Conformational adaptation of UNCG loops upon crowding.

If the A-form helix is the major structural motif found in RNA, the loops that cap them constitute the second most important family of motifs. Among those, two are overrepresented, GNRA and UNCG tetraloops. Recent surveys of RNA structures deposited in the PDB show that GNRA and UNCG tetraloops can adopt tertiary folds that are very different from their canonical conformations, characterized by the presence of a U-turn of a Z-turn, respectively. Crystallographic data from both a lariat-capping (LC) ribozyme and a group II intron ribozyme reveal that a given UUCG tetraloop can adopt a distinct fold depending on its structural environment. Specifically, when the crystal packing applies relaxed constraints on the loop, the canonical Z-turn conformation is observed. In contrast, a highly packed environment induces "squashing" of the tetraloop by distorting its sugar-phosphate backbone in a specific way that expels the first and fourth nucleobases out of the loop, and falls in van der Waals distance of the last base pair of the helix, taking the place of the pair formed between the first and fourth residues in Z-turn loops. The biological relevance of our observations is supported by the presence of similarly deformed loops in the highly packed environment of the ribosome and in a complex between a dsRNA and a RNase III. The finding that Z-turn loops change conformation under higher molecular packing suggests that, in addition to their demonstrated role in stabilizing RNA folding, they may contribute to the three-dimensional structure of RNA by mediating tertiary interactions with distal residues.

3-Hydroxyisobutyrate dehydrogenase (HIBADH) deficiency-A novel disorder of valine metabolism.

3-Hydroxyisobutyric acid (3HiB) is an intermediate in the degradation of the branched-chain amino acid valine. Disorders in valine degradation can lead to 3HiB accumulation and its excretion in the urine. This article describes the first two patients with a new metabolic disorder, 3-hydroxyisobutyrate dehydrogenase (HIBADH) deficiency, its phenotype and its treatment with a low-valine diet. The detected mutation in the HIBADH gene leads to nonsense-mediated mRNA decay of the mutant allele and to a complete loss-of-function of the enzyme. Under strict adherence to a low-valine diet a rapid decrease of 3HiB excretion in the urine was observed. Due to limited patient numbers and intrafamilial differences in phenotype with one affected and one unaffected individual, the clinical phenotype of HIBADH deficiency needs further evaluation.

Understanding the role of intermolecular interactions between lissoclimides and the eukaryotic ribosome.

Natural products that target the eukaryotic ribosome are promising therapeutics to treat a variety of cancers. It is therefore essential to determine their molecular mechanism of action to fully understand their mode of interaction with the target and to inform the development of new synthetic compounds with improved potency and reduced cytotoxicity. Toward this goal, we have previously established a short synthesis pathway that grants access to multiple congeners of the lissoclimide family. Here we present the X-ray co-crystal structure at 3.1 Å resolution of C45, a potent congener with two A-ring chlorine-bearing stereogenic centers with 'unnatural' configurations, with the yeast 80S ribosome, intermolecular interaction energies of the C45/ribosome complex, and single-molecule FRET data quantifying the impact of C45 on both human and yeast ribosomes. Together, these data provide new insights into the role of unusual non-covalent halogen bonding interactions involved in the binding of this synthetic compound to the 80S ribosome.

Use of Different Process Gases for Manufacturing Isolating Alumina Coatings by Flame Spraying with Cords.

Besides conventional industrial demands, thermally sprayed coatings are increasingly used for innovative products. Such an application is the additive manufacturing of electrical components in automotive engineering. In particular, heating units are currently manufactured by a combination of various spray technologies. At present, simpler spraying processes like flame spraying are investigated with regard to their suitability as a future cost-effective alternative for fabricating isolating alumina coatings. In the present study, alumina cords were flame-sprayed using compressed air and argon as atomizing gases. The results demonstrate finely dispersed microstructures and a more regular and partially even higher surface and volume resistivity compared to past investigations in the literature as well as conventionally plasma-sprayed coatings despite a significantly reduced coating thickness. The content of alpha phase is clearly higher than for plasma-sprayed coatings, regardless of the atomizing gas used. Moreover, flame-sprayed coatings using argon reveal a higher resistivity in comparison to coatings sprayed with air. While the atomizing gas is found to mainly influence the ideal stand-off distance, the phase composition is not changed severely. In addition to the phase composition and kinematics, it can finally be concluded that humidity plays a major role in the coating properties.

Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Effectiveness of a Patient Education Class to Enhance Knowledge about Lung Cancer Screening: a Quality Improvement Evaluation.

Best practices to facilitate high-quality shared decision-making for lung cancer screening (LCS) are not well established. In our LCS program, patients are first referred to attend a free group education class on LCS, taught by designated clinician specialists, before a personal shared decision-making visit is scheduled. We conducted an evaluation on  the effectiveness of this class to enhance patient knowledge and shared decision-making about LCS. For quality improvement purposes, participants were asked to complete one-page surveys immediately before and after class to assess knowledge and decision-making capacity regarding LCS. To evaluate knowledge gained, we tabulated the distributions of correct, incorrect, unsure, and missing responses to eight true-false statements included on both pre- and post-class surveys and assessed pre-post differences in the number of correct responses. To evaluate decision-making capacity, we tabulated the distributions of post-class responses to items on decision uncertainty. From June 2017 to August 2018, 680 participants completed both pre- and post-class surveys. Participants had generally poor baseline knowledge about LCS. The proportion who responded correctly to each knowledge-related statement increased pre- to post-class, with a mean difference of 0.9 (paired t test, p < 0.0001) in the total number of correct responses between surveys. About 70% reported having all the information needed to make a screening decision. Our results suggest that a well-designed group education class is an effective system-level approach for initially educating and equipping patients with appropriate knowledge to make informed decisions about LCS.

Competencies required for healthcare improvement positions.

PURPOSE: The purpose of this paper is to examine the competencies that US healthcare organizations require for quality and performance improvement positions. DESIGN/METHODOLOGY/APPROACH: A US healthcare improvement job posting content analysis was conducted using the HQ Essentials competency framework. FINDINGS: The HQ essentials competencies most desired for improvement positions include project management, training, data analysis and applied performance improvement methods. Competency requirements varied somewhat by job focus area: performance, quality, or process improvement, and Lean and Six Sigma. PRACTICAL IMPLICATIONS: Healthcare leaders may use the author's results to understand what competencies may be required for various improvement roles and to identify any gaps in required skills and knowledge areas that may need to be addressed. Educators and policy-makers should consider how these competencies align with employers' needs and what resources or professional development may be needed to address gaps. ORIGINALITY/VALUE: This is the first healthcare improvement competencies analysis based on job postings.

Qualifications and Skills Required for Performance Improvement: A Content Analysis of Job Postings.

EXECUTIVE SUMMARY: Healthcare reform and the implementation of a national quality strategy in the United States have increased emphasis on quality improvement. Additionally, as healthcare organizations focus on value, they are paying more attention to quality in the context of cost and seeking to address these concerns through various performance improvement initiatives. Given the importance of these initiatives, this study analyzed the improvement job positions posted by U.S. healthcare organizations, specifically with respect to the qualifications and skills required. The author conducted a content analysis of improvement job postings using an inductive approach to capture and categorize core information about each posting and a deductive approach to evaluate skills required for selected job areas. The results show that healthcare organizations have invested in a breadth of improvement positions across all levels of the organization. Many positions are tied to strategic initiatives. The greatest number of positions posted were in the performance improvement area, followed by quality improvement, process improvement, Lean, Six Sigma, and Lean Six Sigma. These research results may be useful to healthcare organizations for strategic resource planning purposes and to educators and professional associations to target programs that offer students and members the opportunity to gain needed qualifications and skills.

RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures.

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.

Speciation of a group I intron into a lariat capping ribozyme.

The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms. Despite apparent structural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2',5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes for a conventional mRNA cap in the downstream pre-mRNA encoding a homing endonuclease. We describe the crystal structures of the precleavage and postcleavage LC ribozymes, which suggest that structural features inherited from group I ribozymes have undergone speciation due to profound changes in molecular selection pressure, ultimately giving rise to an original branching ribozyme family. The structures elucidate the role of key elements that regulate the activity of the LC ribozyme by conformational switching and suggest a mechanism by which the signal for branching is transmitted to the catalytic core. The structures also show how conserved interactions twist residues, forming the lariat to join chemical groups involved in branching.

Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors.

Dabigatran and rivaroxaban are novel, vitamin K-independent oral anticoagulants (NOACs) and act via antagonism of the coagulation factor (F) IIa (dabigatran) or FXa (rivaroxaban), respectively. Compared to vitamin-K-antagonists, NOACs have shown non-inferiority of risk and benefit in patients with non valvular atrial fibrillation (AF). In clinical practice there is increasing use of NOACs combined with platelet inhibitors in patients with AF and coronary artery disease. However, whether NOACs affect the function of platelet inhibitors remains incompletely known. This observational study aimed to assess the platelet function in patients receiving dabigatran or rivaroxaban and concomitant platelet inhibitors. A single centre observational study was performed analysing the platelet aggregation of patients treated with dabigatran or rivaroxaban with or without concomitant platelet inhibitors. Measurements before the initiation of NOAC therapy served as the respective control group. Platelet aggregation was measured by multiple electrode aggregometry and was induced with adenosine diphosphate (ADP, 6.5 µM) and arachidonic acid (AA, 0.5 mM), respectively. In order to evaluate whether NOACs interact with platelet inhibition by ASA or the P2Y12-antagonist clopidogrel, 87 patients were grouped according to their concomitant antiplatelet medication. Comparing the ADP- and AA-induced platelet aggregation in patients without concomitant platelet inhibitors (n = 45) no significant differences under therapy with dabigatran (d) or rivaroxaban (r) compared to the control group (c) were observed. In patients taking clopidogrel as a concomitant platelet inhibitor (n = 21), neither dabigatran nor rivaroxaban affected the ADP-induced platelet aggregation (c 20 ± 11, d 21 ± 14, r 18 ± 8 AU*min, p = 0.200). Patients receiving dabigatran or rivaroxaban in combination with ASA (n = 42; 21 ASA only, 21 ASA + clopidogrel) showed no significant differences of the AA-induced aggregation compared to the control group (c 10 ± 8, d 9 ± 7, r 10 ± 8 AU*min, p = 0.810). The antiplatelet effects of ASA and clopidogrel monitored by AA- or ADP-induced platelet aggregation were not affected by NOAC therapy.

High platelet reactivity after P2Y12-inhibition in patients with atrial fibrillation and coronary stenting.

High platelet reactivity (HPR) after P2Y12-inhibition in patients undergoing coronary stenting is associated with an increased risk for thromboembolic events and coronary death. So far it is not known how HPR affects the clinical outcome of different treatment strategies in patients with atrial fibrillation (AF) undergoing coronary stenting. In this single centre, observational study the antiplatelet effect of P2Y12-inhibitors in AF patients undergoing coronary stenting was investigated using impedance aggregometry. Patients received either dual antiplatelet therapy (DAPT) or triple therapy (TT). HPR was defined as the ratio of ADP-to TRAP-induced aggregation (r-ADP-agg) ≥50 %. Thromboembolic and bleeding events were assessed within the first 30 days after stenting. Out of 910 screened patients 167 patients were available for the present analysis. HPR was found in 5 of 43 (12 %) patients treated with DAPT and in 18 of 124 (15 %) patients treated with TT. In patients receiving TT, HPR was not a risk factor for thromboembolic events compared to patients with adequate response to P2Y12-inhibitors (6 vs. 8 %, p = 0.712). There was a trend for less bleeding events in patients with HPR compared to r-ADP-agg <50 % in the TT group (0 vs. 16 %, p = 0.077). Our data suggest that HPR after P2Y12-antagonism in patients receiving TT due to AF and coronary stenting might protect from bleeding without increasing thromboembolic risk. Future studies will need to investigate if patients with AF receiving coronary stenting benefit from a reduction of antithrombotic therapy.

Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides.

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.

Modeling disease mutations by gene targeting in one-cell mouse embryos.

Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool.

CLN3 deficiency leads to neurological and metabolic perturbations during early development.

Juvenile neuronal ceroid lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 yr and is most commonly caused by mutations in the highly conserved CLN3 gene. Here, we generated cln3 morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant cln3 larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and cholesteryl esters, and a global decrease in bis(monoacylglycero)phosphate species, two of which (GPDs and bis(monoacylglycero)phosphates) were previously proposed as potential biomarkers for CLN3 disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for CLN3 Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic CLN3 disease.

Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases.

Gene targeting by homologous recombination in embryonic stem cells is extensively used to generate specific mouse mutants. However, most mammalian species lack tools for targeted gene manipulation. Since double-strand breaks strongly increase the rate of homologous recombination at genomic loci, we explored whether gene targeting can be directly performed in zygotes by the use of zinc-finger nucleases. Here we report that gene targeting is achieved in 1.7-4.5% of murine one-cell embryos upon the coinjection of targeting vectors with zinc-finger nucleases, without preselection. These findings enable the manipulation of the mammalian germ line in a single step in zygotes, independent of ES cells.

A Patient's Journey to Pay a Healthcare Bill: It's Way Too Complicated.

The patient's financial experience is often complex and confusing.Recent regulations support bringing more transparency to healthcare billing processes.A patient journey map illustrates the patient experience in a healthcare process helping to identify improvement opportunities to better support patient-centered care.Price transparency can be increased by communicating the patient's financial responsibility early in the care process as well as simplifying patient communications.Digital health applications can help improve the patient financial experience and ultimately engagement in the payment process.Greater clarity and improved processes may occur if the patient's journey is visualized.

A structural module in RNase P expands the variety of RNA kinks.

RNA structures are built from recurrent modules that can be identified by structural and comparative sequence analysis. In order to assemble sets of helices in compact architectures, modules that introduce bends and kinks are necessary. Among such modules, kink-turns form an important family that presents sequence and structural characteristics. Here, we describe an internal loop in the bacterial type A RNase P RNA that sets helices bound at the junctions exactly in the same relative positions as in kink-turns but without the structural signatures typical of kink-turns. Our work suggests that identifying a structural module in a subset of RNA sequences constitutes a strategy to identify distinct sequential motifs sharing common structural characteristics.