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Background: Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures., Results: For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed., Conclusion: Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.

ABSTRACT: The difference in the adult height of mammals, and hence in endochondral bone formation, is not yet fully understood and may serve to identify targets for bone and cartilage regeneration. In line with this hypothesis, the intra‐species disparity between the adult height of Great Danes and Miniature Poodles was investigated at a transcriptional level. Microarray analysis of the growth plate of five Great Danes and five Miniature Poodles revealed 2,981 unique genes that were differentially expressed, including many genes with an unknown role in skeletal development. A signaling pathway impact analysis indicated activation of the cell cycle, extracellular matrix receptor interaction and the tight junction pathway, and inhibition of pathways associated with inflammation and the complement cascade. In additional validation steps, the gene expression profile of the separate growth plate zones for both dog breeds were determined. Given that the BMP signaling is known for its crucial role in skeletal development and fracture healing, and BMP‐2 is used in orthopaedic and spine procedures for bone augmentation, further investigations concentrated on the BMP pathway.The canonical BMP‐2 and BMP‐6 signaling pathway was activated in the Great Danes compared to Miniature Poodles. In conclusion, investigating the differential expression of genes involved in endochondral bone formation in small and large breed dogs, could be a game changing strategy to provide new insights in growth plate development and identify new targets for bone and cartilage regeneration. © 2017 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:138–148, 2018. [ABSTRACT FROM AUTHOR]

Background: Genetic interactions, or non-additive effects between genes, play a crucial role in many cellular processes and disease. Which mechanisms underlie these genetic interactions has hardly been characterized. Understanding the molecular basis of genetic interactions is crucial in deciphering pathway organization and understanding the relationship between genotype, phenotype and disease. Results: To investigate the nature of genetic interactions between gene-specific transcription factors (GSTFs) in Saccharomyces cerevisiae, we systematically analyzed 72 GSTF pairs by gene expression profiling double and single deletion mutants. These pairs were selected through previously published growth-based genetic interactions as well as through similarity in DNA binding properties. The result is a high-resolution atlas of gene expression-based genetic interactions that provides systems-level insight into GSTF epistasis. The atlas confirms known genetic interactions and exposes new ones. Importantly, the data can be used to investigate mechanisms that underlie individual genetic interactions. Two molecular mechanisms are proposed, "buffering by induced dependency" and "alleviation by derepression". Conclusions: These mechanisms indicate how negative genetic interactions can occur between seemingly unrelated parallel pathways and how positive genetic interactions can indirectly expose parallel rather than same-pathway relationships. The focus on GSTFs is important for understanding the transcription regulatory network of yeast as it uncovers details behind many redundancy relationships, some of which are completely new. In addition, the study provides general insight into the complex nature of epistasis and proposes mechanistic models for genetic interactions, the majority of which do not fall into easily recognizable within- or between-pathway relationships. [ABSTRACT FROM AUTHOR]

Background: The determination of altered expression of genes in specific tumor types and their effect upon cellular processes may create insight in tumorigenesis and help to design better treatments. The Flatcoated retriever is a dog breed with an exceptionally high incidence of histiocytic sarcomas. The breed develops two distinct entities of histiocytic neoplasia, a soft tissue form and a visceral form. Gene expression studies of these tumors have value for comparable human diseases such as histiocytic/dendritic cell sarcoma for which knowledge is difficult to accrue due to their rare occurrence. In addition, such studies may help in the search for genetic aberrations underlying the genetic predisposition in this dog breed. Methods: Microarray analysis and pathway analyses were performed on fresh-frozen tissues obtained from Flatcoated retrievers with localized, soft tissue histiocytic sarcomas (STHS) and disseminated, visceral histiocytic sarcomas (VHS) and on normal canine spleens from various breeds. Expression differences of nine genes were validated with quantitative real-time PCR (qPCR) analyses. Results: QPCR analyses identified the significantly altered expression of nine genes; PPBP, SpiC, VCAM1, ENPEP, ITGAD (down-regulated), and GTSF1, Col3a1, CD90 and LUM (up-regulated) in the comparison of both the soft tissue and the visceral form with healthy spleen. DAVID pathway analyses revealed 24 pathways that were significantly involved in the development of HS in general, most of which were involved in the DNA repair and replication process. Conclusions: This study identified altered expression of nine genes not yet implicated in histiocytic sarcoma manifestations in the dog nor in comparable human histiocytic/dendritic sarcomas. Exploration of the downside effect of canine inbreeding strategies for the study of similar sarcomas in humans might also lead to the identification of genes related to these rare malignancies in the human. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs). Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5Δ and slx8Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein (AIP) and aryl hydrocarbon receptor nuclear translocator (ARNT). The resulting intrahepatic portosystemic shunts (IHPSS) are frequently diagnosed in specific dog breeds, such as the Irish wolfhound. We compared the expression of components of the AHR pathway in healthy Irish wolfhounds and dogs with IHPSS. To this end, we analyzed the mRNA expression in the liver of AHR,AIP, ARNT, and other genes involved in this pathway, namely, those for aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), hypoxia inducible factor 1alpha (HIF1A), heat shock protein 90AA1 (HSP90AA1), cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1), vascular endothelial growth factor A (VEGFA), nitric oxide synthesase 3 (NOS3), and endothelin (EDN1). The observed low expression of AHR mRNA in the Irish wolfhounds is in associated with a LINE-1 insertion in intron 2, for which these dogs were homozygous. Down regulation in Irish wolfhounds was observed for AIP, ARNT2, CYP1A2, CYP1B1 and HSP90AA1 expression, whereas the expression of HIF1A was increased. Immunohistochemistry revealed lower levels of AHR, HIF1A, and VEGFA protein in the nucleus and lower levels of ARNT and HSP90AA1 protein in the cytoplasm of the liver cells of Irish wolfhounds. The impaired expression of HSP90AA1 could trigger the observed differences in mRNA and protein levels and therefore explain the link between two very different functions of AHR: regulation of the closure of the ductus venosus and the response to toxins. [ABSTRACT FROM AUTHOR]

Congenital portosystemic shunts are developmental anomalies of the splanchnic vascular system that cause portal blood to bypass the liver. Large-breed dogs are predisposed for intrahepatic portosystemic shunts (IHPSS) and small-breed dogs for extrahepatic portosystemic shunts (EHPSS). While the phenotype resulting from portal bypass of the liver of the two types of shunt is identical, the genotype and molecular pathways involved are probably different. The aim of this study was to gain insight into the pathways involved in the different types of portosystemic shunting. Microarray analysis of mRNA expression in liver tissue from dogs with EHPSS and IHPSS revealed that the expression of 26 genes was altered in either IHPSS or EHPSS samples compared with that in liver samples from control dogs. Quantitative real-time PCR of these genes in 14 IHPSS, 17 EHPSS, and 8 control liver samples revealed a significant differential expression of ACBP, CCBL1, GPC3, HAMP, PALLD, VCAM1, and WEE1. Immunohistochemistry and Western blotting confirmed an increased expression of VCAM1 in IHPSS but its absence in EHPSS, an increased WEE1 expression in IHPSS but not in EHPSS, and a decreased expression of CCBL1 in both shunt types. Regarding their physiologic functions, these findings may indicate a causative role for VCAM1 in IHPSS and WEE1 for IHPSS. CCBL1 could be an interesting candidate to study not yet elucidated aspects in the pathophysiology of hepatic encephalopathy. [ABSTRACT FROM AUTHOR]

Abstract: Newly hatched chickens are more susceptible to infectious diseases than older birds because of an immature immune system. The aim of this study was to determine to what extent host responses to avian influenza virus (AIV) inoculation are affected by age. Therefore, 1- and 4-week (wk) old birds were inoculated with H9N2 AIV or saline. The trachea and lung were sampled at 0, 8, 16 and 24h post-inoculation (h.p.i.) and gene expression profiles determined using microarray analysis. Firstly, saline controls of both groups were compared to analyse the changes in gene profiles related to development. In 1-wk-old birds, higher expression of genes related to development of the respiratory immune system and innate responses were found, whereas in 4-wk-old birds genes were up regulated that relate to the presence of higher numbers of leukocytes in the respiratory tract. After inoculation with H9N2, gene expression was most affected at 16h.p.i. in 1-wk-old birds and at 16 and 24h.p.i. in 4-wk-old birds in the trachea and especially in the lung. In 1-wk-old birds less immune related genes including innate related genes were induced which might be due to age-dependent reduced functionality of antigen presenting cells (APC), T cells and NK cells. In contrast cytokine and chemokines gene expression was related to viral load in 1-wk-old birds and less in 4-wk-old birds. Expression of cellular host factors that block virus replication by interacting with viral factors was independent of age or tissue for most host factors. These data show that differences in development are reflected in gene expression and suggest that the strength of host responses at transcriptional level may be a key factor in age-dependent susceptibility to infection, and the cellular host factors involved in virus replication are not. [Copyright &y& Elsevier]

We have designed a doxycycline-regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof-of-principle, we have targeted β-catenin in colorectal cancer (CRC) cells,. T-cell factor (TCF) target-gene expression is induced by accumulated β-catenin, and is the main transforming event in these cells. We have shown previously that the disruption of β-catenin/TCF4 activity in CRC cells by the overexpression of dominant-negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of β-catenin. In these CRC cells, TCF reporter-gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genesusing this vector system shows that it should be useful for the inducible knockdown of gene expression. [ABSTRACT FROM AUTHOR]

Expression profiling is a universal tool, with a range of applications that benefit from the accurate determination of differential gene expression. To allow normalization using endogenous transcript levels, current microarray analyses assume that relatively few transcripts vary, or that any changes that occur are balanced. When normalization using endogenous genes is carried out, changes in expression levels are calculated relative to the behaviour of most of the transcripts. This does not reflect absolute changes if global shifts in messenger RNA populations occur. Using external RNA controls, we have set up microarray experiments to monitor global changes. The levels of most mRNAs were found to change during yeast stationary phase and human heat shock when external controls were included. Even small global changes had a significant effect on the number of genes reported as being differentially expressed. This suggests that global mRNA changes occur more frequently than is assumed at present, and shows that monitoring such effects may be important for the accurate determination of changes in gene expression. [ABSTRACT FROM AUTHOR]

DNA microarray technology is a powerful tool for monitoring gene expression or for finding the location of DNA-bound proteins. DNA microarrays can suffer from gene-specific dye bias (GSDB), causing some probes to be affected more by the dye than by the sample. This results in large measurement errors, which vary considerably for different probes and also across different hybridizations. GSDB is not corrected by conventional normalization and has been difficult to address systematically because of its variance. We show that GSDB is influenced by label incorporation efficiency, explaining the variation of GSDB across different hybridizations. A correction method (Gene- And Slide-Specific Correction, GASSCO) is presented, whereby sequence-specific corrections are modulated by the overall bias of individual hybridizations. GASSCO outperforms earlier methods and works well on a variety of publically available datasets covering a range of platforms, organisms and applications, including ChIP on chip. A sequence-based model is also presented, which predicts which probes will suffer most from GSDB, useful for microarray probe design and correction of individual hybridizations. Software implementing the method is publicly available. [ABSTRACT FROM AUTHOR]

Summary: Analyses of biological processes would benefit from accurate definitions of protein complexes. High-throughput mass spectrometry data offer the possibility of systematically defining protein complexes; however, the predicted compositions vary substantially depending on the algorithm applied. We determine consensus compositions for 409 core protein complexes from Saccharomyces cerevisiae by merging previous predictions with a new approach. Various analyses indicate that the consensus is comprehensive and of high quality. For 85 out of 259 complexes not recorded in GO, literature search revealed strong support in the form of coprecipitation. New complexes were verified by an independent interaction assay and by gene expression profiling of strains with deleted subunits, often revealing which cellular processes are affected. The consensus complexes are available in various formats, including a merge with GO, resulting in 518 protein complex compositions. The utility is further demonstrated by comparison with binary interaction data to reveal interactions between core complexes. [Copyright &y& Elsevier]

Summary: Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin. [ABSTRACT FROM AUTHOR]