Global analysis of gene expression reveals mRNA superinduction is required for the inducible immune response to a bacterial pathogen.

The inducible innate immune response to infection requires a concerted process of gene expression that is regulated at multiple levels. Most global analyses of the innate immune response have focused on transcription induced by defined immunostimulatory ligands, such as lipopolysaccharide. However, the response to pathogens involves additional complexity, as pathogens interfere with virtually every step of gene expression. How cells respond to pathogen-mediated disruption of gene expression to nevertheless initiate protective responses remains unclear. We previously discovered that a pathogen-mediated blockade of host protein synthesis provokes the production of specific pro-inflammatory cytokines. It remains unclear how these cytokines are produced despite the global pathogen-induced block of translation. We addressed this question by using parallel RNAseq and ribosome profiling to characterize the response of macrophages to infection with the intracellular bacterial pathogen Legionella pneumophila. Our results reveal that mRNA superinduction is required for the inducible immune response to a bacterial pathogen

Quantitative nucleotide level analysis of regulation of translation in response to depolarization of cultured neural cells

Studies on regulation of gene expression have contributed substantially to understanding mechanisms for the long-term activity-dependent alterations in neural connectivity that are thought to mediate learning and memory. Most of these studies, however, have focused on the regulation of mRNA transcription. Here, we utilized high-throughput sequencing coupled with ribosome footprinting to globally characterize the regulation of translation in primary mixed neuronal-glial cultures in response to sustained depolarization. We identified substantial and complex regulation of translation, with many transcripts demonstrating changes in ribosomal occupancy independent of transcriptional changes. We also examined sequence-based mechanisms that might regulate changes in translation in response to depolarization. We found that these are partially mediated by features in the mRNA sequence—notably upstream open reading frames and secondary structure in the 5′ untranslated region—both of which predict downregulation in response to depolarization. Translationally regulated transcripts are also more likely to be targets of FMRP and include genes implicated in autism in humans. Our findings support the idea that control of mRNA translation plays an important role in response to neural activity across the genome

Profiling of RNAs from Human Islet-Derived Exosomes in a Model of Type 1 Diabetes

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of insulin-producing islet β cells. Biomarkers capable of identifying T1D risk and dissecting disease-related heterogeneity represent an unmet clinical need. Toward the goal of informing T1D biomarker strategies, we profiled coding and noncoding RNAs in human islet-derived exosomes and identified RNAs that were differentially expressed under proinflammatory cytokine stress conditions. Human pancreatic islets were obtained from cadaveric donors and treated with/without IL-1β and IFN-γ. Total RNA and small RNA sequencing were performed from islet-derived exosomes to identify mRNAs, long noncoding RNAs, and small noncoding RNAs. RNAs with a fold change ≥1.3 and a p-value <0.05 were considered as differentially expressed. mRNAs and miRNAs represented the most abundant long and small RNA species, respectively. Each of the RNA species showed altered expression patterns with cytokine treatment, and differentially expressed RNAs were predicted to be involved in insulin secretion, calcium signaling, necrosis, and apoptosis. Taken together, our data identify RNAs that are dysregulated under cytokine stress in human islet-derived exosomes, providing a comprehensive catalog of protein coding and noncoding RNAs that may serve as potential circulating biomarkers in T1D

Duplications of KIAA1549 and BRAF screening by Droplet Digital PCR from formalin-fixed paraffin-embedded DNA is an accurate alternative for KIAA1549-BRAF fusion detection in pilocytic astrocytomas

Pilocytic astrocytomas represent the most common glioma subtype in young patients and account for 5.4% of all gliomas. They are characterized by alterations in the RAS–MAP kinase pathway, the most frequent being a tandem duplication on chromosome 7q34 involving the BRAF gene, resulting in oncogenic BRAF fusion proteins. BRAF fusion involving the KIAA1549 gene is a hallmark of pilocytic astrocytoma, but it has also been recorded in rare cases of gangliogliomas, 1p/19q co-deleted oligodendroglial tumors, and it is also a common feature of disseminated oligodendroglial-like leptomeningeal neoplasm. In some difficult cases, evidence for KIAA1549-BRAF fusion is of utmost importance for the diagnosis. Moreover, because the KIAA1549-BRAF fusion constitutively activates the MAP kinase pathway, it represents a target for drugs such as MEK inhibitors, and therefore, the detection of this genetic abnormality is highly relevant in the context of clinical trials applying such new approaches. In the present study, we aimed to use the high sensitivity of Droplet Digital PCR (DDPCR™) to predict KIAA1549-BRAF fusion on very small amounts of formalin-fixed paraffin-embedded tissue in routine practice. Therefore, we analyzed a training cohort of 55 pilocytic astrocytomas in which the KIAA1549-BRAF fusion status was known by RNA sequencing used as our gold standard technique. Then, we analyzed a prospective cohort of 40 pilocytic astrocytomas, 27 neuroepithelial tumors remaining difficult to classify (pilocytic astrocytoma versus ganglioglioma or diffuse glioma), 15 dysembryoplastic neuroepithelial tumors, and 18 gangliogliomas. We could demonstrate the usefulness and high accuracy (100% sensitivity and specificity when compared to RNA sequencing) of DDPCR™ to assess the KIAA1549-BRAF fusion from very low amounts of DNA isolated from formalin-fixed paraffin-embedded specimens. BRAF duplication is both necessary and sufficient to predict this fusion in most cases and we propose that this single analysis could be used in routine practice to save time, money, and precious tissue

A common phytoene synthase mutation underlies white petal varieties of the California poppy.

The California poppy (Eschscholzia californica) is renowned for its brilliant golden-orange flowers, though white petal variants have been described. By whole-transcriptome sequencing, we have discovered in multiple white petal varieties a single deletion leading to altered splicing and C-terminal truncation of phytoene synthase (PSY), a key enzyme in carotenoid biosynthesis. Our findings underscore the diverse roles of phytoene synthase in shaping horticultural traits, and resolve a longstanding mystery of the regaled golden poppy

RNA Sequencing Reveals Novel Transcripts from Sympathetic Stellate Ganglia During Cardiac Sympathetic Hyperactivity.

Cardiovascular disease is the most prevalent age-related illness worldwide, causing approximately 15 million deaths every year. Hypertension is central in determining cardiovascular risk and is a strong predictive indicator of morbidity and mortality; however, there remains an unmet clinical need for disease-modifying and prophylactic interventions. Enhanced sympathetic activity is a well-established contributor to the pathophysiology of hypertension, however the cellular and molecular changes that increase sympathetic neurotransmission are not known. The aim of this study was to identify key changes in the transcriptome in normotensive and spontaneously hypertensive rats. We validated 15 of our top-scoring genes using qRT-PCR, and network and enrichment analyses suggest that glutamatergic signalling plays a key role in modulating Ca2+ balance within these ganglia. Additionally, phosphodiesterase activity was found to be altered in stellates obtained from the hypertensive rat, suggesting that impaired cyclic nucleotide signalling may contribute to disturbed Ca2+ homeostasis and sympathetic hyperactivity in hypertension. We have also confirmed the presence of these transcripts in human donor stellate samples, suggesting that key genes coupled to neurotransmission are conserved. The data described here may provide novel targets for future interventions aimed at treating sympathetic hyperactivity associated with cardiovascular disease and other dysautonomias

Identification of gene expression logical invariants in Arabidopsis.

Numerous gene expression datasets from diverse tissue samples from the plant variety Arabidopsis thaliana have been already deposited in the public domain. There have been several attempts to do large scale meta-analyses of all of these datasets. Most of these analyses summarize pairwise gene expression relationships using correlation, or identify differentially expressed genes in two conditions. We propose here a new large scale meta-analysis of the publicly available Arabidopsis datasets to identify Boolean logical relationships between genes. Boolean logic is a branch of mathematics that deals with two possible values. In the context of gene expression datasets we use qualitative high and low expression values. A strong logical relationship between genes emerges if at least one of the quadrants is sparsely populated. We pointed out serious issues in the data normalization steps widely accepted and published recently in this context. We put together a web resource where gene expression relationships can be explored online which helps visualize the logical relationships between genes. We believe that this website will be useful in identifying important genes in different biological context. The web link is http://hegemon.ucsd.edu/plant/

Molecular processes underlying synergistic linuron mineralization in a triple-species bacterial consortium biofilm revealed by differential transcriptomics

The proteobacteria Variovorax sp. WDL1, Comamonas testosteroni WDL7, and Hyphomicrobium sulfonivorans WDL6 compose a triple-species consortium that synergistically degrades and grows on the phenylurea herbicide linuron. To acquire a better insight into the interactions between the consortium members and the underlying molecular mechanisms, we compared the transcriptomes of the key biodegrading strains WDL7 and WDL1 grown as biofilms in either isolation or consortium conditions by differential RNAseq analysis. Differentially expressed pathways and cellular systems were inferred using the network-based algorithm PheNetic. Coculturing affected mainly metabolism in WDL1. Significantly enhanced expression of hylA encoding linuron hydrolase was observed. Moreover, differential expression of several pathways involved in carbohydrate, amino acid, nitrogen, and sulfur metabolism was observed indicating that WDL1 gains carbon and energy from linuron indirectly by consuming excretion products from WDL7 and/or WDL6. Moreover, in consortium conditions, WDL1 showed a pronounced stress response and overexpression of cell to cell interaction systems such as quorum sensing, contact-dependent inhibition, and Type VI secretion. Since the latter two systems can mediate interference competition, it prompts the question if synergistic linuron degradation is the result of true adaptive cooperation or rather a facultative interaction between bacteria that coincidentally occupy complementary metabolic niches