Identification and characterisation of novel mutant p53 regulators

© 2018 Dr. Kah Hin TanThe p53 protein is a transcription factor and has been considered as a master tumour suppressor. TP53 gene is frequently mutated in human cancers (more than 50%). Mutation in p53 not only loses its tumour suppressive functions but also gains new oncogenic properties, including increased cell proliferation, higher resistance to therapeutics and enhanced metastasis. These are consistent with the association of mutant p53 cancers with poor clinical prognosis. Importantly, some cancer cells are dependent on mutant p53 expression for survival and proliferation. This opens an exciting therapeutic window for the treatment of multiple cancer types. Despite the prominent roles of mutant p53 in malignant progression, its regulation remains partially understood. The major goal of this study is to gain novel insight into the regulation of mutant p53. Our approach has been to identify novel regulators of mutant p53 by utilising whole-genome RNAi discovery screens. These include protein-coding siRNA, microRNA (miRNA) and long noncoding RNA (lncRNA) screens. We have developed a high content assay for these screens. The libraries were subjected to multi-tiered screens on mutant p53-expressing cell lines, including MDA-MB-468, JH-EsoAd1, MDA-MB-231 and AU565. Of 18,120 protein-coding siRNAs, the screen yielded 50 potential mutant p53 candidate regulators. Six of these were conserved between MDA-MB-468 and JH-EsoAd1, and 44 were cell-line specific. Primary lncRNA screen yielded 154 hits, in which 39 and 44 were validated in MDA-MB- 468 and JH-EsoAd1 cell lines, respectively. The miRNA screen yielded a total of 211 mimics and 46 inhibitor hits. Of these, the top 20 miRNA mimics were pursued further. Three potential candidates were identified to down-regulate mutant p53 expression. One of these is miR-504-5p, which has been previously identified to target and down-regulate wild-type p53. Further investigation of these miRNAs led to the identification of MAP1B as a potential regulator of mutant p53. Our result demonstrated that knockdown of MAP1B sensitises mutant p53-expressing cells. The relevance of MAP1B expression to overall survival (OS) of gastric cancer patients (which harbour high TP53 mutation incidence, up to 77 %) was evaluated. Consistently, high MAP1B mRNA expression correlates with poor overall patient survival. Reciprocally, overall survival is improved in patients with low MAP1B mRNA expression. These results suggest a potential role for MAP1B in the oncogenicity of mutant p53 cancer. The mechanism by which MAP1B affects mutant p53 is yet to be elucidated. Overall, this thesis demonstrates the utility of RNAi screen in the exploration of mutant p53 regulation. This study has identified a list of potential regulators that were not previously shown to regulate mutant p53. Future study of these potential novel mutant p53 regulators will help to dissect the regulatory network of mutant p53 and potentially inform on novel therapeutic approaches

Applying TRIZ to Service Conceptual Design: An Exploratory Study” Creativity annd Innovatıon Management

This paper introduces a new avenue for applying the Theory of Inventive Problem Solving (TRIZ). TRIZ tools may be used in designing new service concepts in the field of new service development (NSD). Up to the present time, the practice of generating new ideas in NSD has been dependent largely on inspiration, luck and flair. One shortcoming in the generation of creative ideas is the psychological inertia or mental block commonly encountered. This research proposes to use TRIZ to help spawn new and perhaps unorthodox ideas and concepts in NSD. A case study on canteen operation demonstrates the feasibility of applying TRIZ in service design

Efficacy and safety of celgosivir in patients with dengue fever (CELADEN): a phase 1b, randomised, double-blind, placebo-controlled, proof-of-concept trial.

BACKGROUND: Dengue infection is the most common mosquito-borne viral disease worldwide, but no suitable antiviral drugs are available. We tested the α-glucosidase inhibitor celgosivir as a treatment for acute dengue fever. METHODS: To establish eligibility for inclusion in a phase 1b, randomised, double-blind, placebo-controlled, proof-of-concept trial, individuals aged 21-65 years who had had a fever (≥38°C) for less than 48 h, met at least two criteria indicating probable dengue infection, and had a positive result on a dengue point-of-care test kit or PCR assay were referred for screening at a centre in Singapore between July 30, 2012, and March 4, 2013. Using a web-based system, we randomly assigned patients who met full inclusion criteria after screening (1:1; random permuted block length four) to celgosivir (initial 400 mg loading dose within 6 h of randomisation, followed by 200 mg every 12 h for a total of nine doses) or matched placebo. Patients and the entire study team were masked to group assignment. The primary endpoints were mean virological log reduction (VLR) from baseline for days 2, 3, and 4, and area under the fever curve (AUC) for a temperature above 37°C from 0 h to 96 h. Efficacy analyses were by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01619969. FINDINGS: We screened 69 patients and randomly assigned 50 (24 to celgosivir, 26 to placebo). Mean VLR was greater in the celgosivir group (-1·86, SD 1·07) than in the placebo group (-1·64, 0·75), but the difference was non-significant (-0·22, 90% CI -0·65 to 0·22; one-sided p=0·203). The mean AUC was also higher in the celgosivir group (54·92, SD 31·04) than in the placebo group (40·72, 18·69), but again the difference was non-significant (14·20, 90% CI 2·16-26·25; one-sided p=0·973). We noted similar incidences of adverse events between groups. INTERPRETATION: Although generally safe and well tolerated, celgosivir does not seem to reduce viral load or fever burden in patients with dengue. FUNDING: STOP Dengue Translational Clinical Research

A TRIZ-based method for new service design

10.1177/1094670505276683Journal of Service Research8148-6

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to < 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of & GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P < 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo

Guidelines for the use and interpretation of assays for monitoring autophagy

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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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. 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. 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

Guidelines for the use and interpretation of assays for monitoring autophagy

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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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. 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. 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