Small RNA Sorting in Drosophila Produces Chemically Distinct Functional RNA-Protein Complexes: A Dissertation

Small interfering RNAs (siRNAs), microRNAs (miRNAs), and piRNAs (piRNA) are conserved classes of small single-stranded ~21-30 nucleotide (nt) RNA guides that repress eukaryotic gene expression using distinct RNA Induced Silencing Complexes (RISCs). At its core, RISC is composed of a single-stranded small RNA guide bound to a member of the Argonaute protein family, which together bind and repress complementary target RNA. miRNAs target protein coding mRNAs—a function essential for normal development and broadly involved in pathways of human disease; small interfering RNAs (siRNA) defend against viruses, but can also be engineered to direct experimental or therapeutic gene silencing; piwi associated RNAs (piRNAs) protect germline genomes from expansion of parasitic nucleic acids such as transposons. Using the fruit fly, Drosophila melanogaster, as a model organism we seek to understand how small silencing RNAs are made and how they function. In Drosophila, miRNAs and siRNAs are proposed to have parallel, but separate biogenesis and effector machinery. miRNA duplexes are excised from imperfectly paired hairpin precursors by Dicer1 and loaded into Ago1; siRNA duplexes are hewn from perfectly paired long dsRNA by Dicer2 and loaded into Ago2. Contrary to this model we found one miRNA, miR-277, is made by Dicer1, but partitions between Ago1 and Ago2 RISCs. These two RISCs are functionally distinct—Ago2 could silence a perfectly paired target, but not a centrally bulged target; Ago1 could silence a bulged target, but not a perfect target. This was surprising since both Ago1 and Ago2 have endonucleolytic cleavage activity necessary for perfect target cleavage in vitro. Our detailed kinetic studies suggested why—Ago2 is a robust multiple turnover enzyme, but Ago1 is not. Along with a complementary in vitro study our data supports a duplex sorting mechanism in which Diced duplexes are released, and rebind to Ago1 or Ago2 loading machinery, regardless of which Dicer produced them. This allows structural information embedded in small RNA duplexes to direct small RNA loading into Ago1 and/or Ago2, resulting in distinct regulatory outputs. Small RNA sorting also has chemical consequences for the small RNA guide. Although siRNAs were presumed to have the signature 2′, 3′ hydroxyl ends left by Dicer, we found that small RNAs loaded into Ago2 or Piwi proteins, but not Ago1, are modified at their 3´ ends by the RNA 2´-O-methyltransferase DmHen1. In plants Hen1 modifies the 3´ ends all small RNAs duplexs, protecting and stabilizing them. Implying a similar function in flies, piRNAs are smaller, less abundant, and their function is perturbed in hen1 mutants. But unlike plants, small RNAs are modified as single-strands in RISC rather than as duplexes. This nicely explains why the dsRNA binding domain in plant Hen1 was discarded in animals, and why both dsRNA derived siRNAs and ssRNA derived piRNAs are modified. The recent discovery that both piRNAs and siRNAs target transposons links terminal modification and transposon silencing, suggesting that it is specialized for this purpose

Statins in the Treatment of Chronic Heart Failure: A Systematic Review

BACKGROUND: The efficacy of statin therapy in patients with established chronic heart failure (CHF) is a subject of much debate. METHODS AND FINDINGS: We conducted three systematic literature searches to assess the evidence supporting the prescription of statins in CHF. First, we investigated the participation of CHF patients in randomized placebo-controlled clinical trials designed to evaluate the efficacy of statins in reducing major cardiovascular events and mortality. Second, we assessed the association between serum cholesterol and outcome in CHF. Finally, we evaluated the ability of statin treatment to modify surrogate endpoint parameters in CHF. Using validated search strategies, we systematically searched PubMed for our three queries. In addition, we searched the reference lists from eligible studies, used the “see related articles” feature for key publications in PubMed, consulted the Cochrane Library, and searched the ISI Web of Knowledge for papers citing key publications. Search 1 resulted in the retrieval of 47 placebo-controlled clinical statin trials involving more than 100,000 patients. CHF patients had, however, been systematically excluded from these trials. Search 2 resulted in the retrieval of eight studies assessing the relationship between cholesterol levels and outcome in CHF patients. Lower serum cholesterol was consistently associated with increased mortality. Search 3 resulted in the retrieval of 18 studies on the efficacy of statin treatment in CHF. On the whole, these studies reported favorable outcomes for almost all surrogate endpoints. CONCLUSIONS: Since CHF patients have been systematically excluded from randomized, controlled clinical cholesterol-lowering trials, the effect of statin therapy in these patients remains to be established. Currently, two large, randomized, placebo-controlled statin trials are under way to evaluate the efficacy of statin treatment in terms of reducing clinical endpoints in CHF patients in particular

Routine Laboratory Results and Thirty Day and One-Year Mortality Risk Following Hospitalization with Acute Decompensated Heart Failure

INTRODUCTION: Several blood tests are performed uniformly in patients hospitalized with acute decompensated heart failure and are predictive of the outcomes: complete blood count, electrolytes, renal function, glucose, albumin and uric acid. We sought to evaluate the relationship between routine admission laboratory tests results, patient characteristics and 30-day and one-year mortality of patients admitted for decompensated heart failure and to construct a simple mortality prediction tool. METHODS: A retrospective population based study. Data from seven tertiary hospitals on all admissions with a principal diagnosis of heart failure during the years 2002-2005 throughout Israel were captured. RESULTS: 8,246 patients were included in the study cohort. Thirty day mortality rate was 8.5% (701 patients) and one-year mortality rate was 28.7% (2,365 patients). Addition of five routine laboratory tests results (albumin, sodium, blood urea, uric acid and WBC) to a set of clinical and demographic characteristics improved c-statistics from 0.76 to 0.81 for 30-days and from 0.72 to 0.76 for one-year mortality prediction (both p-values <0.0001). Three dichotomized abnormal laboratory results with highest odds ratio for one-year mortality (hypoalbuminaemia, hyponatremia and elevated blood urea) were used to construct a simple prediction score, capable of discriminating from 1.1% to 21.4% in 30-day and from 11.6% to 55.6% in one-year mortality rates between patients with a score of 0 (1,477 patients) vs. score of 3 (544 patients). DISCUSSION: A small set of abnormal routine laboratory results upon admission can risk-stratify and independently predict 30-day and one-year mortality in patients hospitalized with acute decompensated heart failure

High sensitive troponin T and heart fatty acid binding protein: Novel biomarker in heart failure with normal ejection fraction?: A cross-sectional study

Background: High sensitive troponin T (hsTnT) and heart fatty acid binding protein (hFABP) are both markers of myocardial injury and predict adverse outcome in patients with systolic heart failure (SHF). We tested whether hsTnT and hFABP plasma levels are elevated in patients with heart failure with normal ejection fraction (HFnEF). Methods: We analyzed hsTnT, hFABP and N-terminal brain natriuretic peptide in 130 patients comprising 49 HFnEF patients, 51 patients with asymptomatic left ventricular diastolic dysfunction (LVDD), and 30 controls with normal diastolic function. Patients were classified to have HFnEF when the diagnostic criteria as recommended by the European Society of Cardiology were met. Results: Levels of hs TnT and hFABP were significantly higher in patients with asymptomatic LVDD and HFnEF (both p < 0.001) compared to controls. The hsTnT levels were 5.6 [0.0-9.8] pg/ml in LVDD vs. 8.5 [3.9-17.5] pg/ml in HFnEF vs. < 0.03 [< 0.03-6.4] pg/ml in controls; hFABP levels were 3029 [2533-3761] pg/ml in LVDD vs. 3669 [2918-4839] pg/ml in HFnEF vs. 2361 [1860-3081] pg/ml in controls. Furthermore, hsTnT and hFABP levels were higher in subjects with HFnEF compared to LVDD (p = 0.015 and p = 0.022). Conclusion: In HFnEF patients, hsTnT and hFABP are elevated independent of coronary artery disease, suggesting that ongoing myocardial damage plays a critical role in the pathophysiology. A combination of biomarkers and echocardiographic parameters might improve diagnostic accuracy and risk stratification of patients with HFnEF

Efficient tumour formation by single human melanoma cells

A fundamental question in cancer biology is whether cells with tumorigenic potential are common or rare within human cancers. Studies on diverse cancers, including melanoma, have indicated that only rare human cancer cells ( 0.1 - 0.0001%) form tumours when transplanted into non- obese diabetic/ severe combined immunodeficiency ( NOD/ SCID) mice. However, the extent to which NOD/ SCID mice underestimate the frequency of tumorigenic human cancer cells has been uncertain. Here we show that modified xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/ SCID interleukin- 2 receptor gamma chain null (Il2rg(-/-)) mice, can increase the detection of tumorigenic melanoma cells by several orders of magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumours under these more permissive conditions. In single- cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumours. Modifications to xenotransplantation assays can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.Howard Hughes Medical Institute ; Allen H. Blondy Research Fellowship ; Lewis and Lillian Becker ; University of Michigan Comprehensive Cancer Center ; National Institutes of Health [CA46592]; University of Michigan Flow Cytometry Core Facility ; N. McAnsh and the University of Michigan Cancer Centre Histology Core ; National Institute of Diabetes, Digestive, and Kidney Diseases [NIH5P60- DK20572]; Michigan Diabetes Research and Training Center ; Spanish Ministry of Education ; Marie Curie Outgoing International Fellowship from the European Commission ; Australian National Health and Medical Research Council ; Human Frontiers Science Program and Australia PostThis work was supported by the Howard Hughes Medical Institute and by the Allen H. Blondy Research Fellowship. The University of Michigan Melanoma Bank was supported by a gift from Lewis and Lillian Becker. Flow cytometry was partly supported by the University of Michigan Comprehensive Cancer Center grant from the National Institutes of Health CA46592. We thank: D. Adams, M. White and the University of Michigan Flow Cytometry Core Facility for support; N. McAnsh and the University of Michigan Cancer Centre Histology Core for histological studies; G. K. Smyth for assistance with statistics; and Z. Azizan for support with tissue collection. Antibody production was supported in part by the National Institute of Diabetes, Digestive, and Kidney Diseases, grant NIH5P60- DK20572 to the Michigan Diabetes Research and Training Center. Some antibodies were provided by Caltag or by eBioscience to screen for cancer stem- cell markers. Human primary melanocyte cultures were provided by M. Soengas. Human mesenchymal stem cells were provided by Z. Wang and P. Krebsbach. E. Q. was supported by the Spanish Ministry of Education and the Marie Curie Outgoing International Fellowship from the European Commission. M. S. was supported by the Australian National Health and Medical Research Council, the Human Frontiers Science Program and Australia Post.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62970/1/nature07567.pd

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells

MicroRNAs (miRNAs), approximately 22-nt RNAs that mediate post-transcriptional regulation of mRNAs in animals and plants, are a diverse class of regulatory genes whose specific biological functions are largely unknown. Here we detail a protocol to design and introduce into cultured Drosophila and human cells sequence-specific antisense oligonucleotides (ASOs) that block the function of individual miRNAs. Coupled with recent studies that catalog the miRNAs expressed in diverse cultured cells, our method offers a rapid (\u3c1 \u3eweek) approach to validate miRNA targets and to study the cellular functions of individual human and Drosophila miRNAs. ASO-based inactivation of miRNAs is faster and simpler than comparable genetic or \u27sponge\u27-based approaches, for which extensive recombinant DNA manipulation is required. We present our ASO design principles and an optimized transfection protocol in which transfection efficiency of Drosophila Schneider 2 cells can approach 100%. Our 3\u27-cholesterol-modified ASOs have enhanced potency, allowing miRNA inhibition for at least 7 d from a single transfection