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Dengue virus (DENV) is a re-emerging arthropod borne flavivirus that infects more than 300 million people worldwide, leading to 50,000 deaths annually. Because dendritic cells (DC) in the skin and blood are the first target cells for DENV, we sought to investigate the early molecular events involved in the host response to the virus in primary human monocyte-derived dendritic cells (Mo-DC). Using a genome-wide transcriptome analysis of DENV2-infected human Mo-DC, three major responses were identified within hours of infection - the activation of IRF3/7/STAT1 and NF-κB-driven antiviral and inflammatory networks, as well as the stimulation of an oxidative stress response that included the stimulation of an Nrf2-dependent antioxidant gene transcriptional program. DENV2 infection resulted in the intracellular accumulation of reactive oxygen species (ROS) that was dependent on NADPH-oxidase (NOX). A decrease in ROS levels through chemical or genetic inhibition of the NOX-complex dampened the innate immune responses to DENV infection and facilitated DENV replication; ROS were also essential in driving mitochondrial apoptosis in infected Mo-DC. In addition to stimulating innate immune responses to DENV, increased ROS led to the activation of bystander Mo-DC which up-regulated maturation/activation markers and were less susceptible to viral replication. We have identified a critical role for the transcription factor Nrf2 in limiting both antiviral and cell death responses to the virus by feedback modulation of oxidative stress. Silencing of Nrf2 by RNA interference increased DENV-associated immune and apoptotic responses. Taken together, these data demonstrate that the level of oxidative stress is critical to the control of both antiviral and apoptotic programs in DENV-infected human Mo-DC and highlight the importance of redox homeostasis in the outcome of DENV infection.

Background Social-environmental data obtained from the US Census is an important resource for understanding health disparities, but rarely is the full dataset utilized for analysis. A barrier to incorporating the full data is a lack of solid recommendations for variable selection, with researchers often hand-selecting a few variables. Thus, we evaluated the ability of empirical machine learning approaches to identify social-environmental factors having a true association with a health outcome. Methods We compared several popular machine learning methods, including penalized regressions (e.g. lasso, elastic net), and tree ensemble methods. Via simulation, we assessed the methods’ ability to identify census variables truly associated with binary and continuous outcomes while minimizing false positive results (10 true associations, 1000 total variables). We applied the most promising method to the full census data ( p  = 14,663 variables) linked to prostate cancer registry data ( n  = 76,186 cases) to identify social-environmental factors associated with advanced prostate cancer. Results In simulations, we found that elastic net identified many true-positive variables, while lasso provided good control of false positives. Using a combined measure of accuracy, hierarchical clustering based on Spearman’s correlation with sparse group lasso regression performed the best overall. Bayesian Adaptive Regression Trees outperformed other tree ensemble methods, but not the sparse group lasso. In the full dataset, the sparse group lasso successfully identified a subset of variables, three of which replicated earlier findings. Conclusions This analysis demonstrated the potential of empirical machine learning approaches to identify a small subset of census variables having a true association with the outcome, and that replicate across empiric methods. Sparse clustered regression models performed best, as they identified many true positive variables while controlling false positive discoveries.

Squamous cell carcinomas of the head and neck (SCCHN) affect anatomical sites including the oral cavity, nasal cavity, pharynx, and larynx. Laryngeal cancers are characterized by high recurrence and poor overall survival, and currently lack robust molecular prognostic biomarkers for treatment stratification. Using an algorithm for integrative clustering that simultaneously assesses gene expression, somatic mutation, copy number variation, and methylation, we for the first time identify laryngeal cancer subtypes with distinct prognostic outcomes, and differing from the non-prognostic laryngeal subclasses reported by The Cancer Genome Atlas (TCGA). Although most common laryngeal gene mutations are found in both subclasses, better prognosis is strongly associated with damaging mutations of the methyltransferases NSD1 and NSD2 , with findings confirmed in an independent validation cohort consisting of 63 laryngeal cancer patients. Intriguingly, NSD1/2 mutations are not prognostic for nonlaryngeal SCCHN. These results provide an immediately useful clinical metric for patient stratification and prognostication. The authors use an integrative clustering approach to identify two laryngeal cancer clusters with distinct prognosis and show that mutations damaging the NSD1 and NSD2 methyltransferases segregate to the cluster with favorable prognosis, and independently predict longer survival in patients with laryngeal, but not other head and neck cancers.

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17βestradiol (E2) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E2 paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E2-induced apoptosis by analysis of gene expression across time (2—96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E2-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E2 in 5C cells compared with both WS8 and 2A cells and hence, were associated with E2-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E2 inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E2-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E2-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E2 interacted to superadditively induce apoptosis. Therefore, these data indicate that E2 induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

The kinase RIPK3 is a key regulator of cell death responses to a growing number of viral and microbial agents. We have found that influenza A virus (IAV)-mediated cell death is largely reliant on RIPK3 and that RIPK3-deficient mice are notably more susceptible to lethal infection by IAV than their wild-type counterparts. Recent studies demonstrate that RIPK3 also participates in regulating gene transcription programs during host pro-inflammatory and innate-immune responses, indicating that this kinase is not solely an inducer of cell death and that RIPK3-driven transcriptional responses may collaborate with cell death in promoting clearance of IAV. Here, we carried out DNA microarray analyses to determine the contribution of RIPK3 to the IAV-elicited host transcriptional response. We report that RIPK3 does not contribute significantly to the RLR-activated transcriptome or to the induction of type I IFN genes, although, interestingly, IFN-β production at a post-transcriptional step was modestly attenuated in IAV-infected ripk3-/- fibroblasts. Overall, RIPK3 regulated the expression of <5% of the IAV-induced transcriptome, and no genes were found to be obligate RIPK3 targets. IFN-β signaling was also found to be largely normal in the absence of RIPK3. Together, these results indicate that RIPK3 is not essential for the host antiviral transcriptional response to IAV in murine fibroblasts.

The liver has remarkable regenerative capacity owing to the boundless proliferative potential of hepatocytes. During liver injury, sustained regeneration must be balanced by mechanisms limiting overgrowth and tumorigenesis. Epithelial plasticity is frequently observed during liver damage and is thought to mediate production of biliary epithelial cells (BECs) or hepatocytes, depending on tissue needs. Here we show that hepatocytes persisting in plastic states are present in virtually all liver injury contexts, representing the predominant outcome of hepatocyte reprogramming rather than their full BEC conversion. By developing tools to trap mouse hepatocytes in plastic states in vivo and using models of regeneration and transplantation, we show that plastic hepatocytes are refractory to proliferation cues from the microenvironment. Unlike terminally differentiated hepatocytes, plastic hepatocytes resist proliferation driven by endogenous oncogenic stimuli. Thus, acquisition of plastic states represents a protective mechanism that constrains hepatocyte proliferation, limiting overgrowth and tumorigenesis during liver disease. The balance between cell proliferation and cell cycle arrest is essential for liver regeneration. Here the authors report the emergence of partially reprogrammed hepatocytes persisting in plastic states during liver tissue injury, which are resistant to proliferation thereby limiting overgrowth and tumorigenesis.

The majority of renal cell carcinoma (RCC) is now incidentally detected and presents as small renal masses (SRMs) defined as ≤ 4 cm in size. SRMs are heterogeneous comprising several histological types of RCC each with different biology and behavior, and benign tumors mainly oncocytoma. The varied prognosis of the different types of renal tumor has implications for management options. A key epigenetic alteration involved in the initiation and progression of cancer is aberrant methylation in the promoter region of a gene. The hypermethylation is associated with transcriptional repression and is an important mechanism of inactivation of tumor suppressor genes in neoplastic cells. We have determined the genome-wide promoter methylation profiles of 47 pT1a and 2 pT1b clear cell, papillary or chromophobe RCC, 25 benign renal oncocytoma ≤ 4 cm and 4 normal renal parenchyma specimens by Infinium HumanMethylation27 beadchip technology. We identify gene promoter hypermethylation signatures that distinguish clear cell and papillary from each other, from chromophobe and oncocytoma, and from normal renal cells. Pairwise comparisons revealed genes aberrantly hypermethylated in a tumor type but unmethylated in normal, and often unmethylated in the other renal tumor types. About 0.4% to 1.7% of genes comprised the promoter methylome in SRMs. The Infinium methylation score for representative genes was verified by gold standard technologies. The genes identified as differentially methylated implicate pathways involved in metabolism, tissue response to injury, epithelial to mesenchymal transition (EMT), signal transduction and G-protein coupled receptors (GPCRs), cancer, and stem cell regulation in the biology of RCC. Our findings contribute towards an improved understanding of the development of RCC, the different biology and behavior of histological types, and discovery of molecular subtypes. The differential methylation signatures may have utility in early detection and particularly differential diagnosis for prognostic stratification as well as identify novel gene and pathway targets for therapeutic intervention.

Background Full-term pregnancy (FTP) at an early age confers long-term protection against breast cancer. Previously, we reported that a FTP imprints a specific gene expression profile in the breast of postmenopausal women. Herein, we evaluated gene expression changes induced by parity in the breast of premenopausal women. Methods Gene expression profiling of normal breast tissue from 30 nulliparous (NP) and 79 parous (P) premenopausal volunteers was performed using Affymetrix microarrays. In addition to a discovery/validation analysis, we conducted an analysis of gene expression differences in P vs. NP women as a function of time since last FTP. Finally, a laser capture microdissection substudy was performed to compare the gene expression profile in the whole breast biopsy with that in the epithelial and stromal tissues. Results Discovery/validation analysis identified 43 differentially expressed genes in P vs. NP breast. Analysis of expression as a function of time since FTP revealed 286 differentially expressed genes (238 up- and 48 downregulated) comparing all P vs. all NP, and/or P women whose last FTP was less than 5 years before biopsy vs. all NP women. The upregulated genes showed three expression patterns: (1) transient: genes upregulated after FTP but whose expression levels returned to NP levels. These genes were mainly related to immune response, specifically activation of T cells. (2) Long-term changing: genes upregulated following FTP, whose expression levels decreased with increasing time since FTP but did not return to NP levels. These were related to immune response and development. (3) Long-term constant: genes that remained upregulated in parous compared to nulliparous breast, independently of time since FTP. These were mainly involved in development/cell differentiation processes, and also chromatin remodeling. Lastly, we found that the gene expression in whole tissue was a weighted average of the expression in epithelial and stromal tissues. Conclusions Genes transiently activated by FTP may have a role in protecting the mammary gland against neoplastically transformed cells through activation of T cells. Furthermore, chromatin remodeling and cell differentiation, represented by the genes that are maintained upregulated long after the FTP, may be responsible for the lasting preventive effect against breast cancer.

Online surveys are a valuable tool for social science research, but the perceived anonymity provided by online administration may lead to problematic behaviors from study participants. Particularly, if a study offers incentives, some participants may attempt to enroll multiple times. We propose a method to identify clusters of non-independent enrollments in a web-based study, motivated by an analysis of survey data which tests the effectiveness of an online skin-cancer risk reduction program. To identify groups of enrollments, we used a hierarchical clustering algorithm based on the Euclidean distance matrix formed by participant responses to a series of Likert-type eligibility questions. We then systematically identified clusters that are unusual in terms of both size and similarity, by repeatedly simulating datasets from the empirical distribution of responses under the assumption of independent enrollments. By performing the clustering algorithm on the simulated datasets, we determined the distribution of cluster size and similarity under independence, which is then used to identify groups of outliers in the observed data. Next, we assessed 12 other quality indicators, including previously proposed and study-specific measures. We summarized the quality measures by cluster membership, and compared the cluster groupings to those found when using the quality indicators with latent class modeling. When we excluded the clustered enrollments and/or lower-quality latent classes from the analysis of study outcomes, the estimates of the intervention effect were larger. This demonstrates how including repeat or low quality participants can introduce bias into a web-based study. As much as is possible, web-based surveys should be designed to verify participant quality. Our method can be used to verify survey quality and identify problematic groups of enrollments when necessary.

The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (Mtap(lacZ)) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten(+/-) . Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of Mtap(lacZ/+) and Mtap (+/+) mice. Survival in both models was significantly decreased in Mtap(lacZ/+) compared to Mtap(+/+) mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap(+/+) mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc Mtap(lacZ/+) and Eµ-myc Mtap(+/+) animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap(+/+) and Mtap(lacZ/+) animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer. Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.