Multi-step screening of neoantigens' HLA- and TCR-interfaces improves prediction of survival.

Improvement of risk stratification through prognostic biomarkers may enhance the personalization of cancer patient monitoring and treatment. We used Ancer, an immunoinformatic CD8, CD4, and regulatory T cell neoepitope screening system, to perform an advanced neoantigen analysis of genomic data derived from the urothelial cancer cohort of The Cancer Genome Atlas. Ancer demonstrated improved prognostic stratification and five-year survival prediction compared to standard analyses using tumor mutational burden or neoepitope identification using NetMHCpan and NetMHCIIpan. The superiority of Ancer, shown in both univariate and multivariate survival analyses, is attributed to the removal of neoepitopes that do not contribute to tumor immunogenicity based on their homology with self-epitopes. This analysis suggests that the presence of a higher number of unique, non-self CD8- and CD4-neoepitopes contributes to cancer survival, and that prospectively defining these neoepitopes using Ancer is a novel prognostic or predictive biomarker.

GR and ER Coactivation Alters the Expression of Differentiation Genes and Associates with Improved ER+ Breast Cancer Outcome.

UNLABELLED: In estrogen receptor (ER)-negative breast cancer, high tumor glucocorticoid receptor (GR) expression has been associated with a relatively poor outcome. In contrast, using a meta-analysis of several genomic datasets, here we find that tumor GR mRNA expression is associated with improved ER(+) relapse-free survival (RFS; independently of progesterone receptor expression). To understand the mechanism by which GR expression is associated with a better ER(+) breast cancer outcome, the global effect of GR-mediated transcriptional activation in ER(+) breast cancer cells was studied. Analysis of GR chromatin immunoprecipitation followed by high-throughput sequencing in ER(+)/GR(+) MCF-7 cells revealed that upon coactivation of GR and ER, GR chromatin association became enriched at proximal promoter regions. Furthermore, following ER activation, increased GR chromatin association was observed at ER, FOXO, and AP1 response elements. In addition, ER associated with GR response elements, suggesting that ER and GR interact in a complex. Coactivation of GR and ER resulted in increased expression (relative to ER activation alone) of transcripts that encode proteins promoting cellular differentiation (e.g., KDM4B, VDR) and inhibiting the Wnt signaling pathway (IGFBP4). Finally, expression of these individual prodifferentiation genes was associated with significantly improved RFS in ER(+) breast cancer patients. Together, these data suggest that the coexpression and subsequent activity of tumor cell GR and ER contribute to the less aggressive natural history of early-stage breast cancer by coordinating the altered expression of genes favoring differentiation. IMPLICATIONS: The interaction between ER and GR activity highlights the importance of context-dependent nuclear receptor function in cancer. Mol Cancer Res; 14(8); 707-19. ©2016 AACR.

Novel method for analysis of allele specific expression in triploid Oryzias latipes reveals consistent pattern of allele exclusion.

Assessing allele-specific gene expression (ASE) on a large scale continues to be a technically challenging problem. Certain biological phenomena, such as X chromosome inactivation and parental imprinting, affect ASE most drastically by completely shutting down the expression of a whole set of alleles. Other more subtle effects on ASE are likely to be much more complex and dependent on the genetic environment and are perhaps more important to understand since they may be responsible for a significant amount of biological diversity. Tools to assess ASE in a diploid biological system are becoming more reliable. Non-diploid systems are, however, not uncommon. In humans full or partial polyploid states are regularly found in both healthy (meiotic cells, polynucleated cell types) and diseased tissues (trisomies, non-disjunction events, cancerous tissues). In this work we have studied ASE in the medaka fish model system. We have developed a method for determining ASE in polyploid organisms from RNAseq data and we have implemented this method in a software tool set. As a biological model system we have used nuclear transplantation to experimentally produce artificial triploid medaka composed of three different haplomes. We measured ASE in RNA isolated from the livers of two adult, triploid medaka fish that showed a high degree of similarity. The majority of genes examined (82%) shared expression more or less evenly among the three alleles in both triploids. The rest of the genes (18%) displayed a wide range of ASE levels. Interestingly the majority of genes (78%) displayed generally consistent ASE levels in both triploid individuals. A large contingent of these genes had the same allele entirely suppressed in both triploids. When viewed in a chromosomal context, it is revealed that these genes are from large sections of 4 chromosomes and may be indicative of some broad scale suppression of gene expression.

RNA-Seq reveals complex genetic response to Deepwater Horizon oil release in Fundulus grandis.

BACKGROUND: The release of oil resulting from the blowout of the Deepwater Horizon (DH) drilling platform was one of the largest in history discharging more than 189 million gallons of oil and subject to widespread application of oil dispersants. This event impacted a wide range of ecological habitats with a complex mix of pollutants whose biological impact is still not yet fully understood. To better understand the effects on a vertebrate genome, we studied gene expression in the salt marsh minnow Fundulus grandis, which is local to the northern coast of the Gulf of Mexico and is a sister species of the ecotoxicological model Fundulus heteroclitus. To assess genomic changes, we quantified mRNA expression using high throughput sequencing technologies (RNA-Seq) in F. grandis populations in the marshes and estuaries impacted by DH oil release. This application of RNA-Seq to a non-model, wild, and ecologically significant organism is an important evaluation of the technology to quickly assess similar events in the future. RESULTS: Our de novo assembly of RNA-Seq data produced a large set of sequences which included many duplicates and fragments. In many cases several of these could be associated with a common reference sequence using blast to query a reference database. This reduced the set of significant genes to 1,070 down-regulated and 1,251 up-regulated genes. These genes indicate a broad and complex genomic response to DH oil exposure including the expected AHR-mediated response and CYP genes. In addition a response to hypoxic conditions and an immune response are also indicated. Several genes in the choriogenin family were down-regulated in the exposed group; a response that is consistent with AH exposure. These analyses are in agreement with oligonucleotide-based microarray analyses, and describe only a subset of significant genes with aberrant regulation in the exposed set. CONCLUSION: RNA-Seq may be successfully applied to feral and extremely polymorphic organisms that do not have an underlying genome sequence assembly to address timely environmental problems. Additionally, the observed changes in a large set of transcript expression levels are indicative of a complex response to the varied petroleum components to which the fish were exposed.

Genomic and physiological footprint of the Deepwater Horizon oil spill on resident marsh fishes.

The biological consequences of the Deepwater Horizon oil spill are unknown, especially for resident organisms. Here, we report results from a field study tracking the effects of contaminating oil across space and time in resident killifish during the first 4 mo of the spill event. Remote sensing and analytical chemistry identified exposures, which were linked to effects in fish characterized by genome expression and associated gill immunohistochemistry, despite very low concentrations of hydrocarbons remaining in water and tissues. Divergence in genome expression coincides with contaminating oil and is consistent with genome responses that are predictive of exposure to hydrocarbon-like chemicals and indicative of physiological and reproductive impairment. Oil-contaminated waters are also associated with aberrant protein expression in gill tissues of larval and adult fish. These data suggest that heavily weathered crude oil from the spill imparts significant biological impacts in sensitive Louisiana marshes, some of which remain for over 2 mo following initial exposures.

Genome-scale phylogenetic analyses of chikungunya virus reveal independent emergences of recent epidemics and various evolutionary rates.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia, however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics in these two transmission cycles.

Mechanical stability and differentially conserved physical-chemical properties of titin Ig-domains.

The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single-molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I-band of a sarcomere is composed of about 40 Ig-domains and is exposed to force under normal physiological conditions and connects the free-hanging ends of the myosin filaments to the Z-disc. Recent single-molecule force spectroscopy data show a mechanical hierarchy in the I-band domains. Domains near the C-terminus in this region unfold at forces two to three times greater than domains near the beginning of the I-band. Though all of these Ig-domains are thought to share a fold and topology common to members of the Ig-like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I-band Ig domain to specific, conserved physical-chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into "strong" and "weak" families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the beta-sandwich fold, and force sensitive residues are not only confined to the A'-G region.