Notch signaling regulates metabolic heterogeneity in glioblastoma stem cells.

Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments

Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3

Background: Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described. Results: Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates. Conclusion: We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution

Geographic variation and factors associated with female genital mutilation among reproductive age women in Ethiopia: A national population based survey

Background: Female genital mutilation (FGM) is a common traditional practice in developing nations including Ethiopia. It poses complex and serious long-term health risks for women and girls and can lead to death. In Ethiopia, the geographic distribution and factors associated with FGM practices are poorly understood. Therefore, we assessed the spatial distribution and factors associated with FGM among reproductive age women in the country. Method: We used population based national representative surveys. Data from two (2000 and 2005) Ethiopian demographic and health surveys (EDHS) were used in this analysis. Briefly, EDHS used a stratified, two-stage cluster sampling design. A total of 15,367 (from EDHS 2000) and 14,070 (from EDHS 2005) women of reproductive age (15-49 years) were included in the analysis. Three outcome variables were used (prevalence of FGM among women, prevalence of FGM among daughters and support for the continuation of FGM). The data were weighted and descriptive statistics (percentage change), bivariate and multivariable logistic regression analyses were carried out. Multicollinearity of variables was assessed using variance inflation factors (VIF) with a reference value of 10 before interpreting the final output. The geographic variation and clustering of weighted FGM prevalence were analyzed and visualized on maps using ArcGIS. Z-scores were used to assess the statistical difference of geographic clustering of FGM prevalence spots. Result: The trend of FGM weighted prevalence has been decreasing. Being wealthy, Muslim and in higher age categories are associated with increased odds of FGM among women. Similarly, daughters from Muslim women have increased odds of experiencing FGM. Women in the higher age categories have increased odds of having daughters who experience FGM. The odds of FGM among daughters decrease with increased maternal education. Mass media exposure, being wealthy and higher paternal and maternal education are associated with decreased odds of women's support of FGM continuation. FGM prevalence and geographic clustering showed variation across regions in Ethiopia. Conclusion: Individual, economic, socio-demographic, religious and cultural factors played major roles in the existing practice and continuation of FGM. The significant geographic clustering of FGM was observed across regions in Ethiopia. Therefore, targeted and integrated interventions involving religious leaders in high FGM prevalence spot clusters and addressing the socio-economic and geographic inequalities are recommended to eliminate FGM. © 2016 Setegn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.We thank T. Jacks (Kras^LSL-G12D), A. Berns (p53^Fx) and the NIH Mouse repository for mice. We also thank Sam Kleeman and Patricia Ogger for assistance with redox cell profiling and cell viability assays, respectively. We are very thankful to CRUK CI BRU staff for support with in vivo work and all the members of the Martins lab for critical comments and advice. This work was supported by the Medical Research Council.This is the author accepted manuscript. The final version is available at http://www.nature.com/nature/journal/v531/n7592/full/nature16967.html

Global regulation of alternative splicing during myogenic differentiation

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.National Institutes of Health (U.S.) (grant number R01GM076493)Ford Foundation (Predoctoral Diversity Fellowship)Baylor College of Medicine. Graduate School of Biomedical Sciences (Baylor Research Advocates for Student Scientists

Radiation therapy improves survival in rectal small cell cancer - Analysis of Surveillance Epidemiology and End Results (SEER) data

BACKGROUND: Small cell carcinoma of the rectum is a rare neoplasm with scant literature to guide treatment. We used the Surveillance Epidemiology and End Results (SEER) database to investigate the role of radiation therapy in the treatment of this cancer. METHODS: The SEER database (National Cancer Institute) was queried for locoregional cases of small cell rectal cancer. Years of diagnosis were limited to 1988–2010 (most recent available) to reduce variability in staging criteria or longitudinal changes in surgery and radiation techniques. Two month conditional survival was applied to minimize bias by excluding patients who did not survive long enough to receive cancer-directed therapy. Patient demographics between the RT and No_RT groups were compared using Pearson Chi-Square tests. Overall survival was compared between patients who received radiotherapy (RT, n = 43) and those who did not (No_RT, n = 28) using the Kaplan-Meier method. Multivariate Cox proportional hazards model was used to evaluate important covariates. RESULTS: Median survival was significantly longer for patients who received radiation compared to those who were not treated with radiation; 26 mo vs. 8 mo, respectively (log-rank P = 0.009). We also noted a higher 1-year overall survival rate for those who received radiation (71.1% vs. 37.8%). Unadjusted hazard ratio for death (HR) was 0.495 with the use of radiation (95% CI 0.286-0.858). Among surgery, radiotherapy, sex and age at diagnosis, radiation therapy was the only significant factor for overall survival with a multivariate HR for death of 0.393 (95% CI 0.206-0.750, P = 0.005). CONCLUSIONS: Using SEER data, we have identified a significant survival advantage with the use of radiation therapy in the setting of rectal small cell carcinoma. Limitations of the SEER data apply to this study, particularly the lack of information on chemotherapy usage. Our findings strongly support the use of radiation therapy for patients with locoregional small cell rectal cancer

Extraction, integration and analysis of alternative splicing and protein structure distributed information

<p>Abstract</p> <p>Background</p> <p>Alternative splicing has been demonstrated to affect most of human genes; different isoforms from the same gene encode for proteins which differ for a limited number of residues, thus yielding similar structures. This suggests possible correlations between alternative splicing and protein structure. In order to support the investigation of such relationships, we have developed the Alternative Splicing and Protein Structure Scrutinizer (PASS), a Web application to automatically extract, integrate and analyze human alternative splicing and protein structure data sparsely available in the Alternative Splicing Database, Ensembl databank and Protein Data Bank. Primary data from these databases have been integrated and analyzed using the Protein Identifier Cross-Reference, BLAST, CLUSTALW and FeatureMap3D software tools.</p> <p>Results</p> <p>A database has been developed to store the considered primary data and the results from their analysis; a system of Perl scripts has been implemented to automatically create and update the database and analyze the integrated data; a Web interface has been implemented to make the analyses easily accessible; a database has been created to manage user accesses to the PASS Web application and store user's data and searches.</p> <p>Conclusion</p> <p>PASS automatically integrates data from the Alternative Splicing Database with protein structure data from the Protein Data Bank. Additionally, it comprehensively analyzes the integrated data with publicly available well-known bioinformatics tools in order to generate structural information of isoform pairs. Further analysis of such valuable information might reveal interesting relationships between alternative splicing and protein structure differences, which may be significantly associated with different functions.</p

A study of alternative splicing in the pig

<p>Abstract</p> <p>Background</p> <p>Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence.</p> <p>Results</p> <p>The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR). Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific.</p> <p>Conclusions</p> <p>In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue-specificity and experimentally validated splice events in different porcine tissue. This study indicates that a cluster size of around 50 ESTs is optimal for <it>in silico </it>detection of alternative splicing. Although based on a limited number of splice events, the study supports the notion that alternative splicing could have an important impact on species differentiation since 31% of the splice events studied appears to be species specific.</p

Lineage-coupled clonal capture identifies clonal evolution mechanisms and vulnerabilities of BRAF

Targeted cancer therapies have revolutionized treatment but their efficacies are limited by the development of resistance driven by clonal evolution within tumors. We developed CAPTURE , a single-cell barcoding approach to comprehensively trace clonal dynamics and capture live lineage-coupled resistant cells for in-depth multi-omics analysis and functional exploration. We demonstrate that heterogeneous clones, either preexisting or emerging from drug-tolerant persister cells, dominated resistance to vemurafenib in BRA

Identification and Functional Analyses of 11 769 Full-length Human cDNAs Focused on Alternative Splicing

We analyzed diversity of mRNA produced as a result of alternative splicing in order to evaluate gene function. First, we predicted the number of human genes transcribed into protein-coding mRNAs by using the sequence information of full-length cDNAs and 5′-ESTs and obtained 23 241 of such human genes. Next, using these genes, we analyzed the mRNA diversity and consequently sequenced and identified 11 769 human full-length cDNAs whose predicted open reading frames were different from other known full-length cDNAs. Especially, 30% of the cDNAs we identified contained variation in the transcription start site (TSS). Our analysis, which particularly focused on multiple variable first exons (FEVs) formed due to the alternative utilization of TSSs, led to the identification of 261 FEVs expressed in the tissue-specific manner. Quantification of the expression profiles of 13 genes by real-time PCR analysis further confirmed the tissue-specific expression of FEVs, e.g. OXR1 had specific TSS in brain and tumor tissues, and so on. Finally, based on the results of our mRNA diversity analysis, we have created the FLJ Human cDNA Database. From our result, it has been understood mechanisms that one gene produces suitable protein-coding transcripts responding to the situation and the environment