Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.Peer reviewe

Direct observations of a surface eigenmode of the dayside magnetopause

The abrupt boundary between a magnetosphere and the surrounding plasma, the magnetopause, has long been known to support surface waves. It was proposed that impulses acting on the boundary might lead to a trapping of these waves on the dayside by the ionosphere, resulting in a standing wave or eigenmode of the magnetopause surface. No direct observational evidence of this has been found to date and searches for indirect evidence have proved inconclusive, leading to speculation that this mechanism might not occur. By using fortuitous multipoint spacecraft observations during a rare isolated fast plasma jet impinging on the boundary, here we show that the resulting magnetopause motion and magnetospheric ultra-low frequency waves at well-defined frequencies are in agreement with and can only be explained by the magnetopause surface eigenmode. We therefore show through direct observations that this mechanism, which should impact upon the magnetospheric system globally, does in fact occur

The Staphylococcus aureus RNome and Its Commitment to Virulence

Staphylococcus aureus is a major human pathogen causing a wide spectrum of nosocomial and community-associated infections with high morbidity and mortality. S. aureus generates a large number of virulence factors whose timing and expression levels are precisely tuned by regulatory proteins and RNAs. The aptitude of bacteria to use RNAs to rapidly modify gene expression, including virulence factors in response to stress or environmental changes, and to survive in a host is an evolving concept. Here, we focus on the recently inventoried S. aureus regulatory RNAs, with emphasis on those with identified functions, two of which are directly involved in pathogenicity

Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation

Role of calcium channel blocking agents in the prevention of atherosclerosis

Calcium channel blocking agents, although effective and widely used in the symptomatic therapy of hypertension and ischemic heart disease, have an uncertain effect on the development of coronary atherosclerosis, plaque rupture, and postrupture thrombosis. Both nifedipine and nicardipine have been shown to prevent the development of new coronary lesions but not the progression of existing lesions in prospective randomized angiographic studies. Verapamil, in contrast, failed to prevent the development of new coronary lesions and had no significant effect on the progression of existing lesions. Diltiazem, although not studied in patients with coronary atheroscleroses, has been shown to prevent the development of post-transplant coronary vascular disease. Despite the beneficial effects of nifedipine and nicardipine on new coronary lesion development, they have not been shown to reduce the incidence of recurrent ischemic events or mortality in the prospective randomized studies that demonstrated their effect on new coronary lesion development. A relatively new dihydropyridine calcium channel blocking agent, amlodipine, is hypothesized to prevent atherosclerosis due to its calcium channel blocking properties as well as by mechanisms independent of its calcium channel blocking properties. This agent has been selected for evaluation in the Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial (PREVENT) to explore whether the use of amlodipine over 3 years will reduce the incidence of early atherosclerotic lesions and, possibly, the progression of existing lesions in both the coronary and carotid arterial beds. Amlodipine could play an important future role in the secondary prevention of ischemic heart disease, but further study and a demonstration of a beneficial effect on recurrent ischemic events is required before any final conclusions concerning its effectiveness are reached.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44649/1/10557_2004_Article_BF00878569.pd

Stress-Induced Reinstatement of Drug Seeking: 20 Years of Progress

In human addicts, drug relapse and craving are often provoked by stress. Since 1995, this clinical scenario has been studied using a rat model of stress-induced reinstatement of drug seeking. Here, we first discuss the generality of stress-induced reinstatement to different drugs of abuse, different stressors, and different behavioral procedures. We also discuss neuropharmacological mechanisms, and brain areas and circuits controlling stress-induced reinstatement of drug seeking. We conclude by discussing results from translational human laboratory studies and clinical trials that were inspired by results from rat studies on stress-induced reinstatement. Our main conclusions are (1) The phenomenon of stress-induced reinstatement, first shown with an intermittent footshock stressor in rats trained to self-administer heroin, generalizes to other abused drugs, including cocaine, methamphetamine, nicotine, and alcohol, and is also observed in the conditioned place preference model in rats and mice. This phenomenon, however, is stressor specific and not all stressors induce reinstatement of drug seeking. (2) Neuropharmacological studies indicate the involvement of corticotropin-releasing factor (CRF), noradrenaline, dopamine, glutamate, kappa/dynorphin, and several other peptide and neurotransmitter systems in stress-induced reinstatement. Neuropharmacology and circuitry studies indicate the involvement of CRF and noradrenaline transmission in bed nucleus of stria terminalis and central amygdala, and dopamine, CRF, kappa/dynorphin, and glutamate transmission in other components of the mesocorticolimbic dopamine system (ventral tegmental area, medial prefrontal cortex, orbitofrontal cortex, and nucleus accumbens). (3) Translational human laboratory studies and a recent clinical trial study show the efficacy of alpha-2 adrenoceptor agonists in decreasing stress-induced drug craving and stress-induced initial heroin lapse

Spectral analysis of four 'hypervariable' AGN: a microneedle in the haystack?

We analyse four extreme active galactic nuclei (AGN) transients to explore the possibility that they are caused by rare, high-amplitude microlensing events. These previously unknown type-I AGN are located in the redshift range 0.6-1.1 and show changes of > 1.5 mag in the g band on a time-scale of similar to years. Multi-epoch optical spectroscopy, from the William Herschel Telescope, shows clear differential variability in the broad line fluxes with respect to the continuum changes and also evolution in the line profiles. In two cases, a simple point-source, point-lens microlensing model provides an excellent match to the long-term variability seen in these objects. For both models, the parameter constraints are consistent with the microlensing being due to an intervening stellar mass object but as yet there is no confirmation of the presence of an intervening galaxy. The models predict a peak amplification of 10.3/13.5 and an Einstein time-scale of 7.5/10.8 yr, respectively. In one case, the data also allow constraints on the size of the C III] emitting region, with some simplifying assumptions, to be similar to 1.0-6.5 light-days and a lower limit on the size of the MgII emitting region to be > 9 light-days (halflight radii). This C III] radius is perhaps surprisingly small. In the remaining two objects, there is spectroscopic evidence for an intervening absorber but the extra structure seen in the light curves requires a more complex lensing scenario to adequately explain

V3 Loop Sequence Space Analysis Suggests Different Evolutionary Patterns of CCR5- and CXCR4-Tropic HIV

The V3 loop of human immunodeficiency virus type 1 (HIV-1) is critical for coreceptor binding and is the main determinant of which of the cellular coreceptors, CCR5 or CXCR4, the virus uses for cell entry. The aim of this study is to provide a large-scale data driven analysis of HIV-1 coreceptor usage with respect to the V3 loop evolution and to characterize CCR5- and CXCR4-tropic viral phenotypes previously studied in small- and medium-scale settings. We use different sequence similarity measures, phylogenetic and clustering methods in order to analyze the distribution in sequence space of roughly 1000 V3 loop sequences and their tropism phenotypes. This analysis affords a means of characterizing those sequences that are misclassified by several sequence-based coreceptor prediction methods, as well as predicting the coreceptor using the location of the sequence in sequence space and of relating this location to the CD4+ T-cell count of the patient. We support previous findings that the usage of CCR5 is correlated with relatively high sequence conservation whereas CXCR4-tropic viruses spread over larger regions in sequence space. The incorrectly predicted sequences are mostly located in regions in which their phenotype represents the minority or in close vicinity of regions dominated by the opposite phenotype. Nevertheless, the location of the sequence in sequence space can be used to improve the accuracy of the prediction of the coreceptor usage. Sequences from patients with high CD4+ T-cell counts are relatively highly conserved as compared to those of immunosuppressed patients. Our study thus supports hypotheses of an association of immune system depletion with an increase in V3 loop sequence variability and with the escape of the viral sequence to distant parts of the sequence space

2D-PAGE as an effective method of RNA degradome analysis

The continuously growing interest in small regulatory RNA exploration is one of the important factors that have inspired the recent development of new high throughput techniques such as DNA microarrays or next generation sequencing. Each of these methods offers some significant advantages but at the same time each of them is expensive, laborious and challenging especially in terms of data analysis. Therefore, there is still a need to develop new analytical methods enabling the fast, simple and cost-effective examination of the complex RNA mixtures. Recently, increasing attention has been focused on the RNA degradome as a potential source of riboregulators. Accordingly, we attempted to employ a two-dimensional gel electrophoresis as a quick and uncomplicated method of profiling RNA degradome in plant or human cells. This technique has been successfully used in proteome analysis. However, its application in nucleic acids studies has been very limited. Here we demonstrate that two dimensional electrophoresis is a technique which allows one to quickly and cost-effectively identify and compare the profiles of 10–90 nucleotide long RNA accumulation in various cells and organs

Biodiversity of Mineral Nutrient and Trace Element Accumulation in Arabidopsis thaliana

In order to grow on soils that vary widely in chemical composition, plants have evolved mechanisms for regulating the elemental composition of their tissues to balance the mineral nutrient and trace element bioavailability in the soil with the requirements of the plant for growth and development. The biodiversity that exists within a species can be utilized to investigate how regulatory mechanisms of individual elements interact and to identify genes important for these processes. We analyzed the elemental composition (ionome) of a set of 96 wild accessions of the genetic model plant Arabidopsis thaliana grown in hydroponic culture and soil using inductively coupled plasma mass spectrometry (ICP-MS). The concentrations of 17–19 elements were analyzed in roots and leaves from plants grown hydroponically, and leaves and seeds from plants grown in artificial soil. Significant genetic effects were detected for almost every element analyzed. We observed very few correlations between the elemental composition of the leaves and either the roots or seeds. There were many pairs of elements that were significantly correlated with each other within a tissue, but almost none of these pairs were consistently correlated across tissues and growth conditions, a phenomenon observed in several previous studies. These results suggest that the ionome of a plant tissue is variable, yet tightly controlled by genes and gene×environment interactions. The dataset provides a valuable resource for mapping studies to identify genes regulating elemental accumulation. All of the ionomic data is available at www.ionomicshub.org