Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells

<p>Abstract</p> <p>Background</p> <p>Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.</p> <p>Results</p> <p>A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently <it>CASP8AP2</it>/<it>FLASH</it>. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of <it>CASP8AP2</it>/<it>FLASH </it>resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of <it>CASP8AP2</it>/<it>FLASH </it>also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of <it>CASP8AP2</it>/<it>FLASH </it>silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.</p> <p>Conclusions</p> <p>We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene <it>NEFH</it>.</p

Quality indicators for Palliative Day Services: A modified Delphi study

BACKGROUND: The goal of Palliative Day Services is to provide holistic care that contributes to the quality of life of people with life threatening-illness and their families. Quality indicators provide a means by which to describe, monitor and evaluate the quality of Palliative Day Services provision, and act as a starting point for quality improvement. However, currently, there are no published quality indicators for Palliative Day Services. AIM: To develop and provide the first set of quality indicators that describe and evaluate the quality of Palliative Day Services. DESIGN AND SETTING: A modified Delphi technique was used to combine best available research evidence derived from a systematic scoping review with multi-disciplinary expert appraisal of the appropriateness and feasibility of candidate indicators. The resulting indicators were compiled into ‘toolkit’, and tested in five UK Palliative Day Service settings. RESULTS: A panel of experts independently reviewed evidence summaries for 182 candidate indicators and provided ratings on appropriateness, followed by a panel discussion and further independent ratings of appropriateness, feasibility, and necessity. This exercise resulted in the identification of 30 indicators which were used in practice testing. The final indicator set comprised 7 structural indicators, 21 process indicators, and 2 outcome indicators. CONCLUSIONS: The indicators fulfil a previously unmet need among Palliative Day Service providers by delivering an appropriate and feasible means to assess, review, and communicate the quality of care, and to identify areas for quality improvement

Impact of RNA degradation on gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis.</p> <p>Methods</p> <p>To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time-dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences.</p> <p>Results</p> <p>The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5' end.</p> <p>Conclusions</p> <p>Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5' end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings.</p

Genomic imbalances in 5918 malignant epithelial tumors: an explorative meta-analysis of chromosomal CGH data

BACKGROUND: Chromosomal abnormalities have been associated with most human malignancies, with gains and losses on some genomic regions associated with particular entities. METHODS: Of the 15429 cases collected for the Progenetix molecular-cytogenetic database, 5918 malignant epithelial neoplasias analyzed by chromosomal Comparative Genomic Hybridization (CGH) were selected for further evaluation. For the 22 clinico-pathological entities with more than 50 cases, summary profiles for genomic imbalances were generated from case specific data and analyzed. RESULTS: With large variation in overall genomic instability, recurring genomic gains and losses were prominent. Most entities showed frequent gains involving 8q2, while gains on 20q, 1q, 3q, 5p, 7q and 17q were frequent in different entities. Loss "hot spots" included 3p, 4q, 13q, 17p and 18q among others. Related average imbalance patterns were found for clinically distinct entities, e.g. hepatocellular carcinomas (ca.) and ductal breast ca., as well as for histologically related entities (squamous cell ca. of different sites). CONCLUSION: Although considerable case-by-case variation of genomic profiles can be found by CGH in epithelial malignancies, a limited set of variously combined chromosomal imbalances may be typical for carcinogenesis. Focus on the respective regions should aid in target gene detection and pathway deduction

Cell cycle and aging, morphogenesis, and response to stimuli genes are individualized biomarkers of glioblastoma progression and survival

<p>Abstract</p> <p>Background</p> <p>Glioblastoma is a complex multifactorial disorder that has swift and devastating consequences. Few genes have been consistently identified as prognostic biomarkers of glioblastoma survival. The goal of this study was to identify general and clinical-dependent biomarker genes and biological processes of three complementary events: lifetime, overall and progression-free glioblastoma survival.</p> <p>Methods</p> <p>A novel analytical strategy was developed to identify general associations between the biomarkers and glioblastoma, and associations that depend on cohort groups, such as race, gender, and therapy. Gene network inference, cross-validation and functional analyses further supported the identified biomarkers.</p> <p>Results</p> <p>A total of 61, 47 and 60 gene expression profiles were significantly associated with lifetime, overall, and progression-free survival, respectively. The vast majority of these genes have been previously reported to be associated with glioblastoma (35, 24, and 35 genes, respectively) or with other cancers (10, 19, and 15 genes, respectively) and the rest (16, 4, and 10 genes, respectively) are novel associations. <it>Pik3r1</it>, <it>E2f3, Akr1c3</it>, <it>Csf1</it>, <it>Jag2</it>, <it>Plcg1</it>, <it>Rpl37a</it>, <it>Sod2</it>, <it>Topors</it>, <it>Hras</it>, <it>Mdm2, Camk2g</it>, <it>Fstl1</it>, <it>Il13ra1</it>, <it>Mtap </it>and <it>Tp53 </it>were associated with multiple survival events.</p> <p>Most genes (from 90 to 96%) were associated with survival in a general or cohort-independent manner and thus the same trend is observed across all clinical levels studied. The most extreme associations between profiles and survival were observed for <it>Syne1</it>, <it>Pdcd4</it>, <it>Ighg1</it>, <it>Tgfa</it>, <it>Pla2g7</it>, and <it>Paics</it>. Several genes were found to have a cohort-dependent association with survival and these associations are the basis for individualized prognostic and gene-based therapies. <it>C2</it>, <it>Egfr</it>, <it>Prkcb</it>, <it>Igf2bp3</it>, and <it>Gdf10 </it>had gender-dependent associations; <it>Sox10</it>, <it>Rps20</it>, <it>Rab31</it>, and <it>Vav3 </it>had race-dependent associations; <it>Chi3l1</it>, <it>Prkcb</it>, <it>Polr2d</it>, and <it>Apool </it>had therapy-dependent associations. Biological processes associated glioblastoma survival included morphogenesis, cell cycle, aging, response to stimuli, and programmed cell death.</p> <p>Conclusions</p> <p>Known biomarkers of glioblastoma survival were confirmed, and new general and clinical-dependent gene profiles were uncovered. The comparison of biomarkers across glioblastoma phases and functional analyses offered insights into the role of genes. These findings support the development of more accurate and personalized prognostic tools and gene-based therapies that improve the survival and quality of life of individuals afflicted by glioblastoma multiforme.</p

Social norms information for alcohol misuse in university and college students.

To determine whether social norms interventions reduce alcohol-related negative consequences, alcohol misuse or alcohol consumption when compared with a control (ranging from assessment only/no intervention to other educational or psychosocial interventions) among university and college students. Search methods The following electronic databases were searched up to May 2014: the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, PsycINFO and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) (only to March 2008). Reference lists of included studies and review articles were manually searched. Selection criteria Randomised controlled trials or cluster-randomised controlled trials that compared a social normative intervention versus no intervention, alcohol education leaflet or other 'non-normative feedback' alcohol intervention and reported on alcohol consumption or alcohol-related problems in university or college students. Data collection and analysis We used standard methodological procedures as expected by The Cochrane Collaboration. Each outcome was analysed by mode of delivery: mailed normative feedback (MF); Web/computer normative feedback (WF); individual face-to-face normative feedback (IFF); group face-to-face normative feedback (GFF); and normative marketing campaign (MC). Main results A total of 66 studies (43,125 participants) were included in the review, and 59 studies (40,951 participants) in the meta-analyses. Outcomes at 4+ months post intervention were of particular interest to assess when effects were sustained beyond the immediate short term. We have reported pooled effects across delivery modes only for those analyses for which heterogeneity across delivery modes is not substantial (I2 < 50%). Alcohol-related problems at 4+ months: IFF standardised mean difference (SMD) -0.16, 95% confidence interval (CI) -0.31 to -0.01 (participants = 1065; studies = 7; moderate quality of evidence), equivalent to a decrease of 1.5 points in the 69-point alcohol problems scale score. No effects were found for WF or MF. Binge drinking at 4+ months: results pooled across delivery modes: SMD -0.06, 95% CI -0.11 to -0.02 (participants = 11,292; studies = 16; moderate quality of evidence), equivalent to 2.7% fewer binge drinkers if 30-day prevalence is 43.9%. Drinking quantity at 4+ months: results pooled across delivery modes: SMD -0.08, 95% CI -0.12 to -0.05 (participants = 20,696; studies = 33; moderate quality of evidence), equivalent to a reduction of 0.9 drinks consumed each week, from a baseline of 13.7 drinks per week. Drinking frequency at 4+ months: WF SMD -0.12, 95% CI -0.18 to -0.05 (participants = 9456; studies = 9; moderate quality of evidence), equivalent to a decrease of 0.19 drinking days/wk, from a baseline of 2.74 days/wk; IFF SMD -0.21, 95% CI -0.31 to -0.10 (participants = 1464; studies = 8; moderate quality of evidence), equivalent to a decrease of 0.32 drinking days/wk, from a baseline of 2.74 days/wk. No effects were found for GFF or MC. Estimated blood alcohol concentration (BAC) at 4+ months: peak BAC results pooled across delivery modes: SMD -0.08, 95% CI -0.17 to 0.00 (participants = 7198; studies = 13; low quality of evidence), equivalent to a reduction in peak PAC from an average of 0.144% to 0.135%. No effects were found for typical BAC with IFF. Authors' conclusions The results of this review indicate that no substantive meaningful benefits are associated with social norms interventions for prevention of alcohol misuse among college/university students. Although some significant effects were found, we interpret the effect sizes as too small, given the measurement scales used in the studies included in this review, to be of relevance for policy or practice. Moreover, the statistically significant effects are not consistent for all misuse measures, heterogeneity was a problem in some analyses and bias cannot be discounted as a potential cause of these findings