scholarly journals Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

2016 ◽  
Vol 113 (51) ◽  
pp. 14727-14732 ◽  
Author(s):  
Nathaniel E. Clark ◽  
Adam Katolik ◽  
Kenneth M. Roberts ◽  
Alexander B. Taylor ◽  
Stephen P. Holloway ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Nucleophilic Attack ◽  
X Rays ◽  
Turnover Rates ◽  
Optimal Position ◽  
Debranching Enzyme ◽  
Aerobic Conditions ◽  
Bona Fide ◽  
Branched Rna ◽  
Retroviral Infections

Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2′,5′- and 3′,5′-phosphodiester linkages. The 2′,5′-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 fromEntamoeba histolyticaby using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s−1. Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s−1is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.

Accuracy of 11 formulae to guide umbilical arterial catheter tip placement in newborn infants

2017 ◽  
Vol 103 (4) ◽  
pp. F364-F369 ◽  
Author(s):  
Wei Ling Lean ◽  
Jennifer A Dawson ◽  
Peter G Davis ◽  
Christiane Theda ◽  
Marta Thio
Keyword(s):  
Gold Standard ◽  
Newborn Infants ◽  
Body Part ◽  
Accurate Method ◽  
Arterial Catheter ◽  
X Rays ◽  
Success Rates ◽  
Common Procedure ◽  
Optimal Position ◽  
Umbilical Arterial Catheter

BackgroundUmbilical arterial catheter (UAC) insertion is a common procedure in the neonatal intensive care unit (NICU). Correct placement of the tip of the UAC at first attempt minimises handling of the infant and reduces the risk of infection and complications. We aimed to determine the accuracy of 11 published formulae to guide UAC placement.MethodsThis was a one-year prospective observational study in a tertiary NICU. Clinicians used their preferred formula for UAC insertion, with X-rays performed immediately post-procedure to check the tip position. Birth weight and measurements included in the 11 formulae were recorded within 48 hours. The gold standard insertion distance was defined as the distance from the abdominal wall to the mid-descending aorta, at T8 level on X-ray (range T6–T10). Insertion length using the 11 formulae was calculated and compared with this gold standard distance.ResultsOne hundred and three infants were included, with median (IQR) gestational age and weight of 28 (26–33.5) weeks and 980 (780–2045) g, respectively. The predicted value of the 11 formulae to place the UAC in correct position ranged from 51.0% to 73.8%. Formulae that involved direct body part measurements showed the highest predicted success rates, smallest mean difference from T8 and narrowest limits of agreement using the Bland-Altman method.ConclusionSuccess rates for accurate UAC placement are highest when formulae that involve body measurements are used. However, even the most accurate method would result in more than 25% of UACs needing manipulation to achieve an optimal position.

scholarly journals Updated X-ray view of the Hyades cluster

2020 ◽  
Vol 640 ◽  
pp. A66 ◽  
Author(s):  
S. Freund ◽  
J. Robrade ◽  
P. C. Schneider ◽  
J. H. M. M. Schmitt
Keyword(s):  
Main Sequence ◽  
Distribution Functions ◽  
X Rays ◽  
Stellar Rotation ◽  
Detection Rates ◽  
X Ray ◽  
Rotation Periods ◽  
Hyades Cluster ◽  
Bona Fide ◽  
Tidal Tails

Aims. We revisit the X-ray properties of the main sequence Hyades members and the relation between X-ray emission and stellar rotation. Methods. As an input catalog for Hyades members, we combined three recent Hyades membership lists derived from Gaia DR2 data that include the Hyades core and its tidal tails. We searched for X-ray detections of the main sequence Hyades members in the ROSAT all-sky survey, and pointings from ROSAT, the Chandra X-Ray Observatory, and XMM-Newton. Furthermore, we adopted rotation periods derived from Kepler’s K2 mission and other resources. Results. We find an X-ray detection for 281 of 1066 bona fide main sequence Hyades members and provide statistical upper limits for the undetected sources. The majority of the X-ray detected stars are located in the Hyades core because of its generally smaller distance to the Sun. F- and G-type stars have the highest detection fraction (72%), while K- and M-type dwarfs have lower detection rates (22%). The X-ray luminosities of the detected members range from ∼2 × 1027 erg s−1 for late M-type dwarfs to ∼2 × 1030 erg s−1 for active binaries. The X-ray luminosity distribution functions formally differ for the members in the core and tidal tails, which is likely caused by a larger fraction of field stars in our Hyades tails sample. Compared to previous studies, our sample is slightly fainter in X-rays due to differences in the Hyades membership list used; furthermore, we extend the X-ray luminosity distribution to fainter luminosities. The X-ray activity of F- and G-type stars is well defined at FX/Fbol ≈ 10−5. The fractional X-ray luminosity and its spread increases to later spectral types reaching the saturation limit (FX/Fbol ≈ 10−3) for members later than spectral type M3. Confirming previous results, the X-ray flux varies by less than a factor of three between epochs for the 104 Hyades members with multiple epoch data, significantly less than expected from solar-like activity cycles. Rotation periods are found for 204 Hyades members, with about half of them being detected in X-rays. The activity-rotation relation derived for the coeval Hyades members has properties very similar to those obtained by other authors investigating stars of different ages.

scholarly journals A homolog of lariat-debranching enzyme modulates turnover of branched RNA

RNA ◽  
2014 ◽  
Vol 20 (8) ◽  
pp. 1337-1348 ◽  
Author(s):  
Stephen M. Garrey ◽  
Adam Katolik ◽  
Mantas Prekeris ◽  
Xueni Li ◽  
Kerri York ◽  
...  
Keyword(s):  
Debranching Enzyme ◽  
Branched Rna

scholarly journals Design, Synthesis, and Properties of Phosphoramidate 2′,5′-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme

2015 ◽  
Vol 80 (20) ◽  
pp. 10108-10118 ◽  
Author(s):  
Nobuhiro Tago ◽  
Adam Katolik ◽  
Nathaniel E. Clark ◽  
Eric J. Montemayor ◽  
Kohji Seio ◽  
...  
Keyword(s):  
Rational Design ◽  
Design Synthesis ◽  
Debranching Enzyme ◽  
Branched Rna

scholarly journals Microglia and border-associated mouse macrophages maintain their embryonic origin during Alzheimers disease

2021 ◽  
Author(s):  
christiane Ruedl ◽  
Xiaoting Wu ◽  
Takashi Saito ◽  
takaomi Saido ◽  
Anna M Barron
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Bone Marrow Cells ◽  
Brain Regions ◽  
Turnover Rates ◽  
Alzheimers Disease ◽  
Brain Macrophages ◽  
Murine Microglia ◽  
Embryonic Origin ◽  
Bona Fide

Brain microglia and border-associated macrophages (BAMs) display distinct spatial, developmental, and phenotypic features. Although at steady-state, the origins of distinct brain macrophages are well-documented, the dynamics of their replenishment in neurodegenerative disorders remain elusive, particularly for disease-associated microglia (DAMs) and BAMs. In this study, we conducted a comprehensive fate-mapping analysis of murine microglia and BAMs and their turnover kinetics during Alzheimers disease (AD) progression. We used a novel inducible AD mouse model to investigate the contribution of bone marrow cells to the pool of foetal-derived brain macrophages during the development of AD. We demonstrated that microglia and DAMs remain a remarkably stable embryonic-derived population even during the progression of AD pathology, indicating that neither parenchymal macrophage subpopulation originates from, nor are replenished by, monocytes. At the border-associated brain regions, bona fide CD206+ BAMs are minimally replaced by monocytes, and their turnover rates are not accelerated by AD. In contrast, all other myeloid cells are swiftly replenished by bone marrow progenitors. This information further elucidates the turnover kinetics of these cells not only at steady-state, but also in neurodegenerative diseases, which is crucial for identifying potential novel therapeutic targets.

scholarly journals RESPONSE OF POTASSIUM RETENTIVITY AND SURVIVAL OF YEAST TO FAR-ULTRAVIOLET, NEAR-ULTRAVIOLET AND VISIBLE, AND X-RADIATION

1958 ◽  
Vol 41 (4) ◽  
pp. 693-702 ◽  
Author(s):  
A. K. Bruce
Keyword(s):  
Yeast Cell ◽  
Dose Response ◽  
X Rays ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Differential Effects ◽  
Near Ultraviolet ◽  
X Irradiation ◽  
Far Ultraviolet ◽  
Control Survival

Potassium retentivity and survival of yeast were studied after exposure to various kinds and conditions of irradiation. The radiations used were: 2537 A ultraviolet, 3500 to 4900 A long-ultraviolet and short visible, and 250 kvp1 x-rays. Both potassium retentivity and survival are decreased by these radiations. The dose-response of survival is about 16 times as sensitive as is potassium retentivity after 2537 A irradiation. Potassium retentivity is about twice as sensitive as survival after irradiation of 3500 to 4900 A. Survival after x-irradiation under aerobic conditions is five times as sensitive as potassium retentivity. Survival of cells irradiated with x-rays under anaerobic conditions was about half as sensitive as under aerobic conditions. The response of potassium retentivity to x-radiation at 25°C. under anaerobic conditions is only slightly affected below 160 kr, at which dose the slope abruptly increases to that obtained under aerobic conditions; lowering the temperature to 0°C. moves this point to about 300 kr. These differential effects are indicative of interaction of radiations with the yeast cell at sites that independently control survival and the retention of potassium.

scholarly journals Mechanisms of Late Restriction Induced by an Endogenous Retrovirus

2007 ◽  
Vol 81 (20) ◽  
pp. 11441-11451 ◽  
Author(s):  
Frederick Arnaud ◽  
Pablo R. Murcia ◽  
Massimo Palmarini
Keyword(s):  
Endogenous Retrovirus ◽  
Dominant Negative ◽  
Endogenous Retroviruses ◽  
Restriction Factors ◽  
Recycling Endosome ◽  
Recycling Endosomes ◽  
Viral Particles ◽  
Bona Fide ◽  
Retroviral Infections ◽  
Late Stages

ABSTRACT The host has developed during evolution a variety of “restriction factors” to fight retroviral infections. We investigated the mechanisms of a unique viral block acting at late stages of the retrovirus replication cycle. The sheep genome is colonized by several copies of endogenous retroviruses, known as enJSRVs, which are highly related to the oncogenic jaagsiekte sheep retrovirus (JSRV). enJS56A1, one of the enJSRV proviruses, can act as a restriction factor by blocking viral particles release of the exogenous JSRV. We show that in the absence of enJS56A1 expression, the JSRV Gag (the retroviral internal structural polyprotein) targets initially the pericentriolar region, in a dynein and microtubule-dependent fashion, and then colocalizes with the recycling endosomes. Indeed, by inhibiting the endocytosis and trafficking of recycling endosomes we hampered JSRV exit from the cell. Using a variety of approaches, we show that enJS56A1 and JSRV Gag interact soon after synthesis and before pericentriolar/recycling endosome targeting of the latter. The transdominant enJS56A1 induces intracellular Gag accumulation in microaggregates that colocalize with the aggresome marker GFP-250 but develop into bona fide aggresomes only when the proteasomal machinery is inhibited. The data argue that dominant-negative proteins can modify the overall structure of Gag multimers/viral particles hampering the interaction of the latter with the cellular trafficking machinery.

scholarly journals Evidence for magnetic activity at starbirth: a powerful X-ray flare from the Class 0 protostar HOPS 383

2020 ◽  
Vol 638 ◽  
pp. L4 ◽  
Author(s):  
Nicolas Grosso ◽  
Kenji Hamaguchi ◽  
David A. Principe ◽  
Joel H. Kastner
Keyword(s):  
Near Infrared ◽  
Infrared Imaging ◽  
Direct Proof ◽  
Magnetic Activity ◽  
Evolutionary Stage ◽  
X Rays ◽  
X Ray ◽  
Order Of Magnitude ◽  
Solar Type ◽  
Bona Fide

Context. Class 0 protostars represent the earliest evolutionary stage of solar-type stars, during which the majority of the system mass resides in an infalling envelope of gas and dust and is not yet in the central, nascent star. Although X-rays are a key signature of magnetic activity in more evolved protostars and young stars, whether such magnetic activity is present at the Class 0 stage is still debated. Aims. We aim to detect a bona fide Class 0 protostar in X-rays. Methods. We observed HOPS 383 in 2017 December in X-rays with the Chandra X-ray Observatory (∼84 ks) and in near-infrared imaging with the Southern Astrophysical Research telescope. Results. HOPS 383 was detected in X-rays during a powerful flare. This hard (E >  2 keV) X-ray counterpart was spatially coincident with the northwest 4 cm component of HOPS 383, which would be the base of the radio thermal jet launched by HOPS 383. The flare duration was ∼3.3 h; at the peak, the X-ray luminosity reached ∼4 × 1031 erg s−1 in the 2−8 keV energy band, a level at least an order of magnitude larger than that of the undetected quiescent emission from HOPS 383. The X-ray flare spectrum is highly absorbed (NH ∼ 7 × 1023 cm−2), and it displays a 6.4 keV emission line with an equivalent width of ∼1.1 keV, arising from neutral or low-ionization iron. Conclusions. The detection of a powerful X-ray flare from HOPS 383 constitutes direct proof that magnetic activity can be present at the earliest formative stages of solar-type stars.

High-quality quasi-parallel X-ray beam obtained by a parabolic monocapillary X-ray lens with a square beam stop

2021 ◽  
pp. 1-13
Author(s):  
Peng Zhou ◽  
Jingduo Cui ◽  
Zelin Du ◽  
Tao Zhang ◽  
Zhiguo Liu
Keyword(s):  
Optical Path ◽  
X Rays ◽  
High Quality ◽  
Optimal Position ◽  
X Ray ◽  
Proposed Model ◽  
Beam Stop ◽  
Path System ◽  
New Applications ◽  
Study Designs

Parabolic monocapillary X-ray lens (PMXRL) is an ideal optical device for constraining the point divergent X-ray beams to quasi-parallel beams, but the overlap of direct X-rays and reflected X-rays through PMXRL deteriorates the outgoing beam divergence. Aiming to solve this problem, this study designs and tests a square-shaped lead occluder (SSLO) embedded in PMXRL to block the direct X-rays passing through the PMXRL. Python simulations are employed to determine the geometric parameters of the SSLO as well as the optimal position of the SSLO in the PMXRL according to our proposed model. The PMXRL with a conic parameter p of 0.000939 mm and a length L of 60.8 mm is manufactured and the SSLO with a size of 0.472 mm×0.472 mm×3.4 mm is embedded into it. An optical path system based on this PMXRL is built to measure the divergence of the outgoing X-ray beam. The experimental results show that the quasi-parallel X-ray beam reaches a divergence of 0.36 mrad in the range from 15–45 mm at the PMXRL outlet. This divergence is 10 times lower than the theoretical divergence without SSLO. Our work provides an alternative method for obtaining highly parallel X-ray beam and is beneficial to generate or facilitate new applications of monocapillary optics in X-ray technology.

scholarly journals A Biochemical Screen for Identification of Small-Molecule Regulators of the Wnt Pathway Using Xenopus Egg Extracts

2011 ◽  
Vol 16 (9) ◽  
pp. 995-1006 ◽  
Author(s):  
Curtis A. Thorne ◽  
Bonnie Lafleur ◽  
Michelle Lewis ◽  
Alison J. Hanson ◽  
Kristin K. Jernigan ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Mammalian Cells ◽  
Wnt Pathway ◽  
Renilla Luciferase ◽  
Signal Distribution ◽  
Egg Extracts ◽  
Bona Fide ◽  
Xenopus Egg ◽  
The Stability ◽  
Xenopus Egg Extracts

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.

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