Skeletal muscle cells possess a 'memory' of acute early life TNF-α exposure: role of epigenetic adaptation.

Sufficient quantity and quality of skeletal muscle is required to maintain lifespan and healthspan into older age. The concept of skeletal muscle programming/memory has been suggested to contribute to accelerated muscle decline in the elderly in association with early life stress such as fetal malnutrition. Further, muscle cells in vitro appear to remember the in vivo environments from which they are derived (e.g. cancer, obesity, type II diabetes, physical inactivity and nutrient restriction). Tumour-necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is chronically elevated in sarcopenia and cancer cachexia. Higher TNF-α levels are strongly correlated with muscle loss, reduced strength and therefore morbidity and earlier mortality. We have extensively shown that TNF-α impairs regenerative capacity in mouse and human muscle derived stem cells [Meadows et al. (J Cell Physiol 183(3):330-337, 2000); Foulstone et al. (J Cell Physiol 189(2):207-215, 2001); Foulstone et al. (Exp Cell Res 294(1):223-235, 2004); Stewart et al. (J Cell Physiol 198(2):237-247, 2004); Al-Shanti et al. (Growth factors (Chur, Switzerland) 26(2):61-73, 2008); Saini et al. (Growth factors (Chur, Switzerland) 26(5):239-253, 2008); Sharples et al. (J Cell Physiol 225(1):240-250, 2010)]. We have also recently established an epigenetically mediated mechanism (SIRT1-histone deacetylase) regulating survival of myoblasts in the presence of TNF-α [Saini et al. (Exp Physiol 97(3):400-418, 2012)]. We therefore wished to extend this work in relation to muscle memory of catabolic stimuli and the potential underlying epigenetic modulation of muscle loss. To enable this aim; C2C12 myoblasts were cultured in the absence or presence of early TNF-α (early proliferative lifespan) followed by 30 population doublings in the absence of TNF-α, prior to the induction of differentiation in low serum media (LSM) in the absence or presence of late TNF-α (late proliferative lifespan). The cells that received an early plus late lifespan dose of TNF-α exhibited reduced morphological (myotube number) and biochemical (creatine kinase activity) differentiation vs. control cells that underwent the same number of proliferative divisions but only a later life encounter with TNF-α. This suggested that muscle cells had a morphological memory of the acute early lifespan TNF-α encounter. Importantly, methylation of myoD CpG islands were increased in the early TNF-α cells, 30 population doublings later, suggesting that even after an acute encounter with TNF-α, the cells have the capability of retaining elevated methylation for at least 30 cellular divisions. Despite these fascinating findings, there were no further increases in myoD methylation or changes in its gene expression when these cells were exposed to a later TNF-α dose suggesting that this was not directly responsible for the decline in differentiation observed. In conclusion, data suggest that elevated myoD methylation is retained throughout muscle cells proliferative lifespan as result of early life TNF-α treatment and has implications for the epigenetic control of muscle loss

Radioecological risk analysis of ANSTO's monthly effluent releases, 2006-07.

In line with its ISO 14000 initiatives, ANSTO endeavours to identify its environmental impacts. Part of this process includes the assessment of its releases of radioactivity into the wider environment. Liquid effluent with low levels of radioactivity is routinely released via Sydney Water's sewerage system, treated to tertiary standard in the Cronulla Sewage Treatment Plant (CSTP) and finally discharged into the marine environment at Potter Point (Figure 1). These releases occur approximately four times a week and, given the delay and mixing that occurs in transit before discharge into the ocean, can be considered to be a relatively constant release. This raises the question as to whether the releases have the potential to cause unintended environmental impacts in the receiving environment. A comprehensive overview of the potential adverse effects of enhanced environmental radioactivity is given in Copplestone et al. (2001). In this study, the possible radiological dose-rates to biota arising from the radioactivity in the ANSTO effluent have been evaluated and compared to international criteria of acceptability to assess the potential ramifications of release

Longevity and skeletal muscle mass: the role of IGF signalling, the sirtuins, dietary restriction and protein intake.

Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating socio-economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin-like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS-1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging

Testosterone enables growth and hypertrophy in fusion impaired myoblasts that display myotube atrophy: deciphering the role of androgen and IGF-I receptors

We have previously highlighted the ability of testosterone to improve differentiation and myotube hypertrophy in fusion impaired myoblasts that display reduced myotube hypertrophy at 72hrs (after transfer to low serum media) via multiple population doublings (PD) vs. their parental controls (CON); an observation which is abrogated via PI3K/Akt inhibition (Deane et al. 2013). However, whether the most predominant molecular mechanism responsible for T induced hypertrophy occurs directly via androgen receptor or indirectly via IGF-IR/PI3K/Akt pathway is currently debated. PD and CON C2C12 muscle cells were exposed to low serum conditions in the presence or absence of T (100 nM) ± inhibitors of AR (flutamide/F, 40 μm) and IGF-IR (Picropodophyllin/PPP, 150 nM) for 72 hrs and 7 days (early/late muscle differentiation respectively). T increased AR and Akt abundance, myogenin expression, and myotube hypertrophy, but not ERK1/2 activity in both CON and PD cell types. Akt activity was not increased significantly in either cell type with T. Testosterone was unable to promote early differentiation in the presence of IGF-IR inhibitor (PPP) yet still able to promote appropriate later increases in myotube hypertrophy and AR abundance despite IGF-IR inhibition. The addition of the AR inhibitor powerfully attenuated all T induced increases in differentiation and myotube hypertrophy with corresponding reductions in AR abundance, phosphorylated Akt, ERK1/2 and gene expression of IGF-I, myoD and myogenin with increases in myostatin mRNA both cell types. Interestingly, despite basally reduced differentiation and myotube hypertrophy, PD cells showed larger increased in AR abundance vs. CON cells, a response abrogated in the presence of AR but not IGF-IR inhibitors. Furthermore, T induced increases in Akt abundance were sustained despite the presence of IGF-IR inhibition in PD cells only. However, flutamide alone reduced IGF-IR mRNA in both cell types across time points, with an observed reduction in activity of ERK and Akt, perhaps suggesting that IGF-IR was transcriptionally regulated by AR. However, where testosterone increased AR protein content there was no increases observed in IGF-IR gene expression. Overall, this suggested that sufficient AR was important to enable normal gene expression of IGF-IR and downstream signalling, yet elevated levels of AR due to testosterone had no further effect on IGF-IR, despite testosterone increasing Akt abundance in the presence of IGF-IR inhibitor. In conclusion, testosterones ability to improve differentiation and myotube hypertrophy occurred predominately via increases in AR and Akt abundance in both CON and PD cells, with fusion impaired cells (PD) showing an increased responsiveness to T induced AR levels. Finally, T induced increases in myotube hypertrophy (but not early differentiation) occurred independently of upstream IGF-IR input, however it appears that normal AR function in basal conditions is required for adequate IGF-IR gene expression and downstream Akt abundance

Impaired hypertrophy in myoblasts is improved with testosterone administration

We investigated the ability of testosterone (T) to restore differentiation in multiple population doubled (PD) murine myoblasts, previously shown to have reduced differentiation in monolayer and bioengineered skeletal muscle cultures vs. their parental controls (CON) (Sharples et al., 2011, 2012 [7] and [26]). Cells were exposed to low serum conditions in the presence or absence of T (100 nM) ± PI3K inhibitor (LY294002) for 72 h and 7 days (early and late muscle differentiation respectively). Morphological analyses were performed to determine myotube number, diameter (μm) and myonuclear accretion as indices of differentiation and myotube hypertrophy. Changes in gene expression for myogenin, mTOR and myostatin were also performed. Myotube diameter in CON and PD cells increased from 17.32 ± 2.56 μm to 21.02 ± 1.89 μm and 14.58 ± 2.66 μm to 18.29 ± 3.08 μm (P ≤ 0.05) respectively after 72 h of T exposure. The increase was comparable in both PD (+25%) and CON cells (+21%) suggesting a similar intrinsic ability to respond to exogenous T administration. T treatment also significantly increased myonuclear accretion (% of myotubes expressing 5+ nuclei) in both cell types after 7 days exposure (P ≤ 0.05). Addition of PI3K inhibitor (LY294002) in the presence of T attenuated these effects in myotube morphology (in both cell types) suggesting a role for the PI3K pathway in T stimulated hypertrophy. Finally, PD myoblasts showed reduced responsiveness to T stimulated mRNA expression of mTOR vs. CON cells and T also reduced myostatin expression in PD myoblasts only. The present study demonstrates testosterone administration improves hypertrophy in myoblasts that basally display impaired differentiation and hypertrophic capacity vs. their parental controls, the action of testosterone in this model was mediated by PI3K/Akt pathway

Evaluation of the Liverpool Drink Less Enjoy More intervention

In the UK it is an offence to knowingly sell alcohol to, or purchase alcohol for, a drunk person (Regulated under Section 141 and 142 of the Licensing Act 2003). However, until recent times public awareness, bar server compliance and police enforcement of this legislation has appeared to be low. Critically, UK nightlife environments are often characterised by high levels of intoxication and alcohol-related harms. Excessive alcohol use damages the public’s health, while managing nightlife drunkenness and associated problems such as anti-social behaviour and violence places huge demands on police, local authorities and health services. To reduce such harms an extensive range of policies and interventions have been implemented at local and national levels including high profile policing, changes to licensing laws and environmental measures to improve safety. Whilst there is some evidence to indicate that these measures may contain and manage alcohol-related harms, they do little to reduce levels of intoxication or address harmful and pervasive cultures of nightlife drunkenness. A study conducted in Liverpool in 2013 found that 84% of alcohol purchase attempts by pseudo-intoxicated actors in pubs, bars and nightclubs were successful (i.e. alcohol was sold to the actor; Hughes et al., 2014). Studies conducted elsewhere have suggested that reductions in the service of alcohol to drunks, and associated harms, in nightlife settings can be achieved through the implementation of multi-component interventions that incorporate community mobilisation, enforcement of the laws around the service of alcohol to drunks and responsible bar server training. Thus to address the sale of alcohol to drunks in the city’s nightlife, local partners developed and implemented the multi-component Say No To Drunks pilot intervention. The intervention aimed to: increase awareness of legislation preventing sales of alcohol to drunks; support bar staff compliance with the law; provide a strong deterrence to selling alcohol to drunks; and promote responsible drinking amongst nightlife users. Following an evaluation of Say No To Drunks, the intervention was further refined, broadened and implemented as a second phase in 2015 – rebranded to Drink Less Enjoy More. To inform the continued development of the intervention, the Centre for Public Health at Liverpool John Moores University was commissioned to evaluate the intervention, comparing the results to previous work

Groundwater residence time in the Kulnura-Mangrove Mountain Plateau (Gosford, NSW, Australia)

The Kulnura-Mangrove Mountain plateau consists of the catchments of Mangrove, Narara, Mooney Mooney, and Ourimbah Creeks, and Wyong River. Groundwater plays a key role in sustaining stream flow within these catchments. Estimates indicate up to 50% of annual stream flow is derived from baseflow. The local community water supply relies on the groundwater within the elevated Hawkesbury- Narrabeen sandstone plateau. Furthermore, the Gosford-Wyong Councils’ Water Authority (WSA) is the third largest in NSW and utilises many of the streams flowing from the sandstone plateau for municipal water supply. It is anticipated that the WSA will provide municipal water for 319 000 persons by the year 2010. The increasing volumes of groundwater being extracted and changing land use have the potential to cause damage to the fresh water aquifer through contamination and aquifer depletion. A hydrogeochemical survey (2006-2009) has been conducted in NSW Dept of Water and Energy (DWE) monitoring wells across the plateau in order to determine groundwater residence times. Groundwater was analysed for major ions, minor and trace elements, H2O 18O and 2H, 13CDIC, 87Sr/86Sr, 14CDIC, and 3H, and complemented with mineralogical and isotopic information obtained from soil and drill chips collected during well construction. Water stable isotopes confirm the meteoric origin of the groundwater with most values plotting on the local meteoric water line. Localised evaporative trends suggest recharge with evaporated groundwater stored in ponds. Shallow groundwaters have 3H and 14C activities consistent with modern recharge (Fig 1). Carbon “bomb pulse” signatures of up to 116.8 pmC are found in the central areas of the plateau. The thin soils, lack of carbonates in the intensely weathered near-surface Hawkesbury sandstone, and the shallow depth of the water samples is consistent with the 3H results measured, suggesting minimal dilution of the original 14C. Input of this data into a southern hemisphere bomb pulse model [1] suggest potential recharge during the 1990´s, coinciding with sustained wet conditions and above average rainfalls experienced during this period. Fig. 1. 14C vs 3H plot of groundwater samples in the Kulnura- Mangrove Mountain Plateau Deeper groundwaters have lower 14C and 3H activities in some cases close to background level (Fig. 1). The quantifiable 3H suggests residence times of <70 a. However, non-corrected 14C residence times are submodern (>500 a). This apparent discrepancy can be explained by either mixing with older waters or dissolution of carbonates. The good correlation of total dissolved inorganic carbon (TDIC) and Ca (R2=0.8), 13CTDIC in groundwater and mineralogy results from drill chips suggest that dissolution of dispersed carbonates is taking place. The deepest groundwaters show the most difference in residence time across the study area. The eastern and western plateaus yield old groundwater with 14C corrected residence times of around 9 ka and 4 ka respectively. However, the groundwater at equivalent depths in the central plateau was found to be considerably younger with residence times of <70 a

A comparison between direct and pan-derived measurements of the isotopic composition of atmospheric waters

The stable isotopes of water can be used to examine and quantify the contribution to atmospheric moisture from evaporation, transpiration and surface-waters. However, obtaining extensive and ongoing time series data of the isotopic composition of atmospheric moisture has been difficult. Presented here is an alternate method using an isotope mass balance approach to estimate the isotopic composition of atmospheric moisture using water samples collected from class A evaporation pans. While this evaporation pan method does not provide the high-resolution time series data that can be obtained from an isotope analyser taking in-situ measurements of atmospheric moisture, the method is relatively simple and inexpensive to set-up and maintain. In this preliminary investigation, a comparison between the isotopic composition of atmospheric moisture estimated from the evaporation pan method and in-situ measurements of the isotopic composition of water vapour using a Fourier Transform Infrared (FTIR) spectrometer deployed at the Lucas Heights weather station in New South Wales is undertaken. Through comparison of the two series of hydrogen isotope data, an assessment of the evaporation pan method can be made. Although there was some agreement between the isotopic composition of vapour measured by the FTIR spectrometer and the estimation for the atmospheric moisture (R2 = 0.49), the comparison is sensitive to climatic parameters that vary significantly within a 24-hour period such as the relative humidity of air and the air and pan temperatures. Inverting the model to use the FTIR spectrometer measurements at an hourly resolution improved the performance of the model (R2 =0.57). However, this also revealed that the model produced more depleted values of the evaporation pan water isotopes than those observed. In contrast, there was a variable relationship between the modelled and observed isotope values of atmospheric moisture. These conflicting results will need to be resolved before the evaporation pan method is broadly applied in isotope hydrology. © 2011 The Modelling and Simulation Society of Australia and New Zealand Inc

L-glutamine improves skeletal muscle cell differentiation and prevents myotube atrophy after cytokine (TNF-α) stress via reduced p38 MAPK signal transduction

Tumour Necrosis Factor- Alpha (TNF-α) is chronically elevated in conditions where skeletal muscle loss occurs. As L-glutamine can dampen the effects of inflamed environments, we investigated the role of L-glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF-α (20 ng.ml−1) ± L-glutamine (20 mM).TNF-α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf-I and Igfbp5. Furthermore, when administered to mature myotubes, TNF-α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf-I, Igfbp2 and glutamine synthetase and parallel increases in Fox03, Cfos, p53 and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non-significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF-α. Importantly, 20 mM L-glutamine reduced p38 MAPK activity in TNF-α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. L-glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf-Ir, Myhc2 & 7, Tnfsfr1b, Adra1d and restored atrophic gene expression of Fox03 back to baseline in TNF-α conditions. In conclusion, L-glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. L-glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. This article is protected by copyright. All rights reserve