Preventing complicated transseptal puncture with intracardiac echocardiography: case report

Supravalvular mitral stenosis is a rare condition characterized by an abnormal ridge, with one or two orifices, covering and obstructing the mitral valve. Preoperative diagnosis is difficult with transtoracic echo (TTE), angiography and magnetic resonance imaging (MRI). In this case, a 36-year-old male, was admitted to our Heart department: He experienced progressive dyspnea on effort and at rest. Diagnosis was made by transesophageal echocardiography which showed, on apical 4-chamber section, an anulare structure attached since a membrane to the atrial wall anterior mitral valve leaflet and just proximal to the posterior mitral leaflet. Pre-operative identification of the supravalvular mitral ring is the target for obtaining good surgical results. Cineangiography and MRI both failed in reaching this objective, whereas, transesophageal echocardiography is the best method to identify this congenital heart disease. Using TEE the identification is not only possible but also easier.     

Higher-order structures of chromatin: the elusive 30 nm fiber

Despite progress in understanding chromatin function, the structure of the 30 nm chromatin fiber has remained elusive. However, with the recent crystal structure of a short tetranucleosomal array, the 30 nm fiber is beginning to come into view.     

Vivaldi: Visualization and validation of biomacromolecular NMR structures from the PDB

We describe Vivaldi (VIsualization and VALidation DIsplay; http://pdbe.org/vivaldi ), a web‐based service for the analysis, visualization, and validation of NMR structures in the Protein Data Bank (PDB). Vivaldi provides access to model coordinates and several types of experimental NMR data using interactive visualization tools, augmented with structural annotations and model‐validation information. The service presents information about the modeled NMR ensemble, validation of experimental chemical shifts, residual dipolar couplings, distance and dihedral angle constraints, as well as validation scores based on empirical knowledge and databases. Vivaldi was designed for both expert NMR spectroscopists and casual non‐expert users who wish to obtain a better grasp of the information content and quality of NMR structures in the public archive. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Insights into antifolate resistance from malarial DHFR-TS structures

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide possible approaches for the design of new drugs to overcome antifolate resistance.     

Insights into protein biosynthesis from structures of bacterial ribosomes

Understanding the structural basis of protein biosynthesis on the ribosome remains a challenging problem for cryo-electron microscopy and X-ray crystallography. Recent high-resolution structures of the Escherichia coli 70S ribosome without ligands, and of the Thermus thermophilus and E. coli 70S ribosomes with bound mRNA and tRNAs, reveal many new features of ribosome dynamics and ribosome-ligand interactions. In addition, the first high-resolution structures of the L7/L12 stalk of the ribosome, responsible for translation factor binding and GTPase activation, reveal the structural basis of the high degree of flexibility in this region of the ribosome. These structures provide groundbreaking insights into the mechanism of protein synthesis at the level of ribosome architecture, ligand binding and ribosome dynamics.     

Insights into the decoding mechanism from recent ribosome structures

During the decoding process, tRNA selection by the ribosome is far more accurate than expected from codon-anticodon pairing. Antibiotics such as streptomycin and paromomycin have long been known to increase the error rate of translation, and many mutations that increase or lower accuracy have been characterized. Recent crystal structures show that the specific recognition of base-pairing geometry leads to a closure of the domains of the small subunit around cognate tRNA. This domain closure is likely to trigger subsequent steps in tRNA selection. Many antibiotics and mutations act by making the domain closure more or less favourable. In conjunction with recent cryoelectron microscopy structures of the ribosome, a comprehensive structural understanding of the decoding process is beginning to emerge.     

Visualization of Caenorhabditis elegans cuticular structures using the lipophilic vital dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal(1-4). The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility(4-6). Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer(7). Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development(8). Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages(2,9). Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology(5,6,10,11). While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression(12), antibody staining(13), and electron microscopy(1). Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures(14). Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail(15). We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.     

Obtaining high-quality DNA from elusive small mammals using low-tech hair snares

Noninvasive sampling approaches are becoming increasingly important for enabling genetic studies of wildlife populations. While a number of methods have been described to noninvasively sample hair from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. Here we describe a novel and inexpensive noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We explore the quality of the sample by assessing PCR amplification success of mitochondrial and nuclear DNA fragments across four commercially available DNA isolation kits and two different quantities of hair in a factorial design. Additionally, we determined the sex of the individual samples using PCR–RFLP of ZFX/ZFY loci. We found that our snare is effective in obtaining hair that yield DNA of sufficient quality and quantity to successfully amplify a range of mitochondrial and nuclear fragment sizes. Specifically, we found the greatest success in amplifying mitochondrial DNA, nuclear microsatellites and ZFX/ZFY loci using at least 25 hairs as starting material and the DNA IQ™ system. The hair snares thus provide a cost-effective and minimally intrusive approach to sample elusive or rare small mammals. We anticipate that this approach will be useful to obtain samples for molecular studies of the ecology, evolution and conservation of small, elusive mammals.     

Insights from modeling three-dimensional structures of the human potassium and sodium channels

Ion channels allow the movement of ions across cell membranes. Nearly all cells have membranes spanned by ion channels, without which human nerves simply would not work. Ion channels are formed by the aggregation of subunits into a cylindrical configuration that allows a pore, thus forming a kind of tube for ion trafficking. In the present study, the subunits of the human potassium channel are formed by four identical protein chains, whereas for the case of the human sodium channel, the corresponding subunits are actually four hetero-domains formed by the folding of a very large but single protein chain. Since both of the two ion channels are important targets for drug discovery, the 3D (dimensional) structures of their pore regions were developed. On the basis of the 3D models, some important molecular biological mechanisms were discussed that may stimulate novel strategies for therapeutic treatment of the diseases related to ion channel disorders, such as long QT syndrome and chronic pain.     

Unique floral structures and iterative evolutionary themes in Asparagales: Insights from a morphological cladistic analysis

A morphological cladistic analysis is presented of the lilioid order Asparagales, with emphasis on relationships within the “lower” asparagoids, in the context of recent new data on both floral and vegetative structures. The analysis retrieved a monophyletic “lower” asparagoid clade, in contrast to molecular analyses, in which lower asparagoids invariably form a grade. However, limited outgroup sampling in the current analysis is a significant factor in this “inside-out” topology; if the morphological tree is rerooted with Orchidaceae as the outgroup, the result is a topology broadly similar to the molecular one. The relatively low resolution of the “lower” asparagoid clade identified here is a result of high homoplasy in several characters, which could be regarded as iterative evolutionary themes within Asparagales, notably (among floral characters) epigyny and zygomorphy. Close relationships between some family pairs were inferred, including Orchidaceae and Hypoxidaceae, Boryaceae and Blandfordiaceae, Asphodelaceae and Hemerocallidaceae, and Iridaceae and Doryanthaceae. The small South African genusPauridia, which differs from other Hypoxidaceae in that it lacks the outer stamen whorl, was placed as sister to Orchidaceae rather than being embedded in Hypoxidaceae as in molecular analyses, because despite some significant similarities with other Hypoxidaceae (e.g., mucilage canals), it shares some characters with Orchidaceae, notably the presence of a gynostemium and pontoperculate pollen.Xanthorrhoea andLanaria were wild-card taxa in the context of this analysis, with characters in common with more than one different group.     

Safety of mapping in the sinus of valsalva region under intracardiac echocardiography guidance without angiography

BackgroundRadiofrequency ablation at the region of the sinus of Valsalva carries a risk to the ostia of the coronary arteries. Coronary angiography is usually utilized to document a safe distance for mapping and ablation.ObjectiveTo show that catheter ablation in the aortic root could be guided by phased-array intra cardiac echocardiography (ICE) and electro anatomic mapping without the need for coronary angiography.MethodsWe reviewed all patients referred to our lab that underwent mapping and/or ablation in the sinus of Valsalva region. Procedures were carried out by operators that are skilled in the use of ICE. The need for angiography was documented, also the rate of success along with the immediate and 30-day complications rate.ResultsSeventy patients (average age 48.7 ± 13.8 years; 64.3% males) were referred for ablation of ventricular and atrial arrhythmias. PVC constituted 95.7% of the cases. All patients underwent mapping and/or ablation at the sinus of Valsalva region without the need for coronary angiography to visualize the coronary ostia. Acute and effective ablation was achieved in 57 out of 70 (81.4%) patients partially effective ablation was achieved in 10 (14.3%) patients, and failure to ablate in the remaining 3 patients (4.3%). There was no occurrence of any adverse events, neither immediately or at day 30 after the procedure.ConclusionIn the hands of experienced operators, mapping and radiofrequency ablation in the sinus of Valsalva can be safely and reliably performed using intracardiac echocardiography alone without the need for supplementary catheter coronary angiography.     

Assembly of protein tertiary structures from secondary structures using optimized potentials

We present a simulated annealing-based method for the prediction of the tertiary structures of proteins given knowledge of the secondary structure associated with each amino acid in the sequence. The backbone is represented in a detailed fashion whereas the sidechains and pairwise interactions are modeled in a simplified way, following the LINUS model of Srinivasan and Rose. A perceptron-based technique is used to optimize the interaction potentials for a training set of three proteins. For these proteins, the procedure is able to reproduce the tertiary structures to below 3 A in root mean square deviation (rmsd) from the PDB targets. We present the results of tests on twelve other proteins. For half of these, the lowest energy decoy has a rmsd from the native state below 6 A and, in 9 out of 12 cases, we obtain decoys whose rmsd from the native states are also well below 5 A.