Establishment and Validation of Computational Model for MT1-MMP Dependent ECM Degradation and Intervention Strategies

MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion

Identification of Neural Outgrowth Genes using Genome-Wide RNAi

While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system

Multidimensional needs of patients living and dying with heart failure in Kenya: a serial interview study

Abstract Background Heart failure is an emerging challenge for Sub Saharan Africa. However, research on patients’ needs and experiences of care is scarce with little evidence available to support and develop services. We aimed to explore the experiences of patients living and dying with heart failure in Kenya. Methods We purposively recruited 18 patients admitted with advanced heart failure at a rural district hospital in Kenya. We conducted serial in depth interviews with patients at 0, 3 and 6 months after recruitment, and conducted bereavement interviews with carers. Interviews were recorded, transcribed into English and analyzed using a thematic approach, assisted by Nvivo software package. Results Forty-four interviews were conducted. Patients experienced physical, psychosocial, spiritual and financial distress. They also had unmet needs for information about their illness, how it would affect them and how they could get better. Patients experience of and their interpretation of symptoms influenced health care seeking. Patients with acute symptoms sought care earlier than those with more gradual symptoms which tended to be normalised as part of daily life or assumed to be linked to common treatable conditions. Nearly all patients expected to be cured and were frustrated by a progressive illness poorly responsive to treatment. Accumulating costs was a barrier to continuity of care and caused tensions in social relationships. Patients valued information on the nature of their illness, prognosis, self-care, lifestyle changes and prevention strategies, but this was rarely available. Conclusions This is the first in-depth study to explore the experiences of people living with advanced heart failure in Kenya. This study suggests that patients would benefit from holistic care, such as a palliative approach that is aimed at providing multidimensional symptom management. A palliative approach to services should be provided alongside chronic disease management aimed at primary prevention of risk factors, and early identification and initiation of disease modifying therapy. Further research is needed to determine best practice for integrating palliative care for people living and dying with heart failure