Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.     

Design and synthesis of 3-(azol-1-yl)phenylpropanes as microbicidal spermicides for prophylactic contraception

We designed a series of 25 3-(azol-1-yl)phenylpropanes which yielded 10 compounds (3, 4, 7, 8, 13, 14, 19, 21, 23, 26) that irreversibly immobilized 100% human sperm at 1% (w/v) concentration in 60 s; 12 compounds (8, 9, 15, 16, 19-21, 23-25, 27, 28) that showed potent microbicidal activity at 12.5-50 μg/mL against Trichomonas vaginalis; and 17 compounds (3-11, 13, 15, 19, 21, 23, 26, 28, 30) that exhibited potent anticandida activity with minimum inhibitory concentration (MIC) of 12.5-50 μg/mL. Almost all the compounds exhibited high level of safety towards normal vaginal flora (Lactobacillus) and human cervical (HeLa) cells in comparison to the marketed spermicide nonoxynol-9 (N-9). All the biological activities were evaluated in vitro. Two compounds (4, 8) with good safety profile exhibited multiple (spermicidal, antitrichomonas and anticandida) activities, warranting further lead optimization for furnishing a prophylactic vaginal contraceptive.     

(⁹⁹m)Tc-bisphosphonate-iron oxide nanoparticle conjugates for dual-modality biomedical imaging

The combination of radionuclide-based imaging modalities such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) with magnetic resonance imaging (MRI) is likely to become the next generation of clinical scanners. Hence, there is a growing interest in the development of SPECT- and PET-MRI agents. To this end, we report a new class of dual-modality imaging agents based on the conjugation of radiolabeled bisphosphonates (BP) directly to the surface of superparamagnetic iron oxide (SPIO) nanoparticles. We demonstrate the high potential of BP-iron oxide conjugation using (??m)Tc-dipicolylamine(DPA)-alendronate, a BP-SPECT agent, and Endorem/Feridex, a liver MRI contrast agent based on SPIO. The labeling of SPIOs with (??m)Tc-DPA-alendronate can be performed in one step at room temperature if the SPIO is not coated with an organic polymer. Heating is needed if the nanoparticles are coated, as long as the coating is weakly bound as in the case of dextran in Endorem. The size of the radiolabeled Endorem (??m)Tc-DPA-ale-Endorem) was characterized by TEM (5 nm, Fe?O? core) and DLS (106 ± 60 nm, Fe?O? core + dextran). EDX, Dittmer-Lester, and radiolabeling studies demonstrate that the BP is bound to the nanoparticles and that it binds to the Fe?O? cores of Endorem, and not its dextran coating. The bimodal imaging capabilities and excellent stability of these nanoparticles were confirmed using MRI and nanoSPECT-CT imaging, showing that (??m)Tc and Endorem co-localize in the liver and spleen In Vivo, as expected for particles of the composition and size of (??m)Tc-DPA-ale-Endorem. To the best of our knowledge, this is the first example of radiolabeling SPIOs with BP conjugates and the first example of radiolabeling SPIO nanoparticles directly onto the surface of the iron oxide core, and not its coating. This work lays down the basis for a new generation of SPECT/PET-MR imaging agents in which the BP group could be used to attach functionality to provide targeting, stealth/stability, and radionuclides to Fe?O? nanoparticles using very simple methodology readily amenable to GMP.     

Aromatic aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live cell imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.     

Effects of chronic administration of clenbuterol on function and metabolism of adult rat cardiac muscle

Clenbuterol (Clen), a beta(2)-agonist, is known to produce skeletal and myocardial hypertrophy. This compound has recently been used in combination with left ventricular assist devices for the treatment of end-stage heart failure to reverse or prevent the adverse effects of unloading-induced myocardial atrophy. However, the mechanisms of action of Clen on myocardial cells have not been fully elucidated. In an attempt to clarify this issue, we examined the effects of chronic administration of Clen on Ca(2+) handling and substrate preference in cardiac muscle. Rats were treated with either 2 mg x kg(-1) x day(-1) Clen or saline (Sal) for 4 wk with the use of osmotic minipumps. Ventricular myocytes were enzymatically dissociated. Cells were field stimulated at 0.5, 1, and 2 Hz, and cytoplasmic Ca(2+) transients were monitored with the use of the fluorescent indicator indo-1 acetoxymethyl ester. Two-dimensional surface area and action potentials in current clamp were also measured. We found that in the Clen group there was significant hypertrophy at the organ and cellular levels compared with Sal. In Clen myocytes, the amplitude of the indo-1 ratio transients was significantly increased. Sarcoplasmic reticulum Ca(2+) content, estimated by rapid application of 20 mM caffeine, was significantly increased in the Clen group. The action potential was prolonged in the Clen group compared with Sal. Carbohydrate contribution to the tricarboxylic cycle (Krebs cycle) flux was increased several times in the Clen group. This increase was associated with decreased expression of peroxisome proliferator-activated receptor-alpha. This study shows that chronic administration of Clen induces cellular hypertrophy and increases oxidative carbohydrate utilization together with an increase in sarcoplasmic reticulum Ca(2+) content, which results in increased amplitude of the Ca(2+) transients. These effects could be important when Clen is used in conjunction with left ventricular assist devices treatment.     

Calcium dependence of proteinase-activated receptor 2 and cholecystokinin-mediated amylase secretion from pancreatic acini

Pancreatic acini secrete digestive enzymes in response to a variety of secretagogues including CCK and agonists acting via proteinase-activated receptor-2 (PAR2). We employed the CCK analog caerulein and the PAR2-activating peptide SLIGRL-NH(2) to compare and contrast Ca(2+) changes and amylase secretion triggered by CCK receptor and PAR2 stimulation. We found that secretion stimulated by both agonists is dependent on a rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and that this rise in [Ca(2+)](i) reflects both the release of Ca(2+) from intracellular stores and accelerated Ca(2+) influx. Both agonists, at low concentrations, elicit oscillatory [Ca(2+)](i) changes, and both trigger a peak plateau [Ca(2+)](i) change at high concentrations. Although the two agonists elicit similar rates of amylase secretion, the rise in [Ca(2+)](i) elicited by caerulein is greater than that elicited by SLIGRL-NH(2). In Ca(2+)-free medium, the rise in [Ca(2+)](i) elicited by SLIGRL-NH(2) is prevented by the prior addition of a supramaximally stimulating concentration of caerulein, but the reverse is not true; the rise elicited by caerulein is neither prevented nor reduced by prior addition of SLIGRL-NH(2). Both the oscillatory and the peak plateau [Ca(2+)](i) changes that follow PAR2 stimulation are prevented by the phospholipase C (PLC) inhibitor U73122, but U73122 prevents only the oscillatory [Ca(2+)](i) changes triggered by caerulein. We conclude that 1) both PAR2 and CCK stimulation trigger amylase secretion that is dependent on a rise in [Ca(2+)](i) and that [Ca(2+)](i) rise reflects release of calcium from intracellular stores as well as accelerated influx of extracellular calcium; 2) PLC mediates both the oscillatory and the peak plateau rise in [Ca(2+)](i) elicited by PAR2 but only the oscillatory rise in [Ca(2+)](i) elicited by CCK stimulation; and 3) the rate of amylase secretion elicited by agonists acting via different types of receptors may not correlate with the magnitude of the [Ca(2+)](i) rise triggered by those different types of secretagogue.     

Determining relevant features to recognize electron density patterns in x-ray protein crystallography

High-throughput computational methods in X-ray protein crystallography are indispensable to meet the goals of structural genomics. In particular, automated interpretation of electron density maps, especially those at mediocre resolution, can significantly speed up the protein structure determination process. TEXTAL(TM) is a software application that uses pattern recognition, case-based reasoning and nearest neighbor learning to produce reasonably refined molecular models, even with average quality data. In this work, we discuss a key issue to enable fast and accurate interpretation of typically noisy electron density data: what features should be used to characterize the density patterns, and how relevant are they? We discuss the challenges of constructing features in this domain, and describe SLIDER, an algorithm to determine the weights of these features. SLIDER searches a space of weights using ranking of matching patterns (relative to mismatching ones) as its evaluation function. Exhaustive search being intractable, SLIDER adopts a greedy approach that judiciously restricts the search space only to weight values that cause the ranking of good matches to change. We show that SLIDER contributes significantly in finding the similarity between density patterns, and discuss the sensitivity of feature relevance to the underlying similarity metric.