Pathways of intracellular protein degradation in cultured muscle cells

To investigate mechanisms responsible for the turnover of endogenous muscle protein, lysosomotropic proteinase inhibitors have been employed to elucidate the relative contributions of lysosomal and non-lysosomal degradation pathways functioning under varying nutritional states and for different classes of intracellular proteins. Proteolysis in cultured bovine aortic smooth muscle cells was measured as the percentage of ³H-phenylalanine released per hour from pre-labelled cellular proteins. To reduce background radioactivity, the intracellular ³H-phenylalanine pool was depleted by serial extraction at 37°C, effecting equilibration between the intracellular pool and the phenylalanine-free medium. Reutilization of labelled amino acids during subsequent incubation periods was minimized by the presence of excess non-labelled phenylalanine in the medium. ³H-phenylalanine was released at a constant rate of 1,5 % per hour for at least 4 h, from cells pre-labelled for 16 h ('long-lived' proteins). Leupeptin, an inhibitor of thiol proteinases including cathepsin 8, inhibited degradation by 12 %, whereas the general lysosomal inhibitors chloroquine and NH₄Cl inhibited degradation by 30 %, presumably the contribution by the lysosomal pathway. In the case of 'short-lived' proteins (pre-labelled for 1 hour), the initial degradation rate was 6,5% per hour, which rapidly declined, reaching the basal rate of 1,5 % after 4 h. Chloroquine and NH₄Cl reduced proteolysis by only 12-15% and leupeptin had no significant inhibition, consistent with the view that the majority of short-lived proteins a degraded by non-lysosomal pathways. Proteolysis rates of 'abnormal' proteins containing the arginine-analogue, canavanine, were found to be significantly elevated (80 %) over controls. Leupeptin had no significant inhibition, and chloroquine and NH₄Cl only reduced degradation by 12-16 %, showing that the rapid removal of 'abnormal' intracellular proteins proceeds mainly via extra-lysosomal mechanisms. Incubation of the cells under nutritional step-down conditions, increased the average degradation rate of long-lived proteins to 3% per hour, and chloroquine and NH₄Cl inhibited degradation by 55-60 %, indicating that the accelerated proteolytic condition is due to increased activity of the lysosomes. Nutritional deprivation did not increase the rate of degradation of short-lived proteins. The results were clarified by the parallel use of the well-characterized LDL degradation system in this cell type, known to occur almost exclusively via lysosomes. This allowed the effectiveness of lysosomotropic inhibitors to be tested. Chloroquine inhibited LDL degradation by over 90 % and NH₄Cl inhibited by 80-95 % in all cases. Other proteinase inhibitors such as chymostatin, pepstatin and the chloromethyl ketones were also tested, and of these chymostatin seemed to be the most valuable because of its additivity to the effect of chloroquine, indicating its selective inhibition of non-lysosomal degradative mechanisms. Incubations of smooth muscle cells under anoxic conditions or with metabolic inhibitors such as fluoride, azide and cyanide, resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of chloroquine, i.e. both lysosomal and non-lysosomal degradation pathways have some energy-dependence. The degradation of long-lived proteins appeared to be more sensitive to temperature than that of short-lived proteins, further indicating the activity of distinct proteolytic mechanisms for these two classes of intracellular proteins. Preliminary studies have indicated a role for Ca⁺⁺ in the regulation of proteolysis, since degradation rates were increased by elevated levels of Ca⁺⁺ in the extracellular medium. Inhibition of this increased proteolysis by leupeptin has indicated a role for a thiol proteinase, possibly Ca⁺⁺-activated neutral proteinase. In similar studies with cultured L8 skeletal muscle cells, an average proteolysis rate of 1,2 % per hour was found, which was increased by 50 % under nutritional step-down conditions. Once again, the lysosomal pathway was found to account for only about one-third of basal protein degradation but fully accounted for the increased proteolysis under nutrient deprivation. The degradation characteristics of intracellular smooth and skeletal muscle cell proteins was examined using double isotope labelling. It was found that large molecular weight proteins and glycoproteins tended to be degraded more rapidly than small proteins and non-glycoproteins. In smooth muscle cells, these correlations were markedly reduced or absent under the accelerated proteolysis associated with nutrient deprivation, possibly confirming the increased activity of the non-selective autophagic lysosomal pathway under these conditions. A similar loss of correlations was not so clearly seen for skeletal muscle cell proteins

An evaluation of student perceptions of learning environments across fully on-line versus blended course delivery formats.

The primary focus of this evaluation study was to describe students\u27 perceptions of their course experiences within two distinct groups of students who participated in either a fully online or a hybrid/blended version of an introductory course. The groups differed in course format (hybrid versus online group) and measures used included primarily the seven scale scores on the Distance Education Learning Environments Survey (DELES) (Walker & Fraser, 2005). Additionally students were asked to respond to one open-ended question designed to assess perceptions of the course delivery format specifically. Although findings must be interpreted with great caution, due primarily to low response rates, a sample limited to one community college, and a focus on perceptions alone rather than broader outcomes, the evaluation study leads to a number of preliminary conclusions. First, it appears that one key outcome from the survey is that students desire that instructors provide constant and prompt feedback to students whether it be negative or positive communication. Second, being able to apply the course content to workplace or life situations was seen as valuable to the students in the online section more so than those in the hybrid section. Third, while there was some negativity from the students enrolled in the online section, overall the comments in the open-ended questions portrayed the instructor in a positive light. Suggestions for further research on this topic include accessing broader and more diverse and representative samples of student participants, working to ensure higher response rates, and gaining measures of actual course impacts on learning or other performance outcomes, rather than relying on perceptions alone

State-steered smartmentality in Chinese smart urbanism

This study explores the socio-political shaping of Chinese smart urbanism by examining the power relations between the government (national and municipal), private firms and citizens embedded in smartmentality. Our exploration begins with teasing out key analytical standpoints of Alberto Vanolo’s concept of smartmentality applied in neoliberal practices of smart urbanism. Through this analytical framework, we conceptualise Chinafied smartmentality and illustrate how it is actually playing out in China by undertaking documentary research and in-depth interviews from an inductive case study of the Smart Transportation System (STS) in the city of Shijiazhuang. We observe that the idea of Chinafication extends smartmentality with a focus on the power dynamic. We further argue that this Chinafied smartmentality implies uncritical technological solutionism that is state-steered in nature and citizen participation in digital platforms that is performed with limited roles and power of being included. The paper concludes by calling for future research on the critical examination of value co-creation for shaping a truly citizen-centric mode of governance in Chinese smart urbanism

Development and preliminary evidence for the validity of an instrument assessing implementation of human-factors principles in medication-related decision-support systems—I-MeDeSA

Background Medication-related decision support can reduce the frequency of preventable adverse drug events. However, the design of current medication alerts often results in alert fatigue and high over-ride rates, thus reducing any potential benefits. Methods The authors previously reviewed human-factors principles for relevance to medication-related decision support alerts. In this study, instrument items were developed for assessing the appropriate implementation of these human-factors principles in drug-drug interaction (DDI) alerts. User feedback regarding nine electronic medical records was considered during the development process. Content validity, construct validity through correlation analysis, and inter-rater reliability were assessed. Results The final version of the instrument included 26 items associated with nine human-factors principles. Content validation on three systems resulted in the addition of one principle (Corrective Actions) to the instrument and the elimination of eight items. Additionally, the wording of eight items was altered. Correlation analysis suggests a direct relationship between system age and performance of DDI alerts (p=0.0016). Inter-rater reliability indicated substantial agreement between raters (κ=0.764). Conclusion The authors developed and gathered preliminary evidence for the validity of an instrument that measures the appropriate use of human-factors principles in the design and display of DDI alerts. Designers of DDI alerts may use the instrument to improve usability and increase user acceptance of medication alerts, and organizations selecting an electronic medical record may find the instrument helpful in meeting their clinicians' usability need

Establishing an international laboratory network for neglected tropical diseases: Understanding existing capacity in five WHO regions [version 4; peer review: 2 approved, 1 approved with reservations]

Background. Limited laboratory capacity is a significant bottleneck in meeting global targets for the control and elimination of neglected tropical diseases (NTD). Laboratories are essential for providing clinical data and monitoring data about the status and changes in NTD prevalence, and for detecting early drug resistance. Currently NTD laboratory networks are informal and specialist laboratory expertise is not well publicised, making it difficult to share global expertise and provide training, supervision, and quality assurance for NTD diagnosis and research. This study aimed to identify laboratories within five World Health Organisation regions (South-East Asia, Eastern Mediterranean, Americas, Western Pacific and Europe) that provide NTD services and could be regarded as national or regional reference laboratories, and to conduct a survey to document their networks and capacity to support NTD programmes. Methods. Potential NTD reference laboratories were identified through systematic searches, snowball sampling and key informants. Results. Thirty-two laboratories responded to the survey. The laboratories covered 17 different NTDs and their main regional and national roles were to provide technical support and training, research, test validation and standard setting. Two thirds of the laboratories were based in academic institutions and almost half had less than 11 staff. Although greater than 90 per cent of the laboratories had adequate technical skills to function as an NTD reference laboratory, almost all laboratories lacked systems for external verification that their results met international standards. Conclusions. This study highlights that although  many laboratories believed they could act as a reference laboratory, only a few had all the characteristics required to fulfil this role as they fell short in the standard and quality assurance of laboratory processes. Networks of high quality laboratories are essential for the control and elimination of disease and this study presents a critical first step in the development of such networks for NTDs

Incorporating Medication Indications into the Prescribing Process

Purpose The incorporation of medication indications into the prescribing process to improve patient safety is discussed. Summary Currently, most prescriptions lack a key piece of information needed for safe medication use: the patient-specific drug indication. Integrating indications could pave the way for safer prescribing in multiple ways, including avoiding look-alike/sound-alike errors, facilitating selection of drugs of choice, aiding in communication among the healthcare team, bolstering patient understanding and adherence, and organizing medication lists to facilitate medication reconciliation. Although strongly supported by pharmacists, multiple prior attempts to encourage prescribers to include the indication on prescriptions have not been successful. We convened 6 expert panels to consult high-level stakeholders on system design considerations and requirements necessary for building and implementing an indications-based computerized prescriber order-entry (CPOE) system. We summarize our findings from the 6 expert stakeholder panels, including rationale, literature findings, potential benefits, and challenges of incorporating indications into the prescribing process. Based on this stakeholder input, design requirements for a new CPOE interface and workflow have been identified. Conclusion The emergence of universal electronic prescribing and content knowledge vendors has laid the groundwork for incorporating indications into the CPOE prescribing process. As medication prescribing moves in the direction of inclusion of the indication, it is imperative to design CPOE systems to efficiently and effectively incorporate indications into prescriber workflows and optimize ways this can best be accomplished

Accuracy of Malaria Rapid Diagnostic Tests in Community Studies and their Impact on Treatment of Malaria in an Area with Declining Malaria Burden in North-Eastern Tanzania.

Despite some problems related to accuracy and applicability of malaria rapid diagnostic tests (RDTs), they are currently the best option in areas with limited laboratory services for improving case management through parasitological diagnosis and reducing over-treatment. This study was conducted in areas with declining malaria burden to assess; 1) the accuracy of RDTs when used at different community settings, 2) the impact of using RDTs on anti-malarial dispensing by community-owned resource persons (CORPs) and 3) adherence of CORPs to treatment guidelines by providing treatment based on RDT results. Data were obtained from: 1) a longitudinal study of passive case detection of fevers using CORPs in six villages in Korogwe; and 2) cross-sectional surveys (CSS) in six villages of Korogwe and Muheza districts, north-eastern, Tanzania. Performance of RDTs was compared with microscopy as a gold standard, and factors affecting their accuracy were explored using a multivariate logistic regression model. Overall sensitivity and specificity of RDTs in the longitudinal study (of 23,793 febrile cases; 18,154 with microscopy and RDTs results) were 88.6% and 88.2%, respectively. In the CSS, the sensitivity was significantly lower (63.4%; χ2=367.7, p<0.001), while the specificity was significantly higher (94.3%; χ2=143.1, p<0.001) when compared to the longitudinal study. As determinants of sensitivity of RDTs in both studies, parasite density of<200 asexual parasites/μl was significantly associated with high risk of false negative RDTs (OR≥16.60, p<0.001), while the risk of false negative test was significantly lower among cases with fever (axillary temperature ≥37.5 °C) (OR≤0.63, p≤0.027). The risk of false positive RDT (as a determinant of specificity) was significantly higher in cases with fever compared to afebrile cases (OR≥2.40, p<0.001). Using RDTs reduced anti-malarials dispensing from 98.9% to 32.1% in cases aged ≥5 years. Although RDTs had low sensitivity and specificity, which varied widely depending on fever and parasite density, using RDTs reduced over-treatment with anti-malarials significantly. Thus, with declining malaria prevalence, RDTs will potentially identify majority of febrile cases with parasites and lead to improved management of malaria and non-malaria fevers