of how you got the fame, once you have it and you're hot right now, prepare to be the object of your fans affections. But where does it go too far?
paparazzi by sugarnstyle-visuals on polyvore.com