Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells

available in PMC 2011 November 01.Cellular RNA levels are determined by the interplay of RNA production, processing and degradation. However, because most studies of RNA regulation do not distinguish the separate contributions of these processes, little is known about how they are temporally integrated. Here we combine metabolic labeling of RNA at high temporal resolution with advanced RNA quantification and computational modeling to estimate RNA transcription and degradation rates during the response of mouse dendritic cells to lipopolysaccharide. We find that changes in transcription rates determine the majority of temporal changes in RNA levels, but that changes in degradation rates are important for shaping sharp 'peaked' responses. We used sequencing of the newly transcribed RNA population to estimate temporally constant RNA processing and degradation rates genome wide. Degradation rates vary significantly between genes and contribute to the observed differences in the dynamic response. Certain transcripts, including those encoding cytokines and transcription factors, mature faster. Our study provides a quantitative approach to study the integrative process of RNA regulation.Human Frontier Science Program (Strasbourg, France)Howard Hughes Medical InstituteBurroughs Wellcome Fund (Career Award at the Scientific Interface

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome

Citation: Chapman, J. A., Mascher, M., Buluç, A., Barry, K., Georganas, E., Session, A., . . . Rokhsar, D. S. (2015). A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome. Genome Biology, 16(1). doi:10.1186/s13059-015-0582-8Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population. © 2015 Chapman et al. licensee BioMed Central.Additional Authors: Muehlbauer, G. J.;Stein, N.;Rokhsar, D. S

Organization and Biology of the Porcine Serum Amyloid A (SAA) Gene Cluster: Isoform Specific Responses to Bacterial Infection.

Serum amyloid A (SAA) is a prominent acute phase protein. Although its biological functions are debated, the wide species distribution of highly homologous SAA proteins and their uniform behavior in response to injury or inflammation in itself suggests a significant role for this protein. The pig is increasingly being used as a model for the study of inflammatory reactions, yet only little is known about how specific SAA genes are regulated in the pig during acute phase responses and other responses induced by pro-inflammatory host mediators. We designed SAA gene specific primers and quantified the gene expression of porcine SAA1, SAA2, SAA3, and SAA4 by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in liver, spleen, and lung tissue from pigs experimentally infected with the Gram-negative swine specific bacterium Actinobacillus pleuropneumoniae, as well as from pigs experimentally infected with the Gram-positive bacterium Staphylococcus aureus. Our results show that: 1) SAA1 may be a pseudogene in pigs; 2) we were able to detect two previously uncharacterized SAA transcripts, namely SAA2 and SAA4, of which the SAA2 transcript is primarily induced in the liver during acute infection and presumably contributes to circulating SAA in pigs; 3) Porcine SAA3 transcription is induced both hepatically and extrahepatically during acute infection, and may be correlated to local organ affection; 4) Hepatic transcription of SAA4 is markedly induced in pigs infected with A. pleuropneumoniae, but only weakly in pigs infected with S. aureus. These results for the first time establish the infection response patterns of the four porcine SAA genes which will be of importance for the use of the pig as a model for human inflammatory responses, e.g. within sepsis, cancer, and obesity research

Epigenetic polypharmacology: from combination therapy to multitargeted drugs

The modern drug discovery process has largely focused its attention in the so-called magic bullets, single chemical entities that exhibit high selectivity and potency for a particular target. This approach was based on the assumption that the deregulation of a protein was causally linked to a disease state, and the pharmacological intervention through inhibition of the deregulated target was able to restore normal cell function. However, the use of cocktails or multicomponent drugs to address several targets simultaneously is also popular to treat multifactorial diseases such as cancer and neurological disorders. We review the state of the art with such combinations that have an epigenetic target as one of their mechanisms of action. Epigenetic drug discovery is a rapidly advancing field, and drugs targeting epigenetic enzymes are in the clinic for the treatment of hematological cancers. Approved and experimental epigenetic drugs are undergoing clinical trials in combination with other therapeutic agents via fused or linked pharmacophores in order to benefit from synergistic effects of polypharmacology. In addition, ligands are being discovered which, as single chemical entities, are able to modulate multiple epigenetic targets simultaneously (multitarget epigenetic drugs). These multiple ligands should in principle have a lower risk of drug-drug interactions and drug resistance compared to cocktails or multicomponent drugs. This new generation may rival the so-called magic bullets in the treatment of diseases that arise as a consequence of the deregulation of multiple signaling pathways provided the challenge of optimization of the activities shown by the pharmacophores with the different targets is addressed

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H

Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens

This is the final version of the article. Available from the publisher via the DOI in this record.Background: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses. Results: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses. Conclusions: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.This article is a joint effort of the working group TRANSBEE and an outcome of two workshops kindly supported by sDiv, the Synthesis Centre for Biodiversity Sciences within the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, funded by the German Science Foundation (FZT 118). New datasets were performed thanks to the Insect Pollinators Initiative (IPI grant BB/I000100/1 and BB/I000151/1), with participation of the UK-USA exchange funded by the BBSRC BB/I025220/1 (datasets #4, 11 and 14). The IPI is funded jointly by the Biotechnology and Biological Sciences Research Council, the Department for Environment, Food and Rural Affairs, the Natural Environment Research Council, the Scottish Government and the Wellcome Trust, under the Living with Environmental Change Partnershi

Alzheimer's risk variants in the clusterin gene are associated with alternative splicing

Genetic variation in CLU encoding clusterin has been associated with Alzheimer's disease (AD) through replicated genome-wide studies, but the underlying mechanisms remain unknown. Following earlier reports that tightly regulated CLU alternative transcripts have different functions, we tested CLU single-nucleotide polymorphisms (SNPs), including those associated with AD for quantitative effects on individual alternative transcripts. In 190 temporal lobe samples without pathology, we found that the risk allele of the AD-associated SNP rs9331888 increases the relative abundance of transcript NM_203339 (P=4.3 × 10−12). Using an independent set of 115 AD and control samples, we replicated this result (P=0.0014) and further observed that multiple CLU transcripts are at higher levels in AD compared with controls. The AD SNP rs9331888 is located in the first exon of NM_203339 and therefore, it is a functional candidate for the observed effects. We tested this hypothesis by in vitro dual luciferase assays using SK-N-SH cells and mouse primary cortical neurons and found allelic effects on enhancer function, consistent with our results on post-mortem human brain. These results suggest a biological mechanism for the genetic association of CLU with AD risk and indicate that rs9331888 is one of the functional DNA variants underlying this association

Socioeconomic status, severity of disease and level of family members’ care in adult surgical intensive care patients: the prospective ECSSTASI study

Low socioeconomic status (SES) is associated with increased mortality from cardiovascular disease, cancer and trauma. However, individual-level prospective data on SES in relation to health outcomes among critically ill patients admitted to intensive care units (ICU) are unavailable. In a cohort of 1,006 patients at a 24-bed surgical ICU of an academic tertiary care facility in Germany, we examined levels of SES in relation to disease severity at admission, time period of mechanical ventilation, length of stay and frequency of phone calls and visits by next-of-kin. Patients with low SES had higher risk for Sequential Organ Failure Assessment (SOFA) score greater or equal to 5 [multivariate-adjusted odds ratio (OR) 1.49; 95% confidence interval (CI) 0.95-2.33; p = 0.029] and a trend for higher risk for Simplified Acute Physiology Score (SAPS II) greater or equal to 31 (OR 1.28; 95% CI 0.80-2.05; p = 0.086) at admission as compared with patients with high SES. When compared with men with high SES, those with low SES had greater risk for ICU treatment a parts per thousand yen5 days (multivariate-adjusted OR 1.99; 95% CI 1.06-3.74; p = 0.036) and showed a trend for a low number of visits from next-of-kin (< 0.5 visits per day) (OR 1.85; 95% CI 0.79-4.30; p = 0.054). In women such associations could not be demonstrated. Socioeconomic status is inversely related to severity of disease at admission and to length of stay in ICU, and positively associated with the level of care by next-of-kin. Whether relations differ by gender requires further examination