The docking protein p130Cas regulates cell sensitivity to proteasome inhibition

<p>Abstract</p> <p>Background</p> <p>The focal adhesion protein p130Cas (Cas) activates multiple intracellular signaling pathways upon integrin or growth factor receptor ligation. Full-length Cas frequently promotes cell survival and migration, while its C-terminal fragment (Cas-CT) produced upon intracellular proteolysis is known to induce apoptosis in some circumstances. Here, we have studied the putative role of Cas in regulating cell survival and death pathways upon proteasome inhibition.</p> <p>Results</p> <p>We found that Cas-/- mouse embryonic fibroblasts (MEFs), as well as empty vector-transfected Cas-/- MEFs (Cas-/- (EV)) are significantly resistant to cell death induced by proteasome inhibitors, such as MG132 and Bortezomib. As expected, wild-type MEFs (WT) and Cas-/- MEFs reconstituted with full-length Cas (Cas-FL) were sensitive to MG132- and Bortezomib-induced apoptosis that involved activation of a caspase-cascade, including Caspase-8. Cas-CT generation was not required for MG132-induced cell death, since expression of cleavage-resistant Cas mutants effectively increased sensitivity of Cas-/- MEFs to MG132. At the present time, the domains in Cas and the downstream pathways that are required for mediating cell death induced by proteasome inhibitors remain unknown. Interestingly, however, MG132 or Bortezomib treatment resulted in activation of autophagy in cells that lacked Cas, but not in cells that expressed Cas. Furthermore, autophagy was found to play a protective role in Cas-deficient cells, as inhibition of autophagy either by chemical or genetic means enhanced MG132-induced apoptosis in Cas-/- (EV) cells, but not in Cas-FL cells. Lack of Cas also contributed to resistance to the DNA-damaging agent Doxorubicin, which coincided with Doxorubicin-induced autophagy in Cas-/- (EV) cells. Thus, Cas may have a regulatory role in cell death signaling in response to multiple different stimuli. The mechanisms by which Cas inhibits induction of autophagy and affects cell death pathways are currently being investigated.</p> <p>Conclusion</p> <p>Our study demonstrates that Cas is required for apoptosis that is induced by proteasome inhibition, and potentially by other death stimuli. We additionally show that Cas may promote such apoptosis, at least partially, by inhibiting autophagy. This is the first demonstration of Cas being involved in the regulation of autophagy, adding to the previous findings by others linking focal adhesion components to the process of autophagy.</p

Test-retest reliability of selected items of Health Behaviour in School-aged Children (HBSC) survey questionnaire in Beijing, China

<p>Abstract</p> <p>Background</p> <p>Children's health and health behaviour are essential for their development and it is important to obtain abundant and accurate information to understand young people's health and health behaviour. The Health Behaviour in School-aged Children (HBSC) study is among the first large-scale international surveys on adolescent health through self-report questionnaires. So far, more than 40 countries in Europe and North America have been involved in the HBSC study. The purpose of this study is to assess the test-retest reliability of selected items in the Chinese version of the HBSC survey questionnaire in a sample of adolescents in Beijing, China.</p> <p>Methods</p> <p>A sample of 95 male and female students aged 11 or 15 years old participated in a test and retest with a three weeks interval. Student Identity numbers of respondents were utilized to permit matching of test-retest questionnaires. 23 items concerning physical activity, sedentary behaviour, sleep and substance use were evaluated by using the percentage of response shifts and the single measure Intraclass Correlation Coefficients (ICC) with 95% confidence interval (CI) for all respondents and stratified by gender and age. Items on substance use were only evaluated for school children aged 15 years old.</p> <p>Results</p> <p>The percentage of no response shift between test and retest varied from 32% for the item on computer use at weekends to 92% for the three items on smoking. Of all the 23 items evaluated, 6 items (26%) showed a moderate reliability, 12 items (52%) displayed a substantial reliability and 4 items (17%) indicated almost perfect reliability. No gender and age group difference of the test-retest reliability was found except for a few items on sedentary behaviour.</p> <p>Conclusions</p> <p>The overall findings of this study suggest that most selected indicators in the HBSC survey questionnaire have satisfactory test-retest reliability for the students in Beijing. Further test-retest studies in a large and diverse sample, as well as validity studies, should be considered for the future Chinese HBSC study.</p

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Does corporate reputation matter? Role of social media in consumer intention to purchase innovative food product

The exponential growth of the corporate reputation in food industry has resulted in innovations in every link of its supply chain. There have been studies that have characterized innovation in various industries from the perspective of technology, but far fewer in the area of corporate reputation, consumer perception, and intention towards innovations in food products. This research analyses the innovations in the food industry from the perspective of the consumer and provides a conceptual framework of food innovation stages. The study also investigates the relationship between corporate reputation and intention towards food innovation along with the other components of TPB model with an extension of social media engagement. The results from India and US samples confirm that social media engagement have a significant role to play in creating intention to purchase innovative food products. The study compares the US and Indian samples and identifies differences in subjective norms and perceived behavioural control

Network Analyses Reveal Novel Aspects of ALS Pathogenesis

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons, muscle atrophy and paralysis. Mutations in the human VAMP-associated protein B (hVAPB) cause a heterogeneous group of motor neuron diseases including ALS8. Despite extensive research, the molecular mechanisms underlying ALS pathogenesis remain largely unknown. Genetic screens for key interactors of hVAPB activity in the intact nervous system, however, represent a fundamental approach towards understanding the in vivo function of hVAPB and its role in ALS pathogenesis. Targeted expression of the disease-causing allele leads to neurodegeneration and progressive decline in motor performance when expressed in the adult Drosophila, eye or in its entire nervous system, respectively. By using these two phenotypic readouts, we carried out a systematic survey of the Drosophila genome to identify modifiers of hVAPB-induced neurotoxicity. Modifiers cluster in a diverse array of biological functions including processes and genes that have been previously linked to hVAPB function, such as proteolysis and vesicular trafficking. In addition to established mechanisms, the screen identified endocytic trafficking and genes controlling proliferation and apoptosis as potent modifiers of ALS8-mediated defects. Surprisingly, the list of modifiers was mostly enriched for proteins linked to lipid droplet biogenesis and dynamics. Computational analysis reveals that most modifiers can be linked into a complex network of interacting genes, and that the human genes homologous to the Drosophila modifiers can be assembled into an interacting network largely overlapping with that in flies. Identity markers of the endocytic process were also found to abnormally accumulate in ALS patients, further supporting the relevance of the fly data for human biology. Collectively, these results not only lead to a better understanding of hVAPB function but also point to potentially relevant targets for therapeutic intervention

Nonlinear 3D projection printing of concave hydrogel microstructures for long-term multicellular spheroid and embryoid body culture.

Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propagation. Here, we used a continuous 3D projection printing approach - with an important modification of nonlinear exposure - to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs

Nonlinear 3D projection printing of concave hydrogel microstructures for long-term multicellular spheroid and embryoid body culture

Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propogation. Here, we used a continuous 3D projection printing approach – with an important modification of nonlinear exposure — to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs