Deep Sequencing Analyses of DsiRNAs Reveal the Influence of 3′ Terminal Overhangs on Dicing Polarity, Strand Selectivity, and RNA Editing of siRNAs

25/27 Base duplex RNAs that are substrates for Dicer have been demonstrated to enhance RNA interference (RNAi) potency and efficacy. Since the target sites are not always equally susceptible to suppression by small interfering RNA (siRNA), not all 27-mer duplexes that are processed into the corresponding conventional siRNAs show increased potency. Thus random designing of Dicer-substrate siRNAs (DsiRNAs) may generate siRNAs with poor RNAi due to unpredictable Dicer processing. Previous studies have demonstrated that the 3′-overhang affects dicing cleavage site and the orientation of Dicer entry. Moreover, an asymmetric 27-mer duplex having a 3′ two-nucleotide overhang and 3′-DNA residues on the blunt end has been rationally designed to obtain greater efficacy. This asymmetric structure directs dicing to predictably yield a single primary cleavage product. In the present study, we analyzed the in vitro and intracellular dicing patterns of chemically synthesized duplex RNAs with different 3′-overhangs. Consistent with previous studies, we observed that Dicer preferentially processes these RNAs at a site 21–22 nucleotide (nt) from the two-base 3′-overhangs. We also observed that the direction and ability of human Dicer to generate siRNAs can be partially or completely blocked by DNA residues at the 3′-termimi. To examine the effects of various 3′-end modifications on Dicer processing in cells, we employed Illumina Deep sequencing analyses to unravel the fates of the asymmetric 27-mer duplexes. To validate the strand selection process and knockdown capabilities we also conducted dual-luciferase psiCHECK reporter assays to monitor the RNAi potencies of both the “sense” (S) and “antisense” (AS) strands derived from these DsiRNAs. Consistent with our in vitro Dicer assays, the asymmetric duplexes were predictably processed into desired primary cleavage products of 21–22-mers in cells. We also observed the trimming of the 3′ end, especially when DNA residues were incorporated into the overhangs and this trimming ultimately influenced the Dicer-cleavage site and RNAi potency. Moreover, the observation that the most efficacious strand was the most abundant revealed that the relative frequencies of each “S” or “AS” strand are highly correlated with the silencing activity and strand selectivity. Collectively, our data demonstrate that even though the only differences between a family of DsiRNAs was the 3′ two-nuclotide overhang, dicing polarity and strand selectivity are distinct depending upon the sequence and chemical nature of this overhang. Thus, it is possible to predictably control dicing polarity and strand selectivity via simply changing the 3′-end overhangs without altering the original duplex sequence. These optimal design features of 3′-overhangs might provide a facile approach for rationally designing highly potent 25/27-mer DsiRNAs

Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart

First simultaneous measurements of waves generated at the bow shock in the solar wind, the magnetosphere and on the ground

On 5 September 2002 the Geotail satellite observed the cone angle of the Interplanetary Magnetic Field (IMF) change to values below 30° during a 56 min interval between 18:14 and 19:10 UT. This triggered the generation of upstream waves at the bow shock, 13 <I>R<sub>E</sub></I> downstream of the position of Geotail. Upstream generated waves were subsequently observed by Geotail between 18:30 and 18:48 UT, during times the IMF cone angle dropped below values of 10°. At 18:24 UT all four Cluster satellites simultaneously observed a sudden increase in wave power in all three magnetic field components, independent of their position in the dayside magnetosphere. We show that the 10 min delay between the change in IMF direction as observed by Geotail and the increase in wave power observed by Cluster is consistent with the propagation of the IMF change from the Geotail position to the bow shock and the propagation of the generated waves through the bow shock, magnetosheath and magnetosphere towards the position of the Cluster satellites. We go on to show that the wave power recorded by the Cluster satellites in the component containing the poloidal and compressional pulsations was broadband and unstructured; the power in the component containing toroidal oscillations was structured and shows the existence of multi-harmonic Alfvénic continuum waves on field lines. Model predictions of these frequencies fit well with the observations. An increase in wave power associated with the change in IMF direction was also registered by ground based magnetometers which were magnetically conjunct with the Cluster satellites during the event. To the best of our knowledge we present the first simultaneous observations of waves created by backstreaming ions at the bow shock in the solar wind, the dayside magnetosphere and on the ground

Comparative analysis of CRISPR off-target discovery tools following ex vivo editing of CD34+ hematopoietic stem and progenitor cells.

While a number of methods exist to investigate CRISPR off-target (OT) editing, few have been compared head-to-head in primary cells after clinically relevant editing processes. Therefore, we compared in silico tools (COSMID, CCTop, and Cas-OFFinder) and empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) after ex vivo hematopoietic stem and progenitor cell (HSPC) editing. We performed editing using 11 different gRNAs complexed with Cas9 protein (high-fidelity [HiFi] or wild-type versions), then performed targeted next-generation sequencing of nominated OT sites identified by in silico and empirical methods. We identified an average of less than one OT site per guide RNA (gRNA) and all OT sites generated using HiFi Cas9 and a 20-nt gRNA were identified by all OT detection methods with the exception of SITE-seq. This resulted in high sensitivity for the majority of OT nomination tools and COSMID, DISCOVER-Seq, and GUIDE-Seq attained the highest positive predictive value (PPV). We found that empirical methods did not identify OT sites that were not also identified by bioinformatic methods. This study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity and PPV, thereby enabling more efficient identification of potential OT sites without compromising a thorough examination for any given gRNA

T-cell activation is enhanced by targeting IL-10 cytokine production in toll-like receptor-stimulated macrophages

Toll-like receptor (TLR) agonists represent potentially useful cancer vaccine adjuvants in their ability to stimulate antigen-presenting cells (APCs) and subsequently amplify the cytotoxic T-cell response. The purpose of this study was to characterize APC responses to TLR activation and to determine the subsequent effect on lymphocyte activation. We exposed murine primary bone marrow-derived macrophages to increasing concentrations of agonists to TLRs 2, 3, 4, and 9. This resulted in a dose-dependent increase in production of not only tumor necrosis factor–alpha (TNF-α), a surrogate marker of the proinflammatory response, but also interleukin 10 (IL-10), a well-described inhibitory cytokine. Importantly, IL-10 secretion was not induced by low concentrations of TLR agonists that readily produced TNF-α. We subsequently stimulated lymphocytes with anti-CD3 antibody in the presence of media from macrophages activated with higher doses of TLR agonists and observed suppression of interferon gamma release. Use of both IL-10 knockout macrophages and IL-10 small-interfering RNA (siRNA) ablated this suppressive effect. Finally, IL-10 siRNA was successfully used to suppress CpG-induced IL-10 production in vivo. We conclude that TLR-mediated APC stimulation can induce a paradoxical inhibitory effect on T-cell activation mediated by IL-10

Associating ground magnetometer observations with current or voltage generators

A circuit analogy for magnetosphere‐ionosphere current systems has two extremes for drivers of ionospheric currents: ionospheric electric fields/voltages constant while current/conductivity vary—the “voltage generator”—and current constant while electric field/conductivity vary—the “current generator.” Statistical studies of ground magnetometer observations associated with dayside Transient High Latitude Current Systems (THLCS) driven by similar mechanisms find contradictory results using this paradigm: some studies associate THLCS with voltage generators, others with current generators. We argue that most of this contradiction arises from two assumptions used to interpret ground magnetometer observations: (1) measurements made at fixed position relative to the THLCS field‐aligned current and (2) negligible auroral precipitation contributions to ionospheric conductivity. We use observations and simulations to illustrate how these two assumptions substantially alter expectations for magnetic perturbations associated with either a current or a voltage generator. Our results demonstrate that before interpreting ground magnetometer observations of THLCS in the context of current/voltage generators, the location of a ground magnetometer station relative to the THLCS field‐aligned current and the location of any auroral zone conductivity enhancements need to be taken into account.Key PointsConductivity and location assumptions used to interpret ground magnetic perturbations yield conflicting resultsHigh‐latitude currents associated with voltage generators may instead be associated with current generators, and vice versaWithout better constraints on conductivity/station location relative to currents, conflicts will not be resolvedPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138366/1/jgra53632.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138366/2/jgra53632_am.pd

The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing

<p>Abstract</p> <p>Background</p> <p>The use of RNAi to analyse gene function <it>in vitro </it>is now widely applied in biological research. However, several difficulties are associated with its use <it>in vivo</it>, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown.</p> <p>Findings</p> <p>We observed that detection of an apparent siRNA-mediated knockdown <it>in vivo </it>was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for <it>RRM1 </it>with equivalent activity <it>in vitro </it>were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of <it>RRM1 </it>mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR.</p> <p>Conclusions</p> <p>Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown <it>in vivo </it>can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR.</p

ANALISIS DETERMINAN KEJADIAN STUNTING PADA BALITA

INTRODUCTION the problem of nutrition the impact on human resources is still going on in Indonesia. The number of children who experience stunting as much as 27.5% in 2016. This means that 1 in 3 children are experiencing a stunting. The purpose of this research is to analyze the factors giving of nutrition, LOW BIRTH WEIGHT, fetal growth and income against Gen. stunting on toddlers. RESEARCH METHODOLOGY the study was observational, analytic. Variables include low birth weight, the height of the fundus uteri, gift of nutrition and income. The sample is selected by sampling a number of exposure fix 150 with comparison 1:2. Data collection using keusioner, and medical record (KIA). The RESULTS there are influences of nutrition (b = 2.54; CI = 1.63 up 3.44; p = 0.001), LBW &lt; (b =-2.10; CI = 1.33-to-2.87; p = 0.001), growth &lt; fetal (b = 5.95; CI = 3.88 to 8.03; p = 0.001) and income &lt; against Gen. stunting in toddlers (b =-1.38; CI = 2.28 to-0.48; p = 0.003). DISCUSSION of the causes of the incidence of stunting in toddlers is a complex thing. The incidence of stunting begins from malnutrition during pregnancy and diperberat with malnutrition in the first five years the growth of toddlers. CONCLUSION there is influence the granting of nutrition, LOW BIRTH WEIGHT, fetal growth and income against Gen. stunting on toddlers. &nbsp;PENDAHULUAN permasalahan gizi yang berdampak pada sumber daya manusia masih terjadi di Indonesia. Jumlah anak yang mengalami stunting sebanyak 27.5% pada tahun 2016. Hal ini dapat diartikan bahwa 1 dari 3 anak mengalami stunting. Tujuan penelitian ini menganalisis faktor pemberian nutrisi, BBLR, pertumbuhan janin dan pendapatan terhadap kejadian stunting pada balita. METODOLOGI PENELITIAN studi ini analitik observasional. Variabel meliputi berat badan lahir rendah, tinggi fundus uteri, pemberian nutrisi dan pendapatan. Sampel dipilih secara fix exposure sampling sejumlah 150 dengan perbandingan 1:2. Teknik pengumpulan data menggunakan keusioner, dan rekam medis (buku KIA). HASIL ada pengaruh nutrisi (b= 2.54; CI= 1.63 sampai 3.44; p= &lt;0.001), BBLR (b= -2.10; CI= -2.87 sampai -1.33; p= &lt;0.001), pertumbuhan janin (b= 5.95; CI= 3.88 sampai 8.03; p= &lt;0.001) dan pendapatan terhadap kejadian stunting pada balita (b= -1.38; CI= 2.28 sampai -0.48; p= 0.003). DISKUSI penyebab kejadian stunting pada balita merupakan hal yang kompleks. Kejadian stunting dimulai dari kondisi kekurangan gizi saat kehamilan dan diperberat dengan kekurangan gizi pada lima tahun pertama pertumbuhan balita. KESIMPULAN ada pengaruh pemberian nutrisi, BBLR, pertumbuhan janin dan pendapatan terhadap kejadian stunting pada balita