Quantitative mapping of mRNA 3' ends in Pseudomonas aeruginosa reveals a pervasive role for premature 3' end formation in response to azithromycin.

Pseudomonas aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including patients with cystic fibrosis. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and quorum sensing (QS). The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using a fast, cost-effective genome-wide approach to quantitate RNA 3' ends (3pMap). We also identified hundreds of P. aeruginosa genes with high incidence of premature 3' end formation indicative of riboregulation in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3' ends indicating premature transcription termination, transcriptional pausing, or accumulation of stable intermediates resulting from the action of nucleases. Reciprocally, AZM reduced premature intragenic 3' end termini in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced changes in 3' end formation alter the expression of central regulators which in turn impairs the expression of QS, biofilm formation and stress response genes, while upregulating genes associated with persistence.

The assembly factor Erb1 functions in multiple remodeling events during 60S ribosomal subunit assembly in S. cerevisiae.

A major gap in our understanding of ribosome assembly is knowing the precise function of each of the ∼200 assembly factors. The steps in subunit assembly in which these factors participate have been examined for the most part by depleting each protein from cells. Depletion of the assembly factor Erb1 prevents stable assembly of seven other interdependent assembly factors with pre-60S subunits, resulting in turnover of early preribosomes, before the ITS1 spacer can be removed from 27SA3 pre-rRNA. To investigate more specific functions of Erb1, we constructed eight internal deletions of 40-60 amino acid residues each, spanning the amino-terminal half of Erb1. The erb1Δ161-200 and erb1Δ201-245 deletion mutations block a later step than depletion of Erb1, namely cleavage of the C2 site that initiates removal of the ITS2 spacer. Two other remodeling events fail to occur in these erb1 mutants: association of twelve different assembly factors with domain V of 25S rRNA, including the neighborhood surrounding the peptidyl transferase center, and stable association of ribosomal proteins with rRNA surrounding the polypeptide exit tunnel. This suggests that successful initiation of construction of these functional centers is a checkpoint for committing to spacer removal.

Nitric Oxide Reverses the Position of the Heart during Embryonic Development.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) plays crucial roles in cardiac homeostasis. Adult cardiomyocyte specific overexpression of eNOS confers protection against myocardial-reperfusion injury. However, the global effects of NO overexpression in developing cardiovascular system is still unclear. We hypothesized that nitric oxide overexpression affects the early migration of cardiac progenitor cells, vasculogenesis and function in a chick embryo. Vehicle or nitric oxide donor DEAN (500 mM) were loaded exogenously through a small window on the broad side of freshly laid egg and embryonic development tracked by live video-microscopy. At Hamburg Hamilton (HH) stage 8, the cardiac progenitor cells (CPC) were isolated and cell migration analysed by Boyden Chamber. The vascular bed structure and heart beats were compared between vehicle and DEAN treated embryos. Finally, expression of developmental markers such as BMP4, Shh, Pitx2, Noggin were measured using reverse transcriptase PCR and in-situ hybridization. The results unexpectedly showed that exogenous addition of pharmacological NO between HH stage 7⁻8 resulted in embryos with situs inversus in 28 out of 100 embryos tested. Embryos treated with NO inhibitor cPTIO did not have situs inversus, however 10 embryos treated with L-arginine showed a situs inversus phenotype. N-acetyl cysteine addition in the presence of NO failed to rescue situs inversus phenotype. The heart beat is normal (120 beats/min) although the vascular bed pattern is altered. Migration of CPCs in DEAN treated embryos is reduced by 60% compared to vehicle. BMP4 protein expression increases on the left side of the embryo compared to vehicle control. The data suggests that the NO levels in the yolk are important in turning of the heart during embryonic development. High levels of NO may lead to situs inversus condition in avian embryo by impairing cardiac progenitor cell migration through the NO-BMP4-cGMP axis.

Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast.

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.

A comparative study of NONOate based NO donors: spermine NONOate is the best suited NO donor for angiogenesis.

Nitric oxide (NO) is a known modulator of angiogenesis. The NONOate subfamily of NO donors has long been used in experimental and clinical studies to promote angiogenesis. However, no studies have been conducted yet to compare the angiogenesis potential of these NO donors in respect to their pattern of NO release. We hypothesize that having different pattern of NO release, each of the NO donors in NONOate subfamily can promote key stages of angiogenesis in differential manner. To verify our hypothesis, NO donors with half life ranging from seconds to several hours and having very different pattern of NO release were selected to evaluate their efficacy in modulating angiogenesis. Endothelial tube formation using EAhy926 cells was maximally increased by Spermine NONOate (SP) treatment. SP treatment maximally induced both ex vivo and in vivo angiogenesis using egg yolk and cotton plug angiogenesis models respectively. Experiment using chick embryo partial ischemia model revealed SP as the best suited NO donor to recover ischemia driven hampered angiogenesis. The present study elaborated that differential release pattern of NO by different NO donors can modulate angiogenesis differentially and also suggested that SP have a unique pattern of NO release that best fits for angiogenesis.

Ion channel modulators mediated alterations in NO-induced free radical generation and neutrophil membrane potential.

The present study investigated the effect of various ion (H+ and K+) channel modulators on nitric oxide (NO) donors (SNP and SNAP) induced free radical generation and on neutrophil membrane potential. Free radical generation was assessed by DCDHF-DA, using flow cytometry, while membrane potential was measured by a fluorescent dye, DiO-C5-(3). Neutrophil suspension in high potassium containing medium or following addition of NO donors (SNP, SNAP) to the neutrophil suspension led to free radical generation and membrane depolarization. DPI (a dual inhibitor of NADPH-oxidase and NOS), ABAH (MPO inhibitor) and BAPTA-AM (calcium chelator) significantly reduced 80 mM KCl or NO mediated free radical generation. Modulators of large (NS1619), intermediate (Chlorzoxazone) and small conductance (Apamin, chlorzoxazone) calcium activated K+ channels (TBA), voltage activated K+ channels (Kv) (4AP, 8Br-cGMP), ATP sensitive K+ channels (K(ATP)) (Glybenclamide, pinacidil), Na+,K+-ATPase (Ouabain) and Na+/H+ exchanger (NHE, Amiloride) altered NO-induced neutrophil free radical generation response and membrane polarity. The results obtained thus suggest an association between rat neutrophil membrane depolarization and NO-dependent free radical generation.