Pharmacological Inhibition of mTOR and ERK1/2 Resulted in Attenuated Protein Synthesis Rates in Differentiated C2C12 Myoblasts in a Similar Fashion to in vivo Rodent Studies.

Fractional protein synthesis rates have long been used as in indicator of acute alterations in the anabolic state of various tissues. Through the use of a number of stable and isotopic tracer methodologies, the measurement of fractional synthesis rates (FSR) in vivo has become a staple of skeletal muscle physiology. Through the application of a deuterium oxide tracer, this project sought to measure pharmacological perturbations in fractional synthesis rates in culture in differentiated C2C12 murine myotubes. PURPOSE: To assess myofibrillar protein FSR in differentiated C2C12 murine myotubes following pharmacological inhibition of rapamycin-sensitive (mTOR) or -insensitive (ERK1/2) pathways, and how signal transduction through these pathways impact FSR as compared to previous in vivo studies of pharmacological inhibition studies in skeletal muscle. METHODS: C2C12 murine myoblasts were cultured in collagen coated 6 well culture dishes, and grown to 60-70% confluency using a high glucose DMEM growth media (GM). Cultures were transitioned to a differentiation media (DM) upon reaching target confluency. DM was changed daily for 4 days to allow for complete differentiation to myotubes. Cultures were randomly assigned treatment conditions of cell control (CC), rapamycin inhibition (RAPA), ERK1/2 inhibition (ERK), and electrical stimulation (ESTIM). Cultures underwent treatment conditions for 24 hours with a 4% deuterium oxide GM supplement. Analysis was carried out using a gas chromatography mass spectrometer. RESULTS: Fractional rates of protein synthesis were significantly lower in the RAPA (p=0.028) and ERK (p=0.029) groups as compared to CC, with no differences between RAPA and ERK groups (p\u3e0.05). Although statistics were not applied to the ESTIM group due to low sample size, electrical pulse stimulation shows promise for the stimulation of FSR in cultured myotubes. CONCLUSION: Diminished FSR in both RAPA and ERK groups are consistent with previous findings from in vivo rodent studies. These results may indicate comparable alterations in skeletal muscle anabolic signaling in cell culture as well as in vivo rodent models. Further investigations into anabolic signaling mechanisms related to the control of protein synthesis are needed

Characterization of Protein Metabolism in Undifferentiated and Differentiated Murine Muscle Tissue

The emergence of cell culture experiments have greatly expanded the understanding of skeletal muscle physiology. However, there is a paucity of data regarding the behaviors of cells grown in culture at various stages versus in vivo. This preliminary set of studies was designed to assess alterations of anabolic responses between undifferentiated and differentiated muscle tissue in [high] and [low] glucose media along with varying dosages of insulin. Purpose: Determine if there is a disparity in fractional synthesis rates (FSR) between C2C12 myoblasts and myotubes with varying levels of insulin and in [high] (4.5g/L) and [low] glucose (2.75 g/L) media. Methods: All cells that were going to be differentiated were started on a [high] glucose differentiation media for 48 hours. The [high] glucose differentiation media was continually applied for the [high] glucose group until harvest of the cells. The [low] glucose media group had the [high] glucose differentiation media removed and [low] glucose differentiation media was applied for 48 hours until the cells were harvested. Both [low] and [high] glucose groups received three different levels of insulin. T-25’s received either 75 µL, 150 µL, or 300 µL. T-75’s 195 µL, 390 µL, and 780 µL. Deuterium oxide was applied 24 hours prior to harvest of the cells at a level of 4%. Results: Preliminary data demonstrates that differentiated murine myotubes have slightly elevated FSR than undifferentiated myoblasts (p\u3c0.013). When insulin was added to the growth media, FSR was found to be elevated in undifferentiated cells compared to controls (p\u3c0.05). Within the differentiated myotubes, the [low] glucose myotubes had higher FSR than myotubes that were incubated in [high] glucose myotubes (p\u3c0.001). There was also no difference in FSR based on flask size for either the undifferentiated (p\u3e0.181) or differentiated (p\u3e0.464) C2C12’s. Conclusion: Future investigators must be aware of the ratio of undifferentiated cells and differentiated myotubes as this ratio could confound results as myoblasts are still present even at later stages of differentiation. Current protocols for differentiation media, regarding insulin addition, provide for optimal anabolic responses. Elevated FSR rates in the myotubes fed [low] glucose media could be explained by the cells having a higher turnover rate of cellular proteins

Do high-risk medicines alerts influence practice?

Background: Medicine-related adverse events are prevalent, costly and mostly preventable. The High Risk Medicines Working Party (Victoria) developed and distributed three highrisk medicines alerts &ndash; wrong route of administration of oral medicines, subcutaneous insulin and unfractionated heparin &ndash; and accompanying audit tools in 2008 and 2009.Aims: To determine the impact of the three high-risk medicines alerts on Victorian health services; to assess the clinical relevance and utility of the audit tools; to identify barriers to implementing recommendations; and to obtain feedback and suggestions for future alert topics.Method: A cross-sectional survey was undertaken from 6 to 31 July 2009 using an online questionnaire. The questionnaire was distributed to 90 metropolitan, regional and rural public health services in Victoria and approximately 200 members of the Quality Use of Medicines Network (Victoria).Results: Most of the 90 respondents were pharmacists (53%) and nurses (31%). 53 (59%) respondents reported making changes as a result of receiving the high-risk medicines alerts &ndash; 21 (40%) concerned the wrong route of administration, 12 (23%) subcutaneous insulin and 7 (13%) unfractionated heparin. Barriers to implementation included time constraints, inadequate staff and resources, excessive paperwork and competing priorities. A minority of respondents indicated some alerts were not relevant to small rural services. Suggestions forimproving the audit tools included making them less labour intensive, enabling electronic responses and ensuring their distribution is coordinated with other medicine-related tools.Conclusion: High-risk medicines alerts and the accompanying audit tools facilitated change but there were some barriers to their implementation, such as time and resource constraints. Not all alerts and audit tools were relevant to all health services.<br /

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives