Structure, Dynamics and Functional Implications of the Eukaryotic Vault Complex.

Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.

High-Flow Oxygen and Other Noninvasive Respiratory Support Therapies in Bronchiolitis: Systematic Review and Network Meta-Analyses.

OBJECTIVES: We present a systematic review on the effectiveness of noninvasive respiratory support techniques in bronchiolitis. DATA SOURCES: Systematic review with pairwise meta-analyses of all studies and network meta-analyses of the clinical trials. STUDY SELECTION: Patients below 24 months old with bronchiolitis who require noninvasive respiratory support were included in randomized controlled trials (RCTs), non-RCT, and cohort studies in which high-flow nasal cannula (HFNC) was compared with conventional low-flow oxygen therapy (LFOT) and/or noninvasive ventilation (NIV). DATA EXTRACTION: Emergency wards and hospitalized patients with bronchiolitis. DATA SYNTHESIS: A total of 3,367 patients were analyzed in 14 RCTs and 8,385 patients in 14 non-RCTs studies. Only in nonexperimental studies, HFNC is associated with a lower risk of invasive mechanical ventilation (MV) than NIV (odds ratio, 0.49; 95% CI, 0.42-0.58), with no differences in experimental studies. There were no differences between HFNC and NIV in other outcomes. HFNC is more effective than LFOT in reducing oxygen days and treatment failure. In the network meta-analyses of clinical trials, NIV was the most effective intervention to avoid invasive MV (surface under the cumulative ranking curve [SUCRA], 57.03%) and to reduce days under oxygen therapy (SUCRA, 79.42%), although crossover effect estimates between interventions showed no significant differences. The included studies show methodological heterogeneity, but it is only statistically significant for the reduction of days of oxygen therapy and length of hospital stay. CONCLUSIONS: Experimental evidence does not suggest that high-flow oxygen therapy has advantages over LFOT as initial treatment nor over NIV as a rescue treatment.

Structure and dsRNA-binding activity of the Birnavirus Drosophila X Virus VP3 protein.

The Birnavirus multifunctional protein VP3 plays an essential role coordinating the virus life cycle, interacting with the capsid protein VP2, with the RNA-dependent RNA polymerase VP1 and with the dsRNA genome. Furthermore, the role of this protein in controlling host cell responses triggered by dsRNA and preventing gene silencing has been recently demonstrated. Here we report the X-ray structure and dsRNA-binding activity of the N-terminal domain of Drosophila X virus (DXV) VP3. The domain folds in a bundle of three α-helices and arranges as a dimer, exposing to the surface a well-defined cluster of basic residues. Site directed mutagenesis combined with Electrophoretic Mobility Shift Assays (EMSA) and Surface Plasmon Resonance (SPR) revealed that this cluster, as well as a flexible and positively charged region linking the first and second globular domains of DXV VP3, are essential for dsRNA-binding. Also, RNA silencing studies performed in insect cell cultures confirmed the crucial role of this VP3 domain for the silencing suppression activity of the protein.IMPORTANCE The Birnavirus moonlighting protein VP3 plays crucial roles interacting with the dsRNA genome segments to form stable ribonucleoprotein complexes and controlling host cell immune responses, presumably by binding to and shielding the dsRNA from recognition by the host silencing machinery. The structural, biophysical and functional data presented in this work has identified the N-terminal domain of VP3 as responsible for the dsRNA-binding and silencing suppression activities of the protein in Drosophila X virus.

Outbreak of Enterovirus Infection with Neurological Presentations in a Pediatric Population in Northern Spain: A Clinical Observational Study.

OBJECTIVE: The study aimed to describe the cases of neurological disease related to the outbreak of enterovirus (EV) in three regions in Northern Spain during 2016. MATERIALS AND METHODS: Multicenter retrospective observational study. Clinical, radiological, and microbiological data were analyzed from patients younger than 15 years with confirmed EV-associated neurological disease admitted to 10 hospitals of Asturias, Cantabria, and Castile and Leon between January 1 and December 31, 2016. RESULTS: Fifty-five patients were included. Median age was 24 months (interquartile range = 18.5 months). Fifteen patients were classified as aseptic meningitis (27.3%). In total, 37 cases presented brainstem encephalitis (67.3%), 25 of them due to EV-A71 with excellent prognosis (84.6% asymptomatic 2 months following the onset). Three cases of acute flaccid myelitis (5.5%) by EV-D68 were reported and presented persistent paresis 2 months following the onset. Microbiological diagnosis by reverse transcriptase polymerase chain reaction was performed in all cases, finding EV in cerebrospinal fluid in meningitis, but not in brainstem encephalitis and acute flaccid myelitis, where EV was found in respiratory or rectal samples. Step therapy was administrated with intravenous immunoglobulin (IVIG; 32.7%), methylprednisolone (10%), and plasmapheresis (3.6%). Four patients received fluoxetine (7.3%). Twenty patients needed to be admitted to pediatric intensive care unit (36.4%). CONCLUSION: Clinical, microbiological, and radiological diagnosis is essential in outbreaks of EV neurological disease, taking into account that it can be difficult to identify EV-A71 and EV-D68 in CSF, requiring throat or rectal samples. There is not specific treatment to these conditions and the efficacy and understanding of the mechanism of action of immune-modulatory treatment (IVIG, corticosteroids, and plasmapheresis) is limited.

Bayes to the Rescue: Continuous Positive Airway Pressure Has Less Mortality Than High-Flow Oxygen.

OBJECTIVES: The merits of high-flow nasal cannula oxygen versus bubble continuous positive airway pressure are debated in children with pneumonia, with suggestions that randomized controlled trials are needed. In light of a previous randomized controlled trial showing a trend for lower mortality with bubble continuous positive airway pressure, we sought to determine the probability that a new randomized controlled trial would find high-flow nasal cannula oxygen superior to bubble continuous positive airway pressure through a "robust" Bayesian analysis. DESING, SETTING, PATIENTS, AND INTERVENTIONS: Sample data were extracted from the trial by Chisti et al, and requisite to "robust" Bayesian analysis, we specified three prior distributions to represent clinically meaningful assumptions. These priors (reference, pessimistic, and optimistic) were used to generate three scenarios to represent the range of possible hypotheses. 1) "Reference": we believe bubble continuous positive airway pressure and high-flow nasal cannula oxygen are equally effective with the same uninformative reference priors; 2) "Sceptic on high-flow nasal cannula oxygen": we believe that bubble continuous positive airway pressure is better than high-flow nasal cannula oxygen (bubble continuous positive airway pressure has an optimistic prior and high-flow nasal cannula oxygen has a pessimistic prior); and 3) "Enthusiastic on high-flow nasal cannula oxygen": we believe that high-flow nasal cannula oxygen is better than bubble continuous positive airway pressure (high-flow nasal cannula oxygen has an optimistic prior and bubble continuous positive airway pressure has a pessimistic prior). Finally, posterior empiric Bayesian distributions were obtained through 100,000 Markov Chain Monte Carlo simulations. MEASUREMENTS AND MAIN RESULTS: In all three scenarios, there was a high probability for more death from high-flow nasal cannula oxygen compared with bubble continuous positive airway pressure (reference, 0.98; sceptic on high-flow nasal cannula oxygen, 0.982; enthusiastic on high-flow nasal cannula oxygen, 0.742). The posterior 95% credible interval on the difference in mortality identified a future randomized controlled trial would be extremely unlikely to find a mortality benefit for high-flow nasal cannula oxygen over bubble continuous positive airway pressure, regardless of the scenario. Interpreting these findings using the "range of practical equivalence" framework would recommend rejecting the hypothesis that high-flow nasal cannula oxygen is superior to bubble continuous positive airway pressure for these children. CONCLUSIONS: For children younger than 5 years with pneumonia, high-flow nasal cannula oxygen has higher mortality than bubble continuous positive airway pressure. A future randomized controlled trial in this population is unlikely to find high-flow nasal cannula oxygen superior to bubble continuous positive airway pressure.

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1.

Proteins containing C2 domains are the sensors for Ca(2+) and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca(2+) binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands ß3-ß4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca(2+)-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.

Mechanical stability and reversible fracture of vault particles.

Vaults are the largest ribonucleoprotein particles found in eukaryotic cells, with an unclear cellular function and promising applications as vehicles for drug delivery. In this article, we examine the local stiffness of individual vaults and probe their structural stability with atomic force microscopy under physiological conditions. Our data show that the barrel, the central part of the vault, governs both the stiffness and mechanical strength of these particles. In addition, we induce single-protein fractures in the barrel shell and monitor their temporal evolution. Our high-resolution atomic force microscopy topographies show that these fractures occur along the contacts between two major vault proteins and disappear over time. This unprecedented systematic self-healing mechanism, which enables these particles to reversibly adapt to certain geometric constraints, might help vaults safely pass through the nuclear pore complex and potentiate their role as self-reparable nanocontainers.

A novel benzonitrile analogue inhibits rhinovirus replication.

OBJECTIVES: To study the characteristics and the mode of action of the anti-rhinovirus compound 4-[1-hydroxy-2-(4,5-dimethoxy-2-nitrophenyl)ethyl]benzonitrile (LPCRW_0005). METHODS: The antiviral activity of LPCRW_0005 was evaluated in a cytopathic effect reduction assay against a panel of human rhinovirus (HRV) strains. To unravel its precise molecular mechanism of action, a time-of-drug-addition study, resistance selection and thermostability assays were performed. The crystal structure of the HRV14/LPCRW_0005 complex was elucidated as well. RESULTS: LPCRW_0005 proved to be a selective inhibitor of the replication of HRV14 (EC(50) of 2 ±â€Š1 µM). Time-of-drug-addition studies revealed that LPCRW_0005 interferes with the earliest stages of virus replication. Phenotypic drug-resistant virus variants were obtained (≥30-fold decrease in susceptibility to the inhibitory effect of LPCRW_0005), which carried either an A150T or A150V amino acid substitution in the VP1 capsid protein. The link between the mutant genotype and drug-resistant phenotype was confirmed by reverse genetics. Cross-resistance studies and thermostability assays revealed that LPCRW_0005 has a similar mechanism of action to the capsid binder pleconaril. Elucidation of the crystal structure of the HRV14/LPCRW_0005 complex revealed the existence of multiple hydrophobic and polar interactions between the VP1 pocket and LPCRW_0005. CONCLUSIONS: LPCRW_0005 is a novel inhibitor of HRV14 replication that acts as a capsid binder. The compound has a chemical structure that is markedly smaller than that of other capsid binders. Structural studies show that LPCRW_0005, in contrast to pleconaril, leaves the toe end of the pocket in VP1 empty. This suggests that extended analogues of LPCRW_0005 that fill the full cavity could be more potent inhibitors of rhinovirus replication.

Transition of human γ-tubulin ring complex into a closed conformation during microtubule nucleation.

Microtubules are essential for intracellular organization and chromosome segregation. They are nucleated by the γ-tubulin ring complex (γTuRC). However, isolated vertebrate γTuRC adopts an open conformation that deviates from the microtubule structure, raising the question of the nucleation mechanism. In this study, we determined cryo-electron microscopy structures of human γTuRC bound to a nascent microtubule. Structural changes of the complex into a closed conformation ensure that γTuRC templates the 13-protofilament microtubules that exist in human cells. Closure is mediated by a latch that interacts with incorporating tubulin, making it part of the closing mechanism. Further rearrangements involve all γTuRC subunits and the removal of the actin-containing luminal bridge. Our proposed mechanism of microtubule nucleation by human γTuRC relies on large-scale structural changes that are likely the target of regulation in cells.

Spontaneous pneumomediastinum: An extra muscular manifestation of anti-MDA5 dermatomyositis. Report of 2 cases.

The use of specific antibodies in inflammatory myopathies has improved the characterization of this disease, identifying different clinical phenotypes. Patients with dermatomyositis (DM) and anti-MDA5 antibodies display typical skin symptoms, lesser muscular involvement, and a prevalence of interstitial lung disease (ILD) of up to 91%. Beyond ILD, spontaneous pneumomediastinum (SN) has been identified as a rare but potentially fatal pulmonary manifestation. Two cases of this complication in patients with anti-MDA5 DM are reported.

New features of vault architecture and dynamics revealed by novel refinement using the deformable elastic network approach.

The vault particle, with a molecular weight of about 10 MDa, is the largest ribonucleoprotein that has been described. The X-ray structure of intact rat vault has been solved at a resolution of 3.5 Å [Tanaka et al. (2009), Science, 323, 384-388], showing an overall barrel-shaped architecture organized into two identical moieties, each consisting of 39 copies of the major vault protein (MVP). The model deposited in the PDB includes 39 MVP copies (half a vault) in the crystal asymmetric unit. A 2.1 Å resolution structure of the seven N-terminal repeats (R1-7) of MVP has also been determined [Querol-Audí et al. (2009), EMBO J. 28, 3450-3457], revealing important discrepancies with respect to the MVP models for repeats R1 and R2. Here, the re-refinement of the vault structure by incorporating the high-resolution information available for the R1-7 domains, using the deformable elastic network (DEN) approach and maintaining strict 39-fold noncrystallographic symmetry is reported. The new refinement indicates that at the resolution presently available the MVP shell can be described well as only one independent subunit organized with perfect D39 molecular symmetry. This refinement reveals that significant rearrangements occur in the N-terminus of MVP during the closing of the two vault halves and that the 39-fold symmetry breaks in the cap region. These results reflect the highly dynamic nature of the vault structure and represent a necessary step towards a better understanding of the biology and regulation of this particle.

Ortner's Syndrome (Cardiovocal Syndrome): A Case Report.

Ortner's syndrome, also known as cardiovocal syndrome, refers to vocal cord paralysis caused by an underlying cardiovascular condition. It is often due to the constriction of the left recurrent laryngeal nerve by the pulmonary artery or left atrium. Recurrent aspiration pneumonia is a frequent complication, which can result in substantial morbidity and mortality. Early recognition and treatment, as well as the resolution of the underlying cause, when feasible, can enhance the otherwise unfavorable prognosis of this condition. In this particular case, a 65-year-old man with idiopathic dilated cardiomyopathy was diagnosed with hoarseness and evidence of left vocal cord palsy.

Psychogenic cough: a diagnosis of exclusion.

Cough is the most common respiratory symptom and a common reason for consultation in both primary care and emergency departments, as a result of family concerns. We report an 11-year-old girl who complains of constant cough episode for 4 hours without any rest. After sequential treatment with nebulized salbutamol and budesonide, dexamethasone, codeine, and midazolam, the patient showed transient improvement, with cough disappearing altogether, but 10 minutes later, it started over with the same intensity from the beginning. When she got distracted and talked, the access decreased, starting again when the word "cough" or similar terms were mentioned in front of her. She remained asymptomatic for 2 hours, after which her symptoms began similarly to the initial, coinciding with taking 1 tablet of clarithromycin, so it was decided to start a continuous infusion of midazolam, with the cough disappearing completely after 15 minutes of starting the infusion. One hour after starting the infusion, the child fell asleep. At waking, the cough had disappeared. She continued treatment with oral codeine for 3 days, showing no relapse. It is important to include psychogenic cough in the differential diagnosis of persistent or recurrent chronic cough and asthma that is difficult to control and communicate that diagnostic criteria are based on indicative symptoms (cough access only when awake), with normal radiology, spirometry, and bronchoscopy, to avoid misdiagnosis and inadequate pharmacological actions. Successful treatment is based on recognizing the underlying cause and use of different forms of cognitive-behavioral therapies that aim to break the habit.

Pulsatile parallel flow of air and a viscoelastic fluid with multiple characteristic times. An application to mucus in the trachea and the frequency of cough.

We study the dynamics of a binary fluid, where the two fluids are flowing parallel to each other in a cylindrical geometry, and driven by a pulsatile pressure gradient. One of the fluids is a low viscosity Newtonian fluid, the other one is viscoelastic. In order to be able to apply the model to different biofluids, we consider that the viscoelastic fluid has several characteristic times. We characterize the dynamics of the fluids as generalized Darcy's laws, with linear response functions to pulsatile pressure gradients, whose parameters are coupled for both fluids through the fluid-fluid boundary conditions. We apply our results to the dynamics of mucus and air in the trachea and find that the frequency that allows for a larger movement of the mucus, coincides with the experimental frequency of cough. This allows us to propose a plausible explanation for the frequency of cough in healthy individuals, a mechanical process to expel noxious substances from the respiratory system.

Symmetry disruption commits vault particles to disassembly.

Vaults are ubiquitous ribonucleoprotein particles involved in a diversity of cellular processes, with promising applications as nanodevices for delivery of multiple cargos. The vault shell is assembled by the symmetrical association of multiple copies of the major vault protein that, initially, generates half vaults. The pairwise, anti-parallel association of two half vaults produces whole vaults. Here, using a combination of vault recombinant reconstitution and structural techniques, we characterized the molecular determinants for the vault opening process. This process commences with a relaxation of the vault waist, causing the expansion of the inner cavity. Then, local disengagement of amino-terminal domains at the vault midsection seeds a conformational change that leads to the aperture, facilitating access to the inner cavity where cargo is hosted. These results inform a hitherto uncharacterized step of the vault cycle and will aid current engineering efforts leveraging vault for tailored cargo delivery.

[CPAP vs oxygen therapy in infants being transported due to acute respiratory failure]. / CPAP vs oxigenoterapia convencional en lactantes trasladados por insuficiencia respiratoria.

OBJECTIVE: The aims of our study are to evaluate the effectiveness and security of CPAP (continuous positive airway pressure) in infants transferred with acute respiratory failure (ARF) and to compare their evolution in PICU between CPAP vs oxygen therapy. MATERIALS AND METHODS: We conducted a retrospective observational and analytical study by reviewing the health records of infants with ARF aged 0 to 12months that required interhospital transfer to the PICU. RESULTS: We included 110 patients: 71 transported with CPAP and 39 with oxygen therapy. The main cause of ARF was acute bronchiolitis (81.8%). The median level of CPAP was 7cmH2O (interquartile range, 6-7). Controlling by the previous values in specific multivariable models, CPAP produced a significant decrease in the Wood-Downes score (beta = -1.08; 95% CI = -1.76 to -0.40; P = .002) and the heart rate (beta = -19.64, 95% CI = -28.46 to -10.81; P < .001). No patients required endotracheal intubation during transport. During the PICU stay, the intubation rate was similar in the CPAP group (7%) and the oxygen therapy group (5.1%) (P=.689). The proportion of patients that required bilevel positive airway pressure within 6hours of admission to the PICU was higher in the oxygen therapy group: 100% (11/11) vs 69.2% (18/26), P=.04. CONCLUSIONS: Early administration of CPAP to infants with ARF was a safe respiratory support intervention during interhospital transport. During patient transport, the use of CPAP achieved greater decreases in the Wood-Downes score and heart rate compared to oxygen therapy.

Tetra-n-butyl-ammonium orotate monohydrate: knowledge-based comparison of the results of accurate and lower-resolution analyses and a non-routine disorder refinement.

The title hydrated mol-ecular salt (systematic name: tetra-n-butyl-ammonium 2,6-dioxo-1,2,3,6-tetra-hydro-pyrimidine-4-carboxyl-ate monohydrate), C16H36N+·C5H3N2O4 -·H2O, crystallizes with N-H⋯O and O-H⋯O hydrogen-bonded double-stranded anti-parallel ribbons consisting of the hydro-philic orotate monoanions and water mol-ecules, separated by the bulky hydro-phobic cations. The hydro-phobic and hydro-philic regions of the structure are joined by weaker non-classical C-H⋯O hydrogen bonds. An accurate structure analysis conducted at T = 100 K is compared to a lower-resolution less accurate determination using data measured at T = 295 K. The results of both analyses are evaluated using a knowledge-based approach, and it is found that the less accurate room-temperature structure analysis provides geometric data that are similar to those derived from the accurate low-temperature analysis, with both sets of results consistent with previously analyzed structures. A minor disorder of one methyl group in the cation at low temperature was found to be slightly more complex at room temperature; while still involving a minor fraction of the structure, the disorder at room temperature was found to require a non-routine treatment, which is described in detail.