El SHAPE de doblamiento de tRNA y del extremo 5' del genoma VIH-1

Desarrolla un método rápido y exacto para determinar la estructura secundaria del ARN y usarlo para entender el doblamiento de ARN y del extremo 5' del genoma de VIH-1. ARN se dobla en estructuras complejas y difíciles de predecir; durante el proceso de doblamiento algunos nucleótidos se hacen flexibles. En el presente trabajo se identificó una molécula que modifica nucleótidos flexibles. Esta reacción fue ligada a extensión con cebadores y este proceso se llama SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension). Se utilizó SHAPE para demostrar que el doblamiento de ARNt involucra interacciones no jerárquicas. Además, se usó SHAPE para observar el genoma de VIH-1 en cuatro estados diferentes.Most RNAs function only once they fold to form difficult-to-predict base-paired helices and other structural elements. As an RNA forms a preferred secondary or tertiary structure, a characteristic set of nucleotides becomes constrained by base pairing and higher-order interactions, while unconconstrained positions remain flexible. Determining local nucleotide flexibility as a function of nucleotide position in a folded RNA provides important information that enables the sequence and structure of an RNA to be related to its biological function. I have developed a technology, termed selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), that can be used to interrogate RNA structure under diverse in vitro and in vivo conditions. SHAPE chemistry can be applied to monitor protein binding events and locate promising sites for primer annealing in arbitrary RNA. SHAPE chemistry is based on the discovery that flexible RNA nucleotides preferentially sample conformations that enhance the nucleophilic reactivity of the 2'-hydroxyl group toward electrophiles, such as N-methylisatoic anhydride. Modified sites are detected as stops in an optimized DNA primer extension reaction, followed by sizing of the extension products. SHAPE chemistry scores local flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained residues with a dynamic range of 20-fold or greater. I have applied SHAPE chemistry to observe equilibrium melting of a model tRNA at single nucleotide resolution. I observed that RNA folding is a complex process involving structural rearrangement and the formation of tertiary structure concurrent with secondary structure. Furthermore, I have employed capillary electrophoresis and sophisticated analysis algorithms to create a high-throughput SHAPE (hSHAPE) experiment that can comprehensively interrogate the flexibility of several hundred nucleotides in a single robust experiment. Using hSHAPE, I analyzed the structure of HIV-1 genomic RNA as a function of 4 different biologically relevant states, including infectious viral particles. Despite many thermodynamically plausible structures, the HIV-1 genome exists in a single conformation. I observed the effects of tRNA primer binding, and the effects of nucleocapsid protein on RNA flexibility. hSHAPE chemistry is a promising, scalable approach that can rapidly and accurately analyze the structure of RNA molecules under biologically relevant conditions.Estados Unidos. University of North Carolina at Chapel Hill: Graduate Student FellowshipTesi

Diagnostic importance of pulmonary interleukin-1beta and interleukin-8 in ventilator-associated pneumonia.

BACKGROUND: Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP. METHODS: A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers. Growth of pathogens at >10(4) colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from "non-VAP". Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves. RESULTS: Seventy-two patients had recoverable lavage-24% had VAP. BALF interleukin-1beta (IL-1beta), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1alpha were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1beta generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1beta <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <10(4) cfu/ml. CONCLUSIONS: BALF IL-1beta and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations

Trial of Dexamethasone for Chronic Subdural Hematoma

BACKGROUND: Chronic subdural hematoma is a common neurologic disorder that is especially prevalent among older people. The effect of dexamethasone on outcomes in patients with chronic subdural hematoma has not been well studied. METHODS: We conducted a multicenter, randomized trial in the United Kingdom that enrolled adult patients with symptomatic chronic subdural hematoma. The patients were assigned in a 1:1 ratio to receive a 2-week tapering course of oral dexamethasone, starting at 8 mg twice daily, or placebo. The decision to surgically evacuate the hematoma was made by the treating clinician. The primary outcome was a score of 0 to 3, representing a favorable outcome, on the modified Rankin scale at 6 months after randomization; scores range from 0 (no symptoms) to 6 (death). RESULTS: From August 2015 through November 2019, a total of 748 patients were included in the trial after randomization - 375 were assigned to the dexamethasone group and 373 to the placebo group. The mean age of the patients was 74 years, and 94% underwent surgery to evacuate their hematomas during the index admission; 60% in both groups had a score of 1 to 3 on the modified Rankin scale at admission. In a modified intention-to-treat analysis that excluded the patients who withdrew consent for participation in the trial or who were lost to follow-up, leaving a total of 680 patients, a favorable outcome was reported in 286 of 341 patients (83.9%) in the dexamethasone group and in 306 of 339 patients (90.3%) in the placebo group (difference, -6.4 percentage points [95% confidence interval, -11.4 to -1.4] in favor of the placebo group; P = 0.01). Among the patients with available data, repeat surgery for recurrence of the hematoma was performed in 6 of 349 patients (1.7%) in the dexamethasone group and in 25 of 350 patients (7.1%) in the placebo group. More adverse events occurred in the dexamethasone group than in the placebo group. CONCLUSIONS: Among adults with symptomatic chronic subdural hematoma, most of whom had undergone surgery to remove their hematomas during the index admission, treatment with dexamethasone resulted in fewer favorable outcomes and more adverse events than placebo at 6 months, but fewer repeat operations were performed in the dexamethasone group. (Funded by the National Institute for Health Research Health Technology Assessment Programme; Dex-CSDH ISRCTN number, ISRCTN80782810.)

Imaging breast cancer using hyperpolarized carbon-13 MRI.

Our purpose is to investigate the feasibility of imaging tumor metabolism in breast cancer patients using 13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13C label exchange between injected [1-13C]pyruvate and the endogenous tumor lactate pool. Treatment-naïve breast cancer patients were recruited: four triple-negative grade 3 cancers; two invasive ductal carcinomas that were estrogen and progesterone receptor-positive (ER/PR+) and HER2/neu-negative (HER2-), one grade 2 and one grade 3; and one grade 2 ER/PR+ HER2- invasive lobular carcinoma (ILC). Dynamic 13C MRSI was performed following injection of hyperpolarized [1-13C]pyruvate. Expression of lactate dehydrogenase A (LDHA), which catalyzes 13C label exchange between pyruvate and lactate, hypoxia-inducible factor-1 (HIF1α), and the monocarboxylate transporters MCT1 and MCT4 were quantified using immunohistochemistry and RNA sequencing. We have demonstrated the feasibility and safety of hyperpolarized 13C MRI in early breast cancer. Both intertumoral and intratumoral heterogeneity of the hyperpolarized pyruvate and lactate signals were observed. The lactate-to-pyruvate signal ratio (LAC/PYR) ranged from 0.021 to 0.473 across the tumor subtypes (mean ± SD: 0.145 ± 0.164), and a lactate signal was observed in all of the grade 3 tumors. The LAC/PYR was significantly correlated with tumor volume (R = 0.903, P = 0.005) and MCT 1 (R = 0.85, P = 0.032) and HIF1α expression (R = 0.83, P = 0.043). Imaging of hyperpolarized [1-13C]pyruvate metabolism in breast cancer is feasible and demonstrated significant intertumoral and intratumoral metabolic heterogeneity, where lactate labeling correlated with MCT1 expression and hypoxia

Trial of Dexamethasone for Chronic Subdural Hematoma

(Trial funded by NIHR, Dex-CSDH Current Controlled Trials number ISRCTN80782810). ACKNOWLEDGEMENTS In memory of Mrs. Kate Massey, who was the patient representative involved in study design. Peter Hutchinson is supported by a Research Professorship and Senior Investigator Award from the NIHR, the NIHR Cambridge Biomedical Research Centre, and the Royal College of Surgeons of England. Ellie Edlmann is supported by the Royal College of Surgeons of England. Angelos Kolias is supported by a Lectureship, School of Clinical Medicine, University of Cambridge and the Royal College of Surgeons of England. SUPPORT This paper presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.Peer reviewedPublisher PD

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Functional antibody and T-cell immunity following SARS-CoV-2 infection, including by variants of concern, in patients with cancer: the CAPTURE study

Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study (NCT03226886) integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2-positive, 94 were symptomatic and 2 patients died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies, 82% had neutralizing antibodies against WT, whereas neutralizing antibody titers (NAbT) against the Alpha, Beta, and Delta variants were substantially reduced. Whereas S1-reactive antibody levels decreased in 13% of patients, NAbT remained stable up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4+ responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment-specific, but presented compensatory cellular responses, further supported by clinical. Overall, these findings advance the understanding of the nature and duration of immune response to SARS-CoV-2 in patients with cancer

The Generation Challenge Programme Platform: Semantic Standards and Workbench for Crop Science

The Generation Challenge programme (GCP) is a global crop research consortium directed toward crop improvement through the application of comparative biology and genetic resources characterization to plant breeding. A key consortium research activity is the development of a GCP crop bioinformatics platform to support GCP research. This platform includes the following: (i) shared, public platform-independent domain models, ontology, and data formats to enable interoperability of data and analysis flows within the platform; (ii) web service and registry technologies to identify, share, and integrate information across diverse, globally dispersed data sources, as well as to access high-performance computational (HPC) facilities for computationally intensive, high-throughput analyses of project data; (iii) platform-specific middleware reference implementations of the domain model integrating a suite of public (largely open-access/-source) databases and software tools into a workbench to facilitate biodiversity analysis, comparative analysis of crop genomic data, and plant breeding decision making