342 research outputs found
Kinetic mechanism of ornithine hydroxylase (PvdA) from Pseudomonas aeruginosa: substrate triggering of O2 addition but not flavin reduction
This publication was made possible by NIH Grant P20 RR-17708-05 from the National Center for Research Resources of the National Institutes of Health. K.M.M. was a recipient of a National Institutes of Health Predoctoral Training Grant Fellowship (GM08545).PvdA catalyzes the hydroxylation of the sidechain primary amine of ornithine in the initial step of the biosynthesis of the Pseudomonas aeruginosa siderophore pyoverdin. The reaction requires FAD, NADPH, and O2. PvdA uses the same co-substrates as several flavin-dependent hydroxylases that differ one from another in the kinetic mechanisms of their oxidative and reductive half-reactions. Therefore, the mechanism of PvdA was determined by absorption stopped-flow experiments. By contrast to some flavin-dependent hydroxylases (notably, p-hydroxybenzoate hydroxylase), binding of the hydroxylation target is not required to trigger reduction of the flavin by NADPH: the reductive half-reaction is equally facile in the presence and absence of ornithine. Reaction of O2 with FADH2 in the oxidative half-reaction is accelerated by ornithine 80-fold, providing a mechanism by which PvdA can ensure coupling of NADPH and ornithine oxidation. In the presence of ornithine, the expected C(4a)-hydroperoxyflavin intermediate with 390-nm absorption accumulates and decays to the C(4a)-hydroxyflavin in a kinetically competent fashion. The slower oxidative half-reaction that occurs in the absence of ornithine involves accumulation of an oxygenated flavin species and two subsequent states that are tentatively assigned as C(4a)-peroxy- and -hydroperoxyflavin intermediates and the oxidized flavin. The enzyme generates stoichiometric hydrogen peroxide in lieu of hydroxyornithine. The data suggest that PvdA employs a kinetic mechanism that is a hybrid of those previously documented for other flavin-dependent hydroxylases
Lethal Mutagenesis of Picornaviruses with N-6-Modified Purine Nucleoside Analogues
RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis
Cluster-Randomized Trial of a Behavioral Intervention to Incorporate a Treat-to-Target Approach to Care of US Patients With Rheumatoid Arthritis
OBJECTIVE: To assess the feasibility and efficacy of implementing a treat-to-target approach versus usual care in a US-based cohort of rheumatoid arthritis patients.
METHODS: In this behavioral intervention trial, rheumatology practices were cluster-randomized to provide treat-to-target care or usual care. Eligible patients with moderate/high disease activity (Clinical Disease Activity Index [CDAI] score \u3e 10) were followed for 12 months. Both treat-to-target and usual care patients were seen every 3 months. Treat-to-target providers were to have monthly visits with treatment acceleration at a minimum of every 3 months in patients with CDAI score \u3e 10; additional visits and treatment acceleration were at the discretion of usual care providers and patients. Coprimary end points were feasibility, assessed by rate of treatment acceleration conditional on CDAI score \u3e 10, and achievement of low disease activity (LDA; CDAI score \u3c /=10) by an intent-to-treat analysis.
RESULTS: A total of 14 practice sites per study arm were included (246 patients receiving treat-to-target and 286 receiving usual care). The groups had similar baseline demographic and clinical characteristics. Rates of treatment acceleration (treat-to-target 47% versus usual care 50%; odds ratio [OR] 0.92 [95% confidence interval (95% CI) 0.64, 1.34]) and achievement of LDA (treat-to-target 57% versus usual care 55%; OR 1.05 [95% CI 0.60, 1.84]) were similar between groups. Treat-to-target providers reported patient reluctance and medication lag time as common barriers to treatment acceleration.
CONCLUSION: This study is the first to examine the feasibility and efficacy of a treat-to-target approach in typical US rheumatology practice. Treat-to-target care was not associated with increased likelihood of treatment acceleration or achievement of LDA, and barriers to treatment acceleration were identified
SeekFusion - A Clinically Validated Fusion Transcript Detection Pipeline for PCR-Based Next-Generation Sequencing of RNA
Detecting gene fusions involving driver oncogenes is pivotal in clinical diagnosis and treatment of cancer patients. Recent developments in next-generation sequencing (NGS) technologies have enabled improved assays for bioinformatics-based gene fusions detection. In clinical applications, where a small number of fusions are clinically actionable, targeted polymerase chain reaction (PCR)-based NGS chemistries, such as the QIAseq RNAscan assay, aim to improve accuracy compared to standard RNA sequencing. Existing informatics methods for gene fusion detection in NGS-based RNA sequencing assays traditionally use a transcriptome-based spliced alignment approach or a de-novo assembly approach. Transcriptome-based spliced alignment methods face challenges with short read mapping yielding low quality alignments. De-novo assembly-based methods yield longer contigs from short reads that can be more sensitive for genomic rearrangements, but face performance and scalability challenges. Consequently, there exists a need for a method to efficiently and accurately detect fusions in targeted PCR-based NGS chemistries. We describe SeekFusion, a highly accurate and computationally efficient pipeline enabling identification of gene fusions from PCR-based NGS chemistries. Utilizing biological samples processed with the QIAseq RNAscan assay and in-silico simulated data we demonstrate that SeekFusion gene fusion detection accuracy outperforms popular existing methods such as STAR-Fusion, TOPHAT-Fusion and JAFFA-hybrid. We also present results from 4,484 patient samples tested for neurological tumors and sarcoma, encompassing details on some novel fusions identified
'To live and die [for] Dixie': Irish civilians and the Confederate States of America
Around 20,000 Irishmen served in the Confederate army in the Civil War. As a result, they left behind, in various Southern towns and cities, large numbers of friends, family, and community leaders. As with native-born Confederates, Irish civilian support was crucial to Irish participation in the Confederate military effort. Also, Irish civilians served in various supporting roles: in factories and hospitals, on railroads and diplomatic missions, and as boosters for the cause. They also, however, suffered in bombardments, sieges, and the blockade. Usually poorer than their native neighbours, they could not afford to become 'refugees' and move away from the centres of conflict. This essay, based on research from manuscript collections, contemporary newspapers, British Consular records, and Federal military records, will examine the role of Irish civilians in the Confederacy, and assess the role this activity had on their integration into Southern communities. It will also look at Irish civilians in the defeat of the Confederacy, particularly when they came under Union occupation. Initial research shows that Irish civilians were not as upset as other whites in the South about Union victory. They welcomed a return to normalcy, and often 'collaborated' with Union authorities. Also, Irish desertion rates in the Confederate army were particularly high, and I will attempt to gauge whether Irish civilians played a role in this. All of the research in this paper will thus be put in the context of the Drew Gilpin Faust/Gary Gallagher debate on the influence of the Confederate homefront on military performance. By studying the Irish civilian experience one can assess how strong the Confederate national experiment was. Was it a nation without a nationalism
LSST Science Book, Version 2.0
A survey that can cover the sky in optical bands over wide fields to faint
magnitudes with a fast cadence will enable many of the exciting science
opportunities of the next decade. The Large Synoptic Survey Telescope (LSST)
will have an effective aperture of 6.7 meters and an imaging camera with field
of view of 9.6 deg^2, and will be devoted to a ten-year imaging survey over
20,000 deg^2 south of +15 deg. Each pointing will be imaged 2000 times with
fifteen second exposures in six broad bands from 0.35 to 1.1 microns, to a
total point-source depth of r~27.5. The LSST Science Book describes the basic
parameters of the LSST hardware, software, and observing plans. The book
discusses educational and outreach opportunities, then goes on to describe a
broad range of science that LSST will revolutionize: mapping the inner and
outer Solar System, stellar populations in the Milky Way and nearby galaxies,
the structure of the Milky Way disk and halo and other objects in the Local
Volume, transient and variable objects both at low and high redshift, and the
properties of normal and active galaxies at low and high redshift. It then
turns to far-field cosmological topics, exploring properties of supernovae to
z~1, strong and weak lensing, the large-scale distribution of galaxies and
baryon oscillations, and how these different probes may be combined to
constrain cosmological models and the physics of dark energy.Comment: 596 pages. Also available at full resolution at
http://www.lsst.org/lsst/sciboo
LSST: from Science Drivers to Reference Design and Anticipated Data Products
(Abridged) We describe here the most ambitious survey currently planned in
the optical, the Large Synoptic Survey Telescope (LSST). A vast array of
science will be enabled by a single wide-deep-fast sky survey, and LSST will
have unique survey capability in the faint time domain. The LSST design is
driven by four main science themes: probing dark energy and dark matter, taking
an inventory of the Solar System, exploring the transient optical sky, and
mapping the Milky Way. LSST will be a wide-field ground-based system sited at
Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m
effective) primary mirror, a 9.6 deg field of view, and a 3.2 Gigapixel
camera. The standard observing sequence will consist of pairs of 15-second
exposures in a given field, with two such visits in each pointing in a given
night. With these repeats, the LSST system is capable of imaging about 10,000
square degrees of sky in a single filter in three nights. The typical 5
point-source depth in a single visit in will be (AB). The
project is in the construction phase and will begin regular survey operations
by 2022. The survey area will be contained within 30,000 deg with
, and will be imaged multiple times in six bands, ,
covering the wavelength range 320--1050 nm. About 90\% of the observing time
will be devoted to a deep-wide-fast survey mode which will uniformly observe a
18,000 deg region about 800 times (summed over all six bands) during the
anticipated 10 years of operations, and yield a coadded map to . The
remaining 10\% of the observing time will be allocated to projects such as a
Very Deep and Fast time domain survey. The goal is to make LSST data products,
including a relational database of about 32 trillion observations of 40 billion
objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures
available from https://www.lsst.org/overvie
Association between depressive symptoms and incident cardiovascular diseases
Importance: It is uncertain whether depressive symptoms are independently associated with subsequent risk of cardiovascular diseases (CVD).
Objective: To characterize the association between depressive symptoms and CVD incidence across the spectrum of lower mood.
Design, setting and participants: A pooled analysis of individual-participant-data from the Emerging Risk Factors Collaboration (ERFC; 162,036 participants; 21 cohorts; baseline surveys, 1960-2008; latest follow-up, March 2020) and UK Biobank (UKB; 401,219 participants; baseline surveys, 2006-2010; latest follow-up, March 2020). Eligible participants had information about self-reported depressive symptoms and no CVD history at baseline.
Exposure: Depressive symptoms were recorded using validated instruments. ERFC scores were harmonized across studies to a scale representative of the Centre for Epidemiological Studies Depression scale (CES-D; range 0-60; â„16 indicates possible depressive disorder). UKB recorded the Patient Health Questionnaire-2 (PHQ-2; range 0-6; â„3 indicates possible depressive disorder).
Main Outcomes and Measures: Primary outcomes were incident fatal/nonfatal coronary heart disease (CHD), stroke and CVD (composite of CHD and stroke). Hazard ratios (HRs) per 1-SD higher log-CES-D or PHQ-2 adjusted for age, sex, smoking and diabetes were reported.
Results: Among 162,036 participants from the ERFC, 73% were female, mean (SD) age at baseline was 63 (9) years, and 5,078 CHD and 3,932 stroke events were recorded (median follow-up, 9.5-years). Associations with CHD, stroke and CVD were log-linear. HRs (95%CI) per 1SD higher depression score for CHD, stroke and CVD respectively were 1.07 (1.03-1.11), 1.05 (1.01-1.10), and 1.06 (1.04-1.08). This reflects, 36 versus 29 CHD events, 28 versus 25 stroke events, and 63 versus 54 CVD events per 1000 individuals over 10 years in the highest versus lowest quintile of CES-D (geometric mean CES-D score, 19 versus 1). Among 401,219 participants from the UKB, 55% were female, mean baseline age was 56 (8) years, and 4607 CHD and 3253 stroke events were recorded (median follow-up, 8.1-years). HRs per 1SD higher depression score for CHD, stroke and CVD respectively were 1.11 (1.08-1.14), 1.10 (1.06-1.14) and 1.10 (1.08-1.13). This reflects, 21 versus 14 CHD events, 15 versus 10 stroke events, and 36 versus 25 CVD events per 1000 individuals over 10 years in those with PHQ2 â„4 versus 0. The magnitude and statistical significance of the HRs were not materially changed after adjustment for additional risk factors.
Conclusions and Relevance: In a pooled analysis of 563,255 participants in 22 cohorts, baseline depressive symptoms were associated with CVD incidence, including at symptom levels below the threshold indicative of a depressive disorder. However, the magnitude of associations was modest.Lisa Pennells, Stephen Kaptoge and Sarah Spackman are funded by a British Heart Foundation Programme Grant (RG/18/13/33946). Steven Bell was funded by the National Institute for Health Research Blood and Transplant Research Unit in Donor Health and Genomics (NIHR BTRU-2014-10024). Tom Bolton is funded by the National Institute for Health Research Blood and Transplant Research Unit in Donor Health and Genomics (NIHR BTRU-2014-10024). Angela Wood is supported by a BHF-Turing Cardiovascular Data Science Award and by the EC-Innovative Medicines Initiative (BigData@Heart). John Danesh holds a British Heart Foundation Professorship and a National Institute for Health Research Senior Investigator Award.*
*The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care
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