Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.

The Cardica C-Port System: clinical and angiographic evaluation of a new device for automated, compliant distal anastomoses in coronary artery bypass grafting surgery--a multicenter prospective clinical trial

OBJECTIVES: The C-Port System (Cardica, Inc, Redwood City, Calif) integrates in one tool all functions necessary to enable rapid automated distal coronary anastomoses. The goal of this prospective, nonrandomized, and multicenter study is to determine the safety and efficacy of this novel anastomotic system. METHODS: Five centers enrolled 133 patients awaiting elective coronary artery bypass grafting surgery. Outcome variables were intraoperative device performance, incidence of device-related adverse events, predischarge and 6-month angiographic graft patency, and 12-month clinical outcome. Independent core laboratories performed qualitative and quantitative angiographic and computed tomographic assessments. RESULTS: The C-Port was used to perform a vein-to-coronary anastomosis in 130 patients. Intraoperative conversion to a hand-sewn anastomosis was necessary in 11 patients because of inadequate target site preparation, inappropriate target vessel selection, or both. Inadequate blood flow related to poor runoff required conversion in 3 additional patients. Three patients died before discharge of causes unrelated to the device. At discharge, 113 patients had a C-Port implant in place, and 104 C-Port anastomoses were studied by means of angiography, resulting in 100 FitzGibbon A, 3 FitzGibbon B, and 1 FitzGibbon 0 classifications. At 6 months, one additional patient died of a device-unrelated cause, and 98 patients were evaluated by means of angiography (n = 89). Overall patency (FitzGibbon A) was 92.1%. Three C-Port anastomoses were rated FitzGibbon B, and 4 were rated FitzGibbon 0. At 12 months, 107 (98.2%) of 109 alive patients were followed up, without any reports of device-related major adverse cardiac events. CONCLUSIONS: The C-Port System allows for a rapid, reliable, and compliant distal anastomosis and yields favorable 6-month angiographic and 12-month clinical results when compared with published studies.

Valved stents for transapical pulmonary valve replacement

OBJECTIVES: Pulmonary valve insufficiency remains a leading cause for reoperations in congenital cardiac surgery. The current percutaneous approach is limited by the size of the access vessel and variable right ventricular outflow tract morphology. This study assesses the feasibility of transapical pulmonary valve replacement based on a new valved stent construction concept. METHODS: A new valved stent design was implanted off-pump under continuous intracardiac echocardiographic and fluoroscopic guidance into the native right ventricular outflow tract in 8 pigs (48.5 +/- 6.0 kg) through the right ventricular apex, and device function was studied by using invasive and noninvasive measures. RESULTS: Procedural success was 100% at the first attempt. Procedural time was 75 +/- 15 minutes. All devices were delivered at the target site with good acute valve function. No valved stents dislodged. No animal had significant regurgitation or paravalvular leaking on intracardiac echocardiographic analysis. All animals had a competent tricuspid valve and no signs of right ventricular dysfunction. The planimetric valve orifice was 2.85 +/- 0.32 cm(2). No damage to the pulmonary artery or structural defect of the valved stents was found at necropsy. CONCLUSIONS: This study confirms the feasibility of direct access valve replacement through the transapical procedure for replacement of the pulmonary valve, as well as validity of the new valved stent design concept. The transapical procedure is targeting a broader patient pool, including the very young and the adult patient. The device design might not be restricted to failing conduits only and could allow for implantation in a larger patient population, including those with native right ventricular outflow tract configurations.

Validation of a magnetic resonance imaging guided stereotactic access to the ovine brainstem.

BackgroundAnatomical differences between humans and domestic mammals preclude the use of reported stereotactic approaches to the brainstem in animals. In animals, brainstem biopsies are required both for histopathological diagnosis of neurological disorders and for research purposes. Sheep are used as a translational model for various types of brain disease and therefore a species-specific approach needs to be developed. The aim of the present study was to establish a minimally invasive, accurate and reproducible stereotactic approach to the brainstem of sheep, using the magnetic resonance imaging guided BrainsightTM frameless stereotactic system.ResultsA transoccipital transcerebellar approach with an entry point in the occipital bone above the vermis between the transverse sinus and the external occipital protuberance was chosen. This approach provided access to the target site in all heads. The overall mean needle placement error was 1.85¿±¿1.22 mm.ConclusionsThe developed transoccipital transcerebellar route is short, provides accurate access to the ovine caudal cranial fossa and is a promising approach to be assessed further in live animals.

Ideal site for ventricular anchoring of artificial chordae in mitral regurgitation

Surgical treatment of mitral leaflet prolapse using artificial neochordae shows excellent outcomes. Upcoming devices attempt the same treatment in a minimally invasive way but target the left ventricular apex as an anchoring point, rather than the tip of the corresponding papillary muscle. In this study, cine cardiac magnetic resonance imaging was used to compare these 2 different anchoring positions and their dynamic relationship with the mitral leaflets.

Swiss Alps Jungfrau Aletsch UNESCO World Heritage Site - The state of this World Heritage region as a starting point for regional monitoring

The UNESCO listing as World Heritage Site confirms the outstanding qualities of the high-mountain region around the Great Aletsch Glacier. The region of the World Heritage Site now faces the responsibility to make these qualities visible and to preserve them for future generations. Consequently the qualities of the site must not be regarded in isolation but in the context of the entire region with its dynamics and developments. Regional monitoring is the observation and evaluation of temporal changes in target variables. It is thus an obligation towards UNESCO, who demands regular reports about the state of the listed World Heritage assets. It also allows statements about sustainable regional development and can be the basis for early recognition of threats to the outstanding qualities. Monitoring programmes face three major challenges: first, great care must be taken in defining the target qualities to be monitored or the monitoring would remain vague. Secondly, the selection of ideal indicators to describe these qualities is impeded by inadequate data quality and availability, compromises are inevitable. Thirdly, there is always an element of insecurity in the interpretation of the results as to what influences and determines the changes in the target qualities. The first survey of the monitoring programme confirmed the exceptional qualities of the region and also highlighted problematic issues.

Coronary plaque composition of culprit/target lesions according to the clinical presentation: a virtual histology intravascular ultrasound analysis

AIMS: To evaluate the plaque composition obtained by virtual histology (VH) IVUS according to the clinical presentation and to compare those data to previously published histopathology data. METHODS AND RESULTS: VH was performed on 95 de novo significant lesions (>75% stenosis) in 85 patients [28 acute coronary syndrome (ACS) patients, 30 lesions; 57 stable angina pectoris (SAP) patients, 65 lesions]. There were a higher prevalence of positive remodelling (47 vs. 22%, P=0.013), thrombus (20 vs. 1.5%, P=0.0037), and echo-lucent area (23.3 vs. 7.7%, P=0.047) in ACS patients. At the minimal lumen site, fibrous plaque area was significantly larger in ACS lesions than in SAP lesions (66.0+/-10.7 vs. 61.4+/-8.9%, P=0.034), whereas necrotic core and dense calcium plaque area were smaller in ACS lesions (Necrotic core: 6.8+/-6.0 vs. 11.0+/-8.3%, P=0.02; Dense calcium: 2.6+/-3.0 vs. 4.9+/-5.8%, P=0.03). No differences in rate of thin cap fibroatheroma, thick fibrotheroma, or for the presence of multiple necrotic core layers were observed between both groups. CONCLUSION: Plaque composition obtained by VH-IVUS shows less necrotic core and more fibrous tissue in ACS compared to SAP lesions, which is in contradiction with previously published histopathologic data.

Use of EORTC Target Definition Guidelines for Dose-Intensified Salvage Radiation Therapy for Recurrent Prostate Cancer: Results of the Quality Assurance Program of the Randomized Trial SAKK 09/10

PURPOSE Different international target volume delineation guidelines exist and different treatment techniques are available for salvage radiation therapy (RT) for recurrent prostate cancer, but less is known regarding their respective applicability in clinical practice. METHODS AND MATERIALS A randomized phase III trial testing 64 Gy vs 70 Gy salvage RT was accompanied by an intense quality assurance program including a site-specific and study-specific questionnaire and a dummy run (DR). Target volume delineation was performed according to the European Organisation for the Research and Treatment of Cancer guidelines, and a DR-based treatment plan was established for 70 Gy. Major and minor protocol deviations were noted, interobserver agreement of delineated target contours was assessed, and dose-volume histogram (DVH) parameters of different treatment techniques were compared. RESULTS Thirty European centers participated, 43% of which were using 3-dimensional conformal RT (3D-CRT), with the remaining centers using intensity modulated RT (IMRT) or volumetric modulated arc technique (VMAT). The first submitted version of the DR contained major deviations in 21 of 30 (70%) centers, mostly caused by inappropriately defined or lack of prostate bed (PB). All but 5 centers completed the DR successfully with their second submitted version. The interobserver agreement of the PB was moderate and was improved by the DR review, as indicated by an increased κ value (0.59 vs 0.55), mean sensitivity (0.64 vs 0.58), volume of total agreement (3.9 vs 3.3 cm(3)), and decrease in the union volume (79.3 vs 84.2 cm(3)). Rectal and bladder wall DVH parameters of IMRT and VMAT vs 3D-CRT plans were not significantly different. CONCLUSIONS The interobserver agreement of PB delineation was moderate but was improved by the DR. Major deviations could be identified for the majority of centers. The DR has improved the acquaintance of the participating centers with the trial protocol.

Molecular analysis of the site for 2-arachidonylglycerol (2-AG) on the β 2 subunit of GABA A receptors

2-arachidonyl glycerol (2-AG) allosterically potentiates GABAA receptors via a binding site located in transmembrane segment M4 of the β2 subunit. Two amino acid residues have been described that are essential for this effect. With the aim to further describe this potential drug target, we performed a cysteine scanning of the entire M4 and part of M3. All four residues in M4 affecting the potentiation here and the two already identified residues locate to the same side of the α-helix. This side is exposed to M3, where further residues were identified. From the fact that the important residues span > 18 Å, we conclude that the hydrophobic tail of the bound 2-AG molecule must be near linear and that the site mainly locates to the inner leaflet but stretches far into the membrane. The influence of the structure of the head group of the ligand molecule on the activity of the molecule was also investigated. We present a model of 2-AG docked to the GABAA receptor.

Synthesis and Characterization of Photoaffinity Probes that Target the 5-HT3 Receptor

Abstract: The 5-HT3 receptor is one of several ion channels responsible for the transmission of nerve impulses in the peripheral and central nervous systems. Until now, it has been difficult to characterize transmembrane receptors with classical structural biology approaches like X-ray crystallography. The use of photoaffinity probes is an alternative approach to identify regions in the protein where small molecules bind. To this end, we present two photoaffinity probes based on granisetron, a well known antagonist of the 5-HT3 receptor. These new probes show nanomolar binding affinity for the orthosteric binding site. In addition, we investigated their reactivity using irradiation experiments.

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules (1). Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii (2). The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production (1). (1) Gebetsberger J. and Polacek N. (2013), RNA Biol. 10:1798-1808 (2) Gebetsberger J. et. al. (2012), Archaea, Article ID 260909

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [1].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-protein-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.