In vitro binding of single-stranded RNA by human Dicer

AbstractWhile Dicer alone has been shown to form stable complexes with double-stranded RNAs and short interfering RNAs, its interactions with single-stranded RNAs (ssRNAs) have not been characterized. Here, we show that recombinant human Dicer alone can bind 21-nt ssRNAs in vitro, independent of their sequence and structure. We also demonstrate that Dicer binds ssRNAs having a 5′-phosphate with greater affinity versus those with a 5′-hydroxyl. In addition, 3′-biotinylated ssRNAs are bound by Dicer with lower affinity than 3′-hydroxyl ssRNAs. The stability of ssRNA–Dicer complexes was found to depend on divalent cations. Together, our results suggest a role for the PAZ domain of Dicer in binding ssRNAs and may indicate roles for Dicer in cellular function beyond those currently known

Inhibition of productive/competitive endocytic pathways enhances siRNA delivery and cell specific targeting

While the use of short interfering RNAs (siRNAs) for laboratory studies is now common practice, development of siRNAs for therapeutic applications has slowed, due in part to a still limited understanding of the endocytosis and intracellular trafficking of siRNA-containing complexes. As a result, it is difficult to design delivery vehicles for specific cell types, resulting in inefficient delivery, cytotoxicity, or immunogenicity when used in vivo. Our aim is to identify which endocytosis and intracellular trafficking pathways lead to active silencing by siRNA-containing complexes. Our work explores the preferential mechanism of endocytosis (whether by clathrin, caveolin, Arf6, Graf1, flotillin, or macropinocytosis) across multiple cell types (HeLa (cervical), H1299 (lung), HEK293 (kidney), and HepG2 (liver)). Using Lipofectamine 2000 (LF2K), fluorescentlylabeled siRNAs were delivered to cells stably expressing green fluorescent protein (GFP). Chemical inhibitors (Filipin, Dynasore, Cytochalasin D, Chlorpromazine, Amiloride, and Methyl-β- cyclodextrin) were used to identify the specific endocytic pathway internalizing the complexes. By measuring the effect of inhibitors on both intracellular levels of siRNA and GFP silencing, we were able to categorize pathways as being productive/competitive according to their functional role in facilitating gene silencing. In productive pathways, siRNAs are actively delivered to a cell and silence a target protein, whereas in competitive pathways, siRNAs are endocytosed but do not lead to silencing. Please click Additional Files below to see the full abstract

Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2002.Includes bibliographical references (p. 165-178).Antisense oligonucleotides have the potential to selectively inhibit the expression of any gene with a known sequence. Antisense-based therapies are under development for the treatment of infectious diseases as well as complex genetic disorders. Although there have been some remarkable successes, realizing this potential is proving difficult because of problems with oligonucleotide stability, specificity, affinity, and delivery. Each of these limitations has been addressed experimentally through the use of chemically-modified oligonucleotides and oligonucleotide conjugates, with much success in enhancing oligonucleotide efficacy. These early studies have shown that selection of target site, once considered a trivial problem, is critical to the success of antisense strategies. It has become clear that the efficacy of antisense oligonucleotides is a strong function of the structure of the target mRNA. Though single-stranded, RNA molecules are typically folded into complex three-dimensional structures, formed primarily by intramolecular Watson-Crick base-pairing. If an oligonucleotide is complementary to a sequence embedded in the three dimensional structure, the oligonucleotide may not be able to bind to its target site and exert its therapeutic effect. Because the majority of the structure of RNA molecules is due to Watson-Crick base-pairing, relatively accurate predictions of these folding interactions can be made from algorithms that locate the structure with the most favorable free energy of folding.(cont.) Taking advantage of the predictability of RNA structures, this thesis addresses the problem of antisense target site selection, first from a theoretical and subsequently an experimental standpoint. A thermodynamic model to predict the binding affinity of oligonucleotides for their target mRNA is described and validated using multiple in vitro and cell-culture based experimental data sets. Subsequently, direct experimental comparisons with theoretical predictions are made on the well-characterized rabbit-[beta]-globin (RBG) mRNA, using a novel, centrifugal, binding affinity assay. The importance of the hybridization kinetics is also explored, as is the role of association kinetics in defining the rate of cleavage by the enzyme ribonuclease H (RNase H). Finally, the applicability of the model in identifying biologically active oligonucleotides is demonstrated.by S. Patrick Walton.Sc.D

Improved asymmetry prediction for short interfering RNA s

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102096/1/febs12599.pd

Tracking Assaultâ injured, Drugâ using Youth in Longitudinal Research: Followâ up Methods

ObjectivesViolence is one of the leading causes of death among youth ages 14 to 24. Hospitalâ and emergency department (ED)â based violence prevention programs are increasingly becoming a critical part of public health efforts; however, evaluation of prevention efforts is needed to create evidenceâ based best practices. Retention of study participants is key to evaluations, although little literature exists regarding optimizing followâ up methods for violently injured youth. This study aims to describe the methods for retention in youth violence studies and the characteristics of hardâ toâ reach participants.MethodsThe Flint Youth Injury (FYI) Study is a prospective study following a cohort of assaultâ injured, drugâ using youth recruited in an urban ED, and a comparison population of drugâ using youth seeking medical or nonâ violenceâ related injury care. Validated survey instruments were administered at baseline and four followâ up time points (6, 12, 18, and 24 months). Followâ up contacts used a variety of strategies and all attempts were coded by type and level of success. Regression analysis was used to predict contact difficulty and followâ up interview completion at 24 months.ResultsA total of 599 patients (ages 14â 24) were recruited from the ED (mean ± SD age = 20.1 ± 2.4 years, 41.2% female, 58.2% African American), with followâ up rates at 6, 12, 18, and 24 months of 85.3%, 83.7% 84.2%, and 85.3%, respectively. Participant contact efforts ranged from two to 53 times per followâ up time frame to complete a followâ up appointment, and more than 20% of appointments were completed off site at community locations (e.g., participantsâ homes, jail/prison). Participants who were younger (p < 0.05) and female (p < 0.01) were more likely to complete their 24â month followâ up interview. Participants who sought care in the ED for assault injury (p < 0.05) and had a substance use disorder (p < 0.01) at baseline required fewer contact attempts to complete their 24â month followâ up, while participants reporting a fight within the immediate 3 months before their 24â month followâ up (p < 0.01) required more intensive contact efforts.ConclusionsThe FYI study demonstrated that achieving high followâ up rates for a difficultâ toâ track, violentlyâ injured ED population is feasible through the use of established contact strategies and a variety of interview locations. Results have implications for followâ up strategies planned as part of other violence prevention studies.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146571/1/acem13495_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146571/2/acem13495.pd

NuSTAR observations of the powerful radio-galaxy Cygnus A

We present NuSTAR observations of the powerful radio galaxy Cygnus A, focusing on the central absorbed active galactic nucleus (AGN). Cygnus A is embedded in a cool-core galaxy cluster, and hence we also examine archival XMM-Newton data to facilitate the decomposition of the spectrum into the AGN and intracluster medium (ICM) components. NuSTAR gives a source-dominated spectrum of the AGN out to >70keV. In gross terms, the NuSTAR spectrum of the AGN has the form of a power law (Gamma~1.6-1.7) absorbed by a neutral column density of N_H~1.6x10^23 cm^-2. However, we also detect curvature in the hard (>10keV) spectrum resulting from reflection by Compton-thick matter out of our line-of-sight to the X-ray source. Compton reflection, possibly from the outer accretion disk or obscuring torus, is required even permitting a high-energy cutoff in the continuum source; the limit on the cutoff energy is E_cut>111keV (90% confidence). Interestingly, the absorbed power-law plus reflection model leaves residuals suggesting the absorption/emission from a fast (15,000-26,000km/s), high column-density (N_W>3x10^23 cm^-2), highly ionized (xi~2,500 erg cm/s) wind. A second, even faster ionized wind component is also suggested by these data. We show that the ionized wind likely carries a significant mass and momentum flux, and may carry sufficient kinetic energy to exercise feedback on the host galaxy. If confirmed, the simultaneous presence of a strong wind and powerful jets in Cygnus A demonstrates that feedback from radio-jets and sub-relativistic winds are not mutually exclusive phases of AGN activity but can occur simultaneously.Comment: 13 pages; accepted for publication in The Astrophysical Journa

Identification of BPIFA1/SPLUNC1 as an epithelium-derived smooth muscle relaxing factor

Asthma is a chronic airway disease characterized by inflammation, mucus hypersecretion and abnormal airway smooth muscle (ASM) contraction. Bacterial permeability family member A1, BPIFA1, is a secreted innate defence protein. Here we show that BPIFA1 levels are reduced in sputum samples from asthmatic patients and that BPIFA1 is secreted basolaterally from healthy, but not asthmatic human bronchial epithelial cultures (HBECs), where it suppresses ASM contractility by binding to and inhibiting the Ca2+ influx channel Orai1. We have localized this effect to a specific, C-terminal α-helical region of BPIFA1. Furthermore, tracheas from Bpifa1−/− mice are hypercontractile, and this phenotype is reversed by the addition of recombinant BPIFA1. Our data suggest that BPIFA1 deficiency in asthmatic airways promotes Orai1 hyperactivity, increased ASM contraction and airway hyperresponsiveness. Strategies that target Orai1 or the BPIFA1 deficiency in asthma may lead to novel therapies to treat this disease

Direct Measurement of Nuclear Dependence of Charged Current Quasielastic-like Neutrino Interactions using MINERvA

Charged-current $\nu_{\mu}$ interactions on carbon, iron, and lead with a final state hadronic system of one or more protons with zero mesons are used to investigate the influence of the nuclear environment on quasielastic-like interactions. The transfered four-momentum squared to the target nucleus, $Q^2$, is reconstructed based on the kinematics of the leading proton, and differential cross sections versus $Q^2$ and the cross-section ratios of iron, lead and carbon to scintillator are measured for the first time in a single experiment. The measurements show a dependence on atomic number. While the quasielastic-like scattering on carbon is compatible with predictions, the trends exhibited by scattering on iron and lead favor a prediction with intranuclear rescattering of hadrons accounted for by a conventional particle cascade treatment. These measurements help discriminate between different models of both initial state nucleons and final state interactions used in the neutrino oscillation experiments

First evidence of coherent K+K^{+} meson production in neutrino-nucleus scattering

Neutrino-induced charged-current coherent kaon production, $\nu_{\mu}A\rightarrow\mu^{-}K^{+}A$, is a rare, inelastic electroweak process that brings a $K^+$ on shell and leaves the target nucleus intact in its ground state. This process is significantly lower in rate than neutrino-induced charged-current coherent pion production, because of Cabibbo suppression and a kinematic suppression due to the larger kaon mass. We search for such events in the scintillator tracker of MINERvA by observing the final state $K^+$, $\mu^-$ and no other detector activity, and by using the kinematics of the final state particles to reconstruct the small momentum transfer to the nucleus, which is a model-independent characteristic of coherent scattering. We find the first experimental evidence for the process at $3\sigma$ significance.Comment: added ancillary file with information about the six kaon candidate