PRFdb: A database of computationally predicted eukaryotic programmed -1 ribosomal frameshift signals

The Programmed Ribosomal Frameshift Database (PRFdb) provides an interface to help researchers identify potential programmed -1 ribosomal frameshift (-1 PRF) signals in eukaryotic genes or sequences of interest. To identify putative -1 PRF signals, sequences are first imported from whole genomes or datasets, e.g. the yeast genome project and mammalian gene collection. They are then filtered through multiple algorithms to identify potential -1 PRF signals as defined by a heptameric slippery site followed by an mRNA pseudoknot. The significance of each candidate -1 PRF signal is evaluated by comparing the predicted thermodynamic stability (ΔG°) of the native mRNA sequence against a distribution of ΔG° values of a pool of randomized sequences derived from the original. The data have been compiled in a user-friendly, easily searchable relational database. The PRFdB enables members of the research community to determine whether genes that they are investigating contain potential -1 PRF signals, and can be used as a metasource of information for cross referencing with other databases. It is available on the web at http://dinmanlab.umd.edu/prfdb .https://doi.org/10.1186/1471-2164-9-33

Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast

Although first discovered in viruses, previous studies have identified operational −1 ribosomal frameshifting (−1 RF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. Here, four yeast −1 RF signals are shown to promote significant mRNA destabilization through the nonsense mediated mRNA decay pathway (NMD), and genetic evidence is presented suggesting that they may also operate through the no-go decay pathway (NGD) as well. Yeast EST2 mRNA is highly unstable and contains up to five −1 RF signals. Ablation of the −1 RF signals or of NMD stabilizes this mRNA, and changes in −1 RF efficiency have opposing effects on the steady-state abundance of the EST2 mRNA. These results demonstrate that endogenous −1 RF signals function as mRNA destabilizing elements through at least two molecular pathways in yeast. Consistent with current evolutionary theory, phylogenetic analyses suggest that −1 RF signals are rapidly evolving cis-acting regulatory elements. Identification of high confidence −1 RF signals in ∼10% of genes in all eukaryotic genomes surveyed suggests that −1 RF is a broadly used post-transcriptional regulator of gene expression

Identification of functional, endogenous programmed −1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae

In viruses, programmed −1 ribosomal frameshifting (−1 PRF) signals direct the translation of alternative proteins from a single mRNA. Given that many basic regulatory mechanisms were first discovered in viral systems, the current study endeavored to: (i) identify −1 PRF signals in genomic databases, (ii) apply the protocol to the yeast genome and (iii) test selected candidates at the bench. Computational analyses revealed the presence of 10 340 consensus −1 PRF signals in the yeast genome. Of the 6353 yeast ORFs, 1275 contain at least one strong and statistically significant −1 PRF signal. Eight out of nine selected sequences promoted efficient levels of PRF in vivo. These findings provide a robust platform for high throughput computational and laboratory studies and demonstrate that functional −1 PRF signals are widespread in the genome of Saccharomyces cerevisiae. The data generated by this study have been deposited into a publicly available database called the PRFdb. The presence of stable mRNA pseudoknot structures in these −1 PRF signals, and the observation that the predicted outcomes of nearly all of these genomic frameshift signals would direct ribosomes to premature termination codons, suggest two possible mRNA destabilization pathways through which −1 PRF signals could post-transcriptionally regulate mRNA abundance

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Host and parasite responses in human diffuse cutaneous leishmaniasis caused by L. amazonensis.

Diffuse cutaneous leishmaniasis (DCL) is a rare form of leishmaniasis where parasites grow uncontrolled in diffuse lesions across the skin. Meta-transcriptomic analysis of biopsies from DCL patients infected with Leishmania amazonensis demonstrated an infiltration of atypical B cells producing a surprising preponderance of the IgG4 isotype. DCL lesions contained minimal CD8+ T cell transcripts and no evidence of persistent TH2 responses. Whereas localized disease exhibited activated (so-called M1) macrophage presence, transcripts in DCL suggested a regulatory macrophage (R-Mϕ) phenotype with higher levels of ABCB5, DCSTAMP, SPP1, SLAMF9, PPARG, MMPs, and TM4SF19. The high levels of parasite transcripts in DCL and the remarkable uniformity among patients afforded a unique opportunity to study parasite gene expression in this disease. Patterns of parasite gene expression in DCL more closely resembled in vitro parasite growth in resting macrophages, in the absence of T cells. In contrast, parasite gene expression in LCL revealed 336 parasite genes that were differently upregulated, relative to DCL and in vitro macrophage growth, and these transcripts may represent transcripts that are produced by the parasite in response to host immune pressure

Loss of rpoE Encoding the δ-Factor of RNA Polymerase Impacts Pathophysiology of the Streptococcus pyogenes M1T1 Strain 5448

Streptococcus pyogenes, also known as the Group A Streptococcus (GAS), is a Gram-positive bacterial pathogen of major clinical significance. Despite remaining relatively susceptible to conventional antimicrobial therapeutics, GAS still causes millions of infections and hundreds of thousands of deaths each year worldwide. Thus, a need for prophylactic and therapeutic interventions for GAS is in great demand. In this study, we investigated the importance of the gene encoding the delta (δ) subunit of the GAS RNA polymerase, rpoE, for its impact on virulence during skin and soft-tissue infection. A defined 5448 mutant with an insertionally-inactivated rpoE gene was defective for survival in whole human blood and was attenuated for both disseminated lethality and lesion size upon mono-culture infection in mouse soft tissue. Furthermore, the mutant had reduced competitive fitness when co-infected with wild type (WT) 5448 in the mouse model. We were unable to attribute this attenuation to any observable growth defect, although colony size and the ability to grow at higher temperatures were both affected when grown with nutrient-rich THY media. RNA-seq of GAS grown in THY to late log phase found that mutation of rpoE significantly impacted (>2-fold) the expression of 429 total genes (205 upregulated, 224 downregulated), including multiple virulence and “housekeeping” genes. The arc operon encoding the arginine deiminase (ADI) pathway was the most upregulated in the rpoE mutant and this could be confirmed phenotypically. Taken together, these findings demonstrate that the delta (δ) subunit of RNA polymerase is vital in GAS gene expression and virulence.https://doi.org/10.3390/microorganisms1008168

Identification and Characterization of vB_PreP_EPr2, a Lytic Bacteriophage of Pan-Drug Resistant <i>Providencia rettgeri</i>

Providencia rettgeri is an emerging opportunistic Gram-negative pathogen with reports of increasing antibiotic resistance. Pan-drug resistant (PDR) P. rettgeri infections are a growing concern, demonstrating a need for the development of alternative treatment options which is fueling a renewed interest in bacteriophage (phage) therapy. Here, we identify and characterize phage vB_PreP_EPr2 (EPr2) with lytic activity against PDR P. rettgeri MRSN 845308, a clinical isolate that carries multiple antibiotic resistance genes. EPr2 was isolated from an environmental water sample and belongs to the family Autographiviridae, subfamily Studiervirinae and genus Kayfunavirus, with a genome size of 41,261 base pairs. Additional phenotypic characterization showed an optimal MOI of 1 and a burst size of 12.3 ± 3.4 PFU per bacterium. EPr2 was determined to have a narrow host range against a panel of clinical P. rettgeri strains. Despite this fact, EPr2 is a promising lytic phage with potential for use as an alternative therapeutic for treatment of PDR P. rettgeri infections