Yaw Shin Ooi
Full Text Available The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ, a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.
Ito, H.; Yamawaki, M.; Yamamoto, T.
The hydrogen absorption properties of U, U 6 Mn and U 6 Ni were investigated at hydrogen pressures below 10 5 Pa. The pressure-composition (P-C) isotherms of U, U 6 Mn and U 6 Ni were obtained and the amounts of absorbed hydrogen for U, U 6 Mn and U 6 Ni were determined to be 3, 16.6 and 16.0 for x in MH x , where M is U, U 6 Mn and U 6 Ni, respectively. The desorption plateau pressures at 573 K decreased in the order: U 6 Mn-H>U 6 Ni-H>U-H. In addition, the results for the amounts of absorbed hydrogen suggests the formation of ternary hydrides U 6 MnH 18 and U 6 NiH 14 . (orig.)
In silico analyses of the draft genome of Diaphorina citri, the Asian citrus psyllid, for genes within the Ribonucleic acid interference(RNAi), pathway was successful. The psyllid is the vector of the plant-infecting bacterium, Candidatus Liberibacter asiaticus (CLas), which is linked to citrus gree...
Federal Laboratory Consortium — Small interfering RNA (siRNA) molecules are pieces of RNA that block the activity of genes through a natural process called RNA interference (RNAi). This process has...
Full Text Available Since the discovery of RNA interference (RNAi, excitement has grown over its potential therapeutic uses. Targeting RNAi pathways provides a powerful tool to change biological processes post-transcriptionally in various health conditions such as cancer or autoimmune diseases. Optimum design of shRNA, siRNA, and miRNA enhances stability and specificity of RNAi-based approaches whereas it has to reduce or prevent undesirable immune responses or off-target effects. Recent advances in understanding pathogenesis of autoimmune diseases have allowed application of these tools in vitro as well as in vivo with some degree of success. Further research on the design and delivery of effectors of RNAi pathway and underlying molecular basis of RNAi would warrant practical use of RNAi-based therapeutics in human applications. This review will focus on the approaches used for current therapeutics and their applications in autoimmune diseases, including rheumatoid arthritis and Sjögren’s syndrome.
Ghosh, Subhanita; Singh, Gatikrushna; Sachdev, Bindiya; Kumar, Ajit; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K
RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line.
Johnathan J Dalzell
Full Text Available While RNA interference (RNAi has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (dsRNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii The Argonautes (AGOs responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii Secondary Argonautes (SAGOs are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research
DeLong, L.E.; Hall, L.N.; Malik, S.K.; Crabtree, G.W.; Lwpl, W.; Gschneidner, K. A. Jr.
Upper critical field data for U 6 Fe and U 6 Co are found to be markedly inconsistent with the weak coupling WHHM model. Nevertheless, excellent quantitative fits of H/sub c/ 2 vs T are obtained by using a modifiction of the WHHM model in which the orbital field is multiplied by a temperature-dependent enhancement factor
Ivashuta, Sergey; Zhang, Yuanji; Wiggins, B Elizabeth; Ramaseshadri, Partha; Segers, Gerrit C; Johnson, Steven; Meyer, Steve E; Kerstetter, Randy A; McNulty, Brian C; Bolognesi, Renata; Heck, Gregory R
Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism. © 2015 Ivashuta et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
The U(6)-phonon model of nuclear collective motion with the semi-direct product structure [HW(21)]U(6) is obtained as a hydrodynamic (macroscopic) limit of the fully microscopic proton–neutron symplectic model (PNSM) with Sp(12, R) dynamical group. The phonon structure of the [HW(21)]U(6) model enables it to simultaneously include the giant monopole and quadrupole, as well as dipole resonances and their coupling to the low-lying collective states. The U(6) intrinsic structure of the [HW(21)]U(6) model, from the other side, gives a framework for the simultaneous shell-model interpretation of the ground state band and the other excited low-lying collective bands. It follows then that the states of the whole nuclear Hilbert space which can be put into one-to-one correspondence with those of a 21-dimensional oscillator with an intrinsic (base) U(6) structure. The latter can be determined in such a way that it is compatible with the proton–neutron structure of the nucleus. The macroscopic limit of the Sp(12, R) algebra, therefore, provides a rigorous mechanism for implementing the unified model ideas of coupling the valence particles to the core collective degrees of freedom within a fully microscopic framework without introducing redundant variables or violating the Pauli principle. (author)
de Vries, Walter; Berkhout, Ben
RNA silencing or RNAi interference (RNAi) serves as an innate antiviral mechanism in plants, fungi and animals. Human viruses, like plant viruses, encode suppressor proteins or RNAs that block or modulate the RNAi pathway. This review summarizes the mechanisms by which pathogenic human viruses
Full Text Available RNA interference (RNAi has been harnessed to produce a new class of drugs for treatment of various diseases. This review summarizes the most important parameters that govern the silencing efficiency and duration of the RNAi effect such as small interfering RNA (siRNA stability and modification, the type of delivery system and particle sizing methods. It also discusses the predominant barriers for siRNA delivery, such as off-target effects and introduces internalization, endosomal escape and mathematical modeling in RNAi therapy and combinatorial RNAi. At present, effective delivery of RNAi therapeutics in vivo remains a challenge although significant progress has been made in this field.
Spracklin, George; Fields, Brandon; Wan, Gang; Becker, Diveena; Wallig, Ashley; Shukla, Aditi; Kennedy, Scott
Gene silencing mediated by dsRNA (RNAi) can persist for multiple generations in Caenorhabditis elegans (termed RNAi inheritance). Here we describe the results of a forward genetic screen in C. elegans that has identified six factors required for RNAi inheritance: GLH-1/VASA, PUP-1/CDE-1, MORC-1, SET-32, and two novel nematode-specific factors that we term here (heritable RNAi defective) HRDE-2 and HRDE-4 The new RNAi inheritance factors exhibit mortal germline (Mrt) phenotypes, which we show is likely caused by epigenetic deregulation in germ cells. We also show that HRDE-2 contributes to RNAi inheritance by facilitating the binding of small RNAs to the inheritance Argonaute (Ago) HRDE-1 Together, our results identify additional components of the RNAi inheritance machinery whose conservation provides insights into the molecular mechanism of RNAi inheritance, further our understanding of how the RNAi inheritance machinery promotes germline immortality, and show that HRDE-2 couples the inheritance Ago HRDE-1 with the small RNAs it needs to direct RNAi inheritance and germline immortality. Copyright © 2017 by the Genetics Society of America.
Shan, Xiu-ying; Liu, Zhao-liang; Wang, Biao; Guo, Guo-xiang; Wang, Mei-shui; Zhuang, Fu-lian; Cai, Chuan-shu; Zhang, Ming-feng; Zhang, Yan-ding
To construct lentivector carrying Tie2-Small interfering RNA (SiRNA), so as to study its influence on malignant melanoma cells. Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with XbaI, ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-I or pNL-EGFP-U6-Tie2-II, and then the electrophoresis clones was sequenced. Plasmids of pNL-EGFP-U6-Tie2-I and pNL-EGFP-U6-Tie2-II were constructed and combined with pVSVG and pHelper, respectively, to constitute lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Tie2- I and pNL-EGFP-U6-Tie2-II lentivirus. Then the supernatant was collected to determine the titer. Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing. And the titer of lentiviral vector was 8.8 x 10(3)/ml, which was determined by 293T cell. The results of Realtime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells (P0.05) between the two lentiviral vectors of Tie2-RNAi. Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly. The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.
Fei Zhaoliang; Chen Zheng; Wang Zhugang; Fei Jian
RNA interference (RNAi) is a powerful technique and is widely used to down-regulate expression of specific genes in cultured cells and in vivo. In this paper, we report our development of a new tetracycline-inducible RNAi expression using a modified TRE-mouse U6 promoter in which the distal sequence element (DSE) was replaced by the tetracycline-responsive element (TRE). The modified TRE-mouse U6 promoter can be activated by a Tet-on version tetracycline-regulated artificial activator rTetOct which was constructed by fusing the rtTA DNA binding domain with the Oct-1 POU activation domain. This rTetOct/TRE-U6 system was successfully applied to conditionally and reversibly down-regulate the expression of endogenous p53 gene in MCF7 cells, and the expression of β-defensin gene (mBin1b) either transiently expressed in COS7 cells or stably expressed in CHO cells
Full Text Available Abstract Background Grasshopper serves as important model system in neuroscience, development and evolution. Representatives of this primitive insect group are also highly relevant targets of pest control efforts. Unfortunately, the lack of genetics or gene specific molecular manipulation imposes major limitations to the study of grasshopper biology. Results We investigated whether juvenile instars of the grasshopper species Schistocerca americana are conducive to gene silencing via the systemic RNAi pathway. Injection of dsRNA corresponding to the eye colour gene vermilion into first instar nymphs triggered suppression of ommochrome formation in the eye lasting through two instars equivalent to 10–14 days in absolute time. QRT-PCR analysis revealed a two fold decrease of target transcript levels in affected animals. Control injections of EGFP dsRNA did not result in detectable phenotypic changes. RT-PCR and in situ hybridization detected ubiquitous expression of the grasshopper homolog of the dsRNA channel protein gene sid-1 in embryos, nymphs and adults. Conclusion Our results demonstrate that systemic dsRNA application elicits specific and long-term gene silencing in juvenile grasshopper instars. The conservation of systemic RNAi in the grasshopper suggests that this pathway can be exploited for gene specific manipulation of juvenile and adult instars in a wide range of primitive insects.
Full Text Available Abstract Background shRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF and cell enzyme linked immunosorbent assays (cell ELISA, and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells. Results Our results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 106 TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 102 TCID50 of FMDV. Conclusions Iαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.
Dalzell, Johnathan J; Warnock, Neil D; McVeigh, Paul; Marks, Nikki J; Mousley, Angela; Atkinson, Louise; Maule, Aaron G
Almost a decade has passed since the first report of RNA interference (RNAi) in a parasitic helminth. Whilst much progress has been made with RNAi informing gene function studies in disparate nematode and flatworm parasites, substantial and seemingly prohibitive difficulties have been encountered in some species, hindering progress. An appraisal of current practices, trends and ideals of RNAi experimental design in parasitic helminths is both timely and necessary for a number of reasons: firstly, the increasing availability of parasitic helminth genome/transcriptome resources means there is a growing need for gene function tools such as RNAi; secondly, fundamental differences and unique challenges exist for parasite species which do not apply to model organisms; thirdly, the inherent variation in experimental design, and reported difficulties with reproducibility undermine confidence. Ideally, RNAi studies of gene function should adopt standardised experimental design to aid reproducibility, interpretation and comparative analyses. Although the huge variations in parasite biology and experimental endpoints make RNAi experimental design standardization difficult or impractical, we must strive to validate RNAi experimentation in helminth parasites. To aid this process we identify multiple approaches to RNAi experimental validation and highlight those which we deem to be critical for gene function studies in helminth parasites.
Lee, Sang-Kyung; Kumar, Priti
RNA interference (RNAi) has the potential to permit the downregulation of virtually any gene. While transgenic RNAi enables stable propagation of the resulting phenotype to progeny, the dominant nature of RNAi limits its use to applications where the continued suppression of gene expression does not disturb normal cell functioning. This is of particular importance when the target gene product is essential for cell survival, development or differentiation. It is therefore desirable that knockdown be externally regulatable. This review is aimed at providing an overview of the approaches for conditional RNAi in mammalian systems, with a special mention of studies employing these approaches to target therapeutically/biologically relevant molecules, their advantages and disadvantages, and a pointer towards approaches best suited for RNAi-based gene therapy.
Yu, Yanping; Zhang, Song; Kong, Weijia
To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human ABCG2 gene, to assay the expression of ABCG2 in a human nasopharyngeal carcinoma (NPC) cell line, CEN-2 cell line, and to detect the RNAi effect of shRNA. Targeting ABCG2 gene sequence, three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and then identified by restriction digestion, PCR and sequencing. The recombinant plasmids were transfected into CEN-2 cells. ABCG2 expression was assayed by real-time quantitative PCR and Western blot. The construction of pGC-silencer-U6/Neo/GFP/ABCG2 was succeed. The shRNA plasmids significantly down-regulated the ABCG2 expression in CEN-2 cells, at both mRNA level and protein level. Recombinant plasmid 1 had the strongest effect compared with plasmids 2 and 3 (P < 0.05), with an inhibition ratio of 75% at the mRNA level and 68% at the protein level. pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells, which could help further studies of ABCG2 functions CEN-2 cell line and contribute to the NPC gene therapy.
Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael
RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.
Dec 29, 2008 ... structure and genome integrity (Hannon, 2002; Grewal and Moazed ... function. COMPONENTS OF RNAi. Among the components of gene silencing process, some serve as .... PTGS technology has many advantages: It is.
Chen, Jing; Xie, Jianping
Jing Chen, Jianping XieInstitute of Modern Biopharmaceuticals, State Key Laboratory Breeding Base of Ministry of Education Eco-Environment of the Three Gorges Reservoir Region, School of Life Sciences, Southwest University, Chongqing, ChinaAbstract: RNA interference (RNAi) is a promising strategy to suppress the expression of disease-relevant genes and induce post-transcriptional gene silencing. Their simplicity and stability endow RNAi with great advantages in molecular medicine. Several RNA...
Vasselon, Thierry; Bouttier, Manuella; Saumet, Anne; Lecellier, Charles-Henri
RNA interference (RNAi) is a potent cellular system against viruses in various organisms. Although common traits are observed in plants, insects, and nematodes, the situation observed in mammals appears more complex. In mammalian somatic cells, RNAi is implicated in endonucleolytic cleavage mediated by artificially delivered small interfering RNAs (siRNAs) as well as in translation repression mediated by microRNAs (miRNAs). Because siRNAs and miRNAs recognize viral mRNAs, RNAi inherently limits virus production and participates in antiviral defense. However, several observations made in the cases of hepatitis C virus and retroviruses (including the human immunodeficiency virus and the primate foamy virus) bring evidence that this relationship is much more complex and that certain components of the RNAi effector complex [called the RNA-induced silencing complex (RISC)], such as AGO2, are also required for viral replication. Here, we summarize recent discoveries that have revealed this dual implication in virus biology. We further discuss their potential implications for the functions of RNAi-related proteins, with special emphasis on retrotransposition and genome stability.
Use of ribonucleic acid interference, RNAi, to reduce plant feeding Hemiptera in fruit tree and grapevines. The successful use of RNAi strategies to reduce insect pests, psyllids and leafhoppers was demonstrated. An RNAi bioassay which absorbs dsRNA into plant tissues provided up to 40 days of act...
Berkhout, Ben; ter Brake, Olivier
Background: RNA interference (RNAi) can be employed as a potent antiviral mechanism Objective: To discuss RNAi approaches to target pathogenic human viruses causing acute or chronic infections, in particular RNAi gene therapy against HIV-1. Methods: A review of relevant literature.
Josefsen, Knud; Lee, Ying Chiu
Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify...
Bakhtizin, R.Z.; Yumaguzin, Yu.M.
The VEhU-6 charnel electron multiplier timing characteristics are experimentally studied. Dependence of monoelectron pulse duration at the VEhU-6 output at different values of channel supply voltage is investigated. The VEhU-6 delay time is measured. Delay time increased from 10 to 30 ns with the increase of channel supply voltage from 2.8 to 3.2 kV (at approximately 10 5 pulse/s loading). Delay time increases with loading decrease
Meyer, M. K.; Wiencek, T. C.; Hayes, S. L.; Hofman, G. L.
Irradiation testing of U 6Mn-Al dispersion fuel miniplates was conducted in the Oak Ridge Research Reactor (ORR). Post-irradiation examination showed that U 6Mn in an unrestrained plate configuration performs similarly to U 6Fe under irradiation, forming extensive and interlinked fission gas bubbles at a fission density of approximately 3×10 27 m-3. Fuel plate failure occurs by fission gas pressure driven `pillowing' on continued irradiation.
Didychuk, Allison L; Montemayor, Eric J; Carrocci, Tucker J; DeLaitsch, Andrew T; Lucarelli, Stefani E; Westler, William M; Brow, David A; Hoskins, Aaron A; Butcher, Samuel E
U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. Here, we report structures of the U6 RNA processing enzyme Usb1 from yeast and a substrate analog bound complex from humans. Unlike the human ortholog, we show that yeast Usb1 has cyclic phosphodiesterase activity that leaves a terminal 3' phosphate which prevents overprocessing. Usb1 processing of U6 RNA dramatically alters its affinity for cognate RNA-binding proteins. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp1, Prp24, Usb1 and Lsm2-8) and anti-cooperative interactions between Prp24 and Lhp1. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome.The mechanism of U6 small nuclear ribonucleoprotein (snRNP) biogenesis is not well understood. Here the authors characterize the enzymatic activities and structures of yeast and human U6 RNA processing enzyme Usb1, reconstitute post-transcriptional assembly of yeast U6 snRNP in vitro, and propose a model for U6 snRNP assembly.
Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W
RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response.
Larruga José M
Full Text Available Abstract Background World-wide phylogeographic distribution of human complete mitochondrial DNA sequences suggested a West Asian origin for the autochthonous North African lineage U6. We report here a more detailed analysis of this lineage, unraveling successive expansions that affected not only Africa but neighboring regions such as the Near East, the Iberian Peninsula and the Canary Islands. Results Divergence times, geographic origin and expansions of the U6 mitochondrial DNA clade, have been deduced from the analysis of 14 complete U6 sequences, and 56 different haplotypes, characterized by hypervariable segment sequences and RFLPs. Conclusions The most probable origin of the proto-U6 lineage was the Near East. Around 30,000 years ago it spread to North Africa where it represents a signature of regional continuity. Subgroup U6a reflects the first African expansion from the Maghrib returning to the east in Paleolithic times. Derivative clade U6a1 signals a posterior movement from East Africa back to the Maghrib and the Near East. This migration coincides with the probable Afroasiatic linguistic expansion. U6b and U6c clades, restricted to West Africa, had more localized expansions. U6b probably reached the Iberian Peninsula during the Capsian diffusion in North Africa. Two autochthonous derivatives of these clades (U6b1 and U6c1 indicate the arrival of North African settlers to the Canarian Archipelago in prehistoric times, most probably due to the Saharan desiccation. The absence of these Canarian lineages nowadays in Africa suggests important demographic movements in the western area of this Continent.
Kruithof Egbert KO
Full Text Available Abstract Background RNA interference (RNAi can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs, used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2 mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death. Results As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers or more increased expression of the oligoadenylate synthase-1 (OAS1 interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing. Conclusions Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained
Full Text Available Genetically modified (GM crops producing double-stranded RNAs (dsRNAs are being investigated largely as an RNA interference (RNAi-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (Anthonomus grandis, we showed that the chimeric protein PTD-DRBD (peptide transduction domain—dsRNA binding domain combined with dsRNA forms a ribonucleoprotein particle (RNP that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.
Gillet, François-Xavier; Garcia, Rayssa A; Macedo, Leonardo L P; Albuquerque, Erika V S; Silva, Maria C M; Grossi-de-Sa, Maria F
Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil ( Anthonomus grandis ), we showed that the chimeric protein PTD-DRBD (peptide transduction domain-dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.
Huang, Yuping; Wang, Yajun; Zeng, Baosheng; Liu, Zhaoxia; Xu, Xuejiao; Meng, Qian; Huang, Yongping; Yang, Guang; Vasseur, Liette; Gurr, Geoff M; You, Minsheng
RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest. Copyright © 2017 Elsevier Ltd. All rights reserved.
Full Text Available RNA interference (RNAi is the process of mRNA degradation induced by double-stranded RNA in a sequence-specific manner. Different types of promoters, such as U6, H1, tRNA, and CMV, have been used to control the inhibitory effect of RNAi expression vectors. In the present study, we constructed two shRNA expression vectors, respectively, controlled by tRNAlys and CMV enhancer-tRNAlys promoters. Compared to the vectors with tRNAlys or U6 promoter, the vector with a CMV enhancer-tRNAlys promoter silenced pokemon more efficiently on both the mRNA and the protein levels. Meanwhile, the silencing of pokemon inhibited the proliferation of MCF7 cells, but the induction of apoptosis of MCF7 cells was not observed. We conclude that the CMV enhancer-tRNAlys promoter may be a powerful tool in driving intracellular expression of shRNA which can efficiently silence targeted gene.
Weiwei, Ma; Zhenhua, Xie; Feng, Liu; Hang, Ning; Yuyang, Jiang
RNA interference (RNAi) is the process of mRNA degradation induced by double-stranded RNA in a sequence-specific manner. Different types of promoters, such as U6, H1, tRNA, and CMV, have been used to control the inhibitory effect of RNAi expression vectors. In the present study, we constructed two shRNA expression vectors, respectively, controlled by tRNAlys and CMV enhancer-tRNAlys promoters. Compared to the vectors with tRNAlys or U6 promoter, the vector with a CMV enhancer-tRNAlys promoter silenced pokemon more efficiently on both the mRNA and the protein levels. Meanwhile, the silencing of pokemon inhibited the proliferation of MCF7 cells, but the induction of apoptosis of MCF7 cells was not observed. We conclude that the CMV enhancer-tRNAlys promoter may be a powerful tool in driving intracellular expression of shRNA which can efficiently silence targeted gene. PMID:19859553
RNAi-based silencing of genes encoding the vacuolar- ATPase subunits a and c in pink bollworm (Pectinophora gossypiella). Ahmed M. A. Mohammed. Abstract. RNA interference is a post- transcriptional gene regulation mechanism that is predominantly found in eukaryotic organisms. RNAi demonstrated a successful ...
RNA interference (RNAi) is a homology-dependent gene silencing technology that is initiated by double stranded RNA (dsRNA). It has emerged as a genetic tool for engineering plants resistance against prokaryotic pathogens such as virus and bacteria. Recent studies broaden the role of RNAi, and many successful ...
Pijlman, Gorben P
Flaviviruses are important human pathogens that are transmitted by invertebrate vectors, mostly mosquitoes and ticks. During replication in their vector, flaviviruses are subject to a potent innate immune response known as antiviral RNA interference (RNAi). This defense mechanism is associated with the production of small interfering (si)RNA that lead to degradation of viral RNA. To what extent flaviviruses would benefit from counteracting antiviral RNAi is subject of debate. Here, the experimental evidence to suggest the existence of flavivirus RNAi suppressors is discussed. I will highlight the putative role of non-coding, subgenomic flavivirus RNA in suppression of RNAi in insect and mammalian cells. Novel insights from ongoing research will reveal how arthropod-borne viruses modulate innate immunity including antiviral RNAi. Copyright © 2014 Elsevier B.V. All rights reserved.
Dowling, Daniel; Pauli, Thomas; Donath, Alexander
RNAinterference (RNAi) refers tothe set ofmolecular processes foundin eukaryotic organisms in which smallRNAmolecules mediate the silencing or down-regulation of target genes. In insects, RNAi serves a number of functions, including regulation of endogenous genes, anti-viral defense, and defense...... against transposable elements. Despite being well studied in model organisms, such as Drosophila, the distribution of core RNAi pathway genes and their evolution in insects is not well understood. Here we present the most comprehensive overview of the distribution and diversity of core RNAi pathway genes...... across 100 insect species, encompassing all currently recognized insect orders. We inferred the phylogenetic origin of insect-specific RNAi pathway genes and also identified several hitherto unrecorded gene expansions using whole-body transcriptome data from the international 1KITE (1000 Insect...
Hayes, D. B.; Hall, C.; Hixson, R. S.
Prior shock experiments on the alloy uranium-niobium-6 wt.% (U6Nb) were absent an elastic precursor when one was expected (A. K. Zurek, et. al., Journal de Physique IV, 10 (#9) p677-682). This was later explained as a consequence of shear stress relaxation from time-dependent twinning that prevented sufficient shear stress for plastic yielding. (D. B. Hayes, et. al., Shock Compression of Condensed Matter-2003, p1177, American Institute of Physics 2004) Pressure was ramped to 13 GPa in 150-ns on eight U6Nb specimens with thicknesses from 0.5 -- 1.1-mm and the back surface velocities were measured with laser interferometry. This pressure load produces a stress wave with sufficiently fast rise time so that, according to the prior work, twins do not have time to form. Four of the U6Nb specimens had been cold-rolled which increased the yield stress. Each velocity history was analyzed with a backward integration analysis to give the stress-strain response of the U6Nb. Comparison of these results with prior Hugoniot measurements shows that the U6Nb in the present experiments responds as an elastic-plastic material and the deduced yield strength of the baseline and of the cold-rolled material agree with static results.
12 years following the discovery of the RNAi mechanism in Man, a number of RNAi therapeutics development candidates have emerged with profiles suggesting that they could become drugs of significant medical importance for diseases like TTR amyloidosis, HBV, solid cancers, and hemophilia. Despite this robust progress, the perception of RNAi therapeutics has been on a roller-coaster ride driven not only by science, but also regulatory trends, the stock markets, and Big Pharma business development decisions . This presentation provides an update on the current state of RNAi therapeutics development with a particular focus on what RNAi delivery can achieve today and key challenges to be overcome to expand therapeutic opportunities. The delivery of RNAi triggers to disease-relevant cell types clearly represents the rate-limiting factor in broadly expanding the applicability of RNAi therapeutics. Today, with at least 3 delivery options (lipid nanoparticles/LNPs, GalNAc-siRNA conjugates, Dynamic PolyConjugates/DPCs) for which profound gene knockdowns have been demonstrated in non-human primates and in the clinic, RNAi therapeutics should in principle be able to address most diseases related to gene expression in the liver. Given the central importance of the liver in systemic physiology, this already represents a significant therapeutic and commercial opportunity rivaling that of e.g. monoclonal antibodies. Beyond the liver, there is a reason to believe that current RNAi therapeutics technologies can address a number of solid tumors (e.g. LNPs), diseases of the eye (e.g. self-delivering RNAi triggers) as well as diseases involving the respiratory epithelium (e.g. aerosolized LNPs), certain phagocytic cells (LNPs), hematopoietic stem cells and their progeny (lentiviral DNA-directed RNAi), vascular endothelial cells (cationic lipoplexes), and certain cell types in the kidney (self-delivering RNAi triggers, DPCs; Table 1). Despite this success, there has been a sense that
Lindbo, John A; Dougherty, William G
This article describes the discovery of RNA-activated sequence-specific RNA degradation, a phenomenon now referred to as RNA silencing or RNA interference (RNAi). From 1992 to 1996, a series of articles were published on virus resistant transgenic plants expressing either translatable or nontranslatable versions of the coat protein gene of Tobacco etch virus (TEV). Certain transgenic plant lines were resistant to TEV but not to closely related viruses. In these plants a surprising correlation was observed: Transgenic plant lines with the highest degree of TEV resistance had actively transcribed transgenes but low steady-state levels of transgene RNA. Molecular analysis of these transgenic plants demonstrated the existence of a cellular-based, sequence-specific, posttranscriptional RNA-degradation system that was programmed by the transgene-encoded RNA sequence. This RNA-degradation activity specifically targeted both the transgene RNA and TEV (viral) RNA for degradation and was the first description of RNA-mediated gene silencing.
Ellermeier, Chad; Higuchi, Emily C; Phadnis, Naina
During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essen......During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes....... Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between...... types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis....
Baake, M.; Reinicke, P.
In this paper, we present the group theory of models with broken u(6/2j+1) supersymmetry described by the chain u(6/2j+1) contains usub(B)(6) x usub(F)(2j+1) contains usub(B)(6) x spsub(F)(2j+1) contains ... contains sosub(B)(3) x susub(F)(2) contains susub(B+F)(2) which has recently been suggested for application to nuclear physics. We present all invariants that are needed for the construction of the general Hamiltonian for this model. (orig.)
Vergnes, M.; Grafeuille, S.; Rotbard, G.
It was suggested recently that the level schemes of the odd Hg isotopes with 193 200 Hg, 198 Hg, 196 Hg(p,d) 199 Hg, 197 Hg, 195 Hg. The comparison of the experimental spectroscopic factors with the ones computed using the U(6/12) model shows that, among the three nuclei studied, 195 Hg and 197 Hg can be considered as reasonably described. The agreement in this case with U(6/12) is better in the U(5) limit than in the O(6) limit
Weinstein, Shiri; Peer, Dan
RNAi, as a novel therapeutic modality, has an enormous potential to bring the era of personalized medicine one step further from notion into reality. However, delivery of RNAi effector molecules into their target tissues and cells remain extremely challenging. Major attempts have been made in recent years to develop sophisticated nanocarriers that could overcome these hurdles. This review will present the recent progress with the challenges and opportunities in this emerging field, focusing mostly on the in vivo applications with special emphasis on the strategies for RNAi delivery into immune cells. (topical review)
Full Text Available ABSTRAK PEMBUATAN SERBUK PADUAN U-6Zr DENGAN PENGKAYAAN URANIUM 19,75 % UNTUK BAHAN BAKAR REAKTOR RISET. Telah dilakukan pembuatan serbuk paduan U-6Zr dengan pengkayaan 19,75 % untuk bahan bakar reaktor riset. Pembuatan bahan bakar U-6Zr ini dalam rangka mencari bahan bakar baru yang mempunyai densitas tinggi untuk mengganti bahan bakar yang sudah ada U3Si2-Al. Tujuan dari percobaan ini untuk mengetahui sifat-sifat serbuk paduan U- 6Zr yang diperoleh dari proses hydriding-dehydriding sebagai kandidat bahan bakar reaktor riset. Serbuk yang diperoleh dari proses hydriding-dehydriding dikenai pengujian, diantaranya pungujian komposisi kimia, densitas, kandungan hidrogen, fasa dan sifat termal. Hasil pengujian komposisi kimia menunjukkan beberapa unsur seperti Al, Ca, Cu, dan Ni melebihi batas yang diijinkan dimana masing-masing unsur terdapat sebesar 202,21 ppm; 214,05 ppm; 61,25 ppm dan 134,53 ppm. Pada pengujian diperolah densitas serbuk U-6Zr sebesar 13,58 g/cm3 dan pada pengujian kandungan hidrogen sisa diperoleh kandungan hidrogen sebesar 0,16 %. Untuk pengujian fasa, diperoleh fasa αU dan δU, sedangkan pada pengujian sifat termal yakni transformasi temperatur terdapat dua puncak yakni puncak pertama terjadi pada temperatur 274 hingga 311 oC dan puncak kedua terjadi pada temperatur 493 hingga 527oC. Puncak pertama terjadi reaksi endotermik dengan menyerap panas sebesar ∆H = 6,23 cal/g tetapi tidak terbentuk fasa baru, sedangkan puncak kedua terjadi reaksi eksotermik dengan mengeluarkan panas sebesar ∆H = -9.34 cal/g dan terbentuk fasa αZr. Sementara itu, dari pengujian kapasitas panas pada temperatur 34 hingga 75 oC, terjadinya penurunan nilai kapasitas panas yang disertai dengan penyerapan panas. Pada temperatur yang lebih tinggi hingga temperatur 437oC nilai kapasitas panas menjadi lebih kecil disertai pengeluaran panas. Reaksi termokimia antara Zr dengan hidrogen sisa menunjukkan terbentuknya fasa αZr yang diindikasikan oleh reaksi
Full Text Available Depletion of amyotrophic lateral sclerosis (ALS-associated transactivation response (TAR RNA/DNA-binding protein 43 kDa (TDP-43 alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function.
Gerbi, Susan A.; Lange, Thilo Sascha
Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3′ end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3′-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3′ hydroxyl of U6 snRNA to a 3′ phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies. PMID:12221120
Casacuberta, Josep M; Devos, Yann; du Jardin, Patrick; Ramon, Matthew; Vaucheret, Hervé; Nogué, Fabien
RNAi offers opportunities to generate new traits in genetically modified (GM) plants. Instead of expressing novel proteins, RNAi-based GM plants reduce target gene expression. Silencing of off-target genes may trigger unintended effects, and identifying these genes would facilitate risk assessment. However, using bioinformatics alone is not reliable, due to the lack of genomic data and insufficient knowledge of mechanisms governing mRNA-small (s)RNA interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.
Mamta, B; Rajam, M V
The insect pests are big threat in meeting the food demands for future generation. The present pest control strategies, including the existing transgenic approaches show certain limitations and are not completely successful in limiting the insect pests. However, the sequence-specific gene silencing via RNA interference (RNAi) holds a great promise for effective management of agricultural pests. RNAi is naturally occurring conserved process responsible for gene regulation and defense against pathogens. The efficacy of RNAi varies among different insect orders and also depends upon various factors, including the target gene selection, method of dsRNAs delivery, expression of dsRNAs and presence of off-target effects. RNAi-mediated silencing of different insect genes involved in various physiological processes was found to be detrimental to insects growth, development and survival. In this article, we have reviewed the potential of RNAi-based strategies for effective management of insect pests. We have also discussed the various parameters, which are to be considered for host-induced RNAi-mediated control of insect pests without producing any effect on non-target organisms and environment.
Full Text Available RNA interference (RNAi is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.
Julia F Pielage
Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.
Department of Homeland Security — HSIP Non-Crossing Ports-of-Entry A Port of Entry is any designated place at which a CBP officer is authorized to accept entries of merchandise to collect duties, and...
Morrison, I.; Jarvis, P.D.
A supersymmetric extension of the interacting boson model (IBM) is constructed to describe high-spin anomalies in both even- and odd-mass spectra of the Hg, Pt region (190<=A<=200). Supergroup chains such as U(6/2j+1)containsOsp(6/2j+1)containsO(6)xSp(2j+1)... or U(6/2j+1)containsU(5/2j+1)xU(1)containsOsp(5/2j+1)... incorporate a single j-shell fermion in addition to the usual 's' and 'd' bosons (L=0 and L=2). The orthosympletic supergroup reflects the strong pairing force in the subspace of the fermion intruder level. The model agrees favourably with experiment and microscopic calculation. (orig.)
Aikin, Jr., Robert M. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
This report describes work to further develop a sound technical basis and best practices for mold design and process parameters for the Direct Casting of U-6wt%Nb components. One major challenge to the production of U-6Nb components is the propensity for niobium segregation during casting and solidification. This is especially true for cylindrical castings where the vertical side walls allow flotation of Nb resulting in severe inverse macrosegregation. In this work, a small (120 mm diameter by 180 mm tall) and large cylinder (250 mm diameter by 310 mm tall) are examined with a focus on reducing, or eliminating, niobium segregation. It is demonstrated that counter gravity casting (top-to-bottom solidification) can be used to minimize segregation in the small cylinder. Attempts to counter gravity cast the large cylinder were unsuccessful, in large part due to size limitations of the current furnace. A path forward for casting of the large cylinders is discussed.
DeLong, L.E.; Crabtree, G.W.; Hall, L.N.; Kierstead, H.; Aoki, H.; Dhar, S.K.; Gschneidner, K.A. Jr.; Junod, A.
High purity U 6 Fe polycrystals with a resistance ratio rrr=9 at T=4 K are found to exhibit a record onset temperature (T 0 >4.0 K) to superconductivity for U materials. Our measured values of the electronic coefficient of heat capacity Csup(*)=(150+-3) mJ/mol.K 2 , the mean Debye temperature THETA(T->0)=116 K and the normalized jump in heat capacity at Tsub(c), ΔC/γsup(*)Tsub(c)=2.3+-0.1, are all in good agreement with previous work, and suggest that U 6 Fe is a strong-coupled superconductor. Resistive upper critical field measurements on high purity samples revealed a pronounced S-shaped curvature of Hsub(c2)(T) and evidence for anisotropy, similar to effects seen in UPt 3 , but not observed in lower purity U 6 Fe samples. We find Hsub(c2)(0)>10 T and transition widths ΔTsub(c)proportionalHsup(1/2) at moderate fields, followed by an abrupt increase in ΔTsub(c) for H>8 T. Our Hsub(c2) data cannot be explained by existing theoretical models. Preliminary heat capacity measurements in magnetic fields 0 6 Fe at T< or approx.110 K. (orig.)
Hayes, D. B.; Gray, G. T. III; Hixson, R. S.; Hall, C. A.
When uranium-niobium (6 wt.%) alloy is shock loaded, the expected elastic precursor is absent. A prior model attributed this absence to shear-induced twinning and the concomitant shear stress reduction that prevented the shocked material from reaching the plastic yield point. In the present study, carefully prepared U6Nb was subjected to shock loading to verify the adequacy of the prior model. Other samples were loaded with a ramp pressure pulse with strain rate large enough so that significant twinning would not occur during the experiment. Backward integration analyses of these latter experiments' back surface motion give stress-strain loading paths in U6Nb that suggest ordinary elastic-plastic flow. Some of the U6Nb was pre-strained by cold rolling in an effort to further ensure that twinning did not affect wave propagation. Shock and ramp loadings yielded similar results to the baseline material except, as expected, they are consistent with a higher yield stress and twinning shear stress threshold
Fluorescence microscopy is one of the most powerful tools to investigate complex cellular processes such as cell division, cell motility, or intracellular trafficking. The availability of RNA interference (RNAi) technology and automated microscopy has opened the possibility to perform cellular imaging in functional genomics and other large-scale applications. Although imaging often dramatically increases the content of a screening assay, it poses new challenges to achieve accurate quantitative annotation and therefore needs to be carefully adjusted to the specific needs of individual screening applications. In this review, we discuss principles of assay design, large-scale RNAi, microscope automation, and computational data analysis. We highlight strategies for imaging-based RNAi screening adapted to different library and assay designs. PMID:20176920
Katharine J Sepp
Full Text Available While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new
RNA interference (RNAi) is a post-transcriptional gene silencing process triggered by double-strand RNA, including synthetic short interfering RNA (siRNA) and endogenous microRNA (miRNA). RNAi has attracted great attention for developing a new class of therapeutics, due to its capability to speci......RNA/miRNA and transport them to the action site in the target cells. This thesis describes the development of various nanocarriers for siRNA/miRNA delivery and investigate their potential biomedical applications including: anti-inflammation, tissue engineering and cancer...
U-6 wt % Nb conventionally is water quenched from 800 0 C in order to obtain a niobium supersaturated α'' structure having good corrosion resistance and high ductility (125% tensile elongation). The high cooling rate associated with the water quench, however, produces undesirable distortion and residual stress. This study was conducted to determine the extent to which the quench rate could be reduced (in order to minimize the distortion and residual stress problems) without sacrificing properties. The results indicate that quench rate can be reduced by as much as a factor of 10 without any loss of ductility, and that a factor of 100 reduction in quench rate (as is produced by air cooling) still produces material with moderate ductility (> 12% tensile elongation). The results also indicate that supersaturated α'' structures are produced at all of these quench rates. This suggests that these reductions in quench rate should not have drastic adverse effects on corrosion resistance. Hence, it should not be possible to substantially reduce the magnitudes of the distortion and residual stress problems while retaining appreciable ductility and corrosion resistance in U-6 wt % Nb
van Mierlo, J.T.; van Cleef, K.W.; Rij, R.P. van
RNA interference (RNAi) is an important pathway to combat virus infections in insects and plants. Hallmarks of antiviral RNAi in these organisms are: (1) an increase in virus replication after inactivation of major actors in the RNAi pathway, (2) production of virus-derived small interfering RNAs
Malík, Radek; Svoboda, Petr
Roč. 134, 1-2 (2012), s. 64-68 ISSN 0378-4320 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : RNAi * oocyte * transgene * silencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.897, year: 2012
Flaviviruses are important human pathogens that are transmitted by invertebrate vectors, mostly mosquitoes and ticks. During replication in their vector, flaviviruses are subject to a potent innate immune response known as antiviral RNA interference (RNAi). This defense mechanism is associated with
RNA interference (RNAi) is a specific technique using only a few double stranded RNA (dsRNA) molecules to stop the expression which has made it one of the important areas in molecular biology. By introducing a gene into the host genome which is highly homologous to an endogenous gene, the RNA silencing is ...
Full Text Available Abstract Background RNA interference (RNAi becomes an increasingly important and effective genetic tool to study the function of target genes by suppressing specific genes of interest. This system approach helps identify signaling pathways and cellular phase types by tracking intensity and/or morphological changes of cells. The traditional RNAi screening scheme, in which one siRNA is designed to knockdown one specific mRNA target, needs a large library of siRNAs and turns out to be time-consuming and expensive. Results In this paper, we propose a conceptual model, called compressed sensing RNAi (csRNAi, which employs a unique combination of group of small interfering RNAs (siRNAs to knockdown a much larger size of genes. This strategy is based on the fact that one gene can be partially bound with several small interfering RNAs (siRNAs and conversely, one siRNA can bind to a few genes with distinct binding affinity. This model constructs a multi-to-multi correspondence between siRNAs and their targets, with siRNAs much fewer than mRNA targets, compared with the conventional scheme. Mathematically this problem involves an underdetermined system of equations (linear or nonlinear, which is ill-posed in general. However, the recently developed compressed sensing (CS theory can solve this problem. We present a mathematical model to describe the csRNAi system based on both CS theory and biological concerns. To build this model, we first search nucleotide motifs in a target gene set. Then we propose a machine learning based method to find the effective siRNAs with novel features, such as image features and speech features to describe an siRNA sequence. Numerical simulations show that we can reduce the siRNA library to one third of that in the conventional scheme. In addition, the features to describe siRNAs outperform the existing ones substantially. Conclusions This csRNAi system is very promising in saving both time and cost for large-scale RNAi
Full Text Available This article intends to contribute to the understanding of Jesus’ entry into Jerusalem. The author studies the entry, which is found in all the Gospels, in its Jewish context. The author argues that Jesus’ entry into Jerusalem on an ass is to be understood as a prophetic sign which was primarily meant to convey a message to the Jews.
Arif, Muhammad Asif; Frank, Wolfgang; Khraiwesh, Basel
RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. 2013 by the authors; licensee MDPI, Basel, Switzerland.
Arif, Muhammad Asif
RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. 2013 by the authors; licensee MDPI, Basel, Switzerland.
Zheng, Xiaomei; Zheng, Ping; Sun, Jibin; Kun, Zhang; Ma, Yanhe
U6 promoters have been used for single guide RNA (sgRNA) transcription in the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) genome editing system. However, no available U6 promoters have been identified in Aspergillus niger, which is an important industrial platform for organic acid and protein production. Two CRISPR/Cas9 systems established in A. niger have recourse to the RNA polymerase II promoter or in vitro transcription for sgRNA synthesis, but these approaches generally increase cloning efforts and genetic manipulation. The validation of functional RNA polymerase II promoters is therefore an urgent need for A. niger . Here, we developed a novel CRISPR/Cas9 system in A. niger for sgRNA expression, based on one endogenous U6 promoter and two heterologous U6 promoters. The three tested U6 promoters enabled sgRNA transcription and the disruption of the polyketide synthase albA gene in A. niger . Furthermore, this system enabled highly efficient gene insertion at the targeted genome loci in A. niger using donor DNAs with homologous arms as short as 40-bp. This study demonstrated that both heterologous and endogenous U6 promoters were functional for sgRNA expression in A. niger . Based on this result, a novel and simple CRISPR/Cas9 toolbox was established in A. niger, that will benefit future gene functional analysis and genome editing.
Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M
RNA interference (RNAi) is a conserved eukaryotic mechanism that uses small RNA molecules to suppress gene expression through sequence-specific messenger RNA degradation, translational repression, or transcriptional inhibition. In filamentous fungi, the protective function of RNAi in the maintenance of genome integrity is well known. However, knowledge of the regulatory role of RNAi in fungi has had to wait until the recent identification of different endogenous small RNA classes, which are generated by distinct RNAi pathways. In addition, RNAi research on new fungal models has uncovered the role of small RNAs and RNAi pathways in the regulation of diverse biological functions. In this review, we give an up-to-date overview of the different classes of small RNAs and RNAi pathways in fungi and their roles in the defense of genome integrity and regulation of fungal physiology and development, as well as in the interaction of fungi with biotic and abiotic environments.
The crystal structures of uranyl minerals and inorganic uranyl compounds are important for understanding the genesis of U deposits, the interaction of U mine and mill tailings with the environment, transport of actinides in soils and the vadose zone, the performance of geological repositories for nuclear waste, and for the development of advanced materials with novel applications. Over the past decade, the number of inorganic uranyl compounds (including minerals) with known structures has more than doubled, and reconsideration of the structural hierarchy of uranyl compounds is warranted. Here, 368 inorganic crystal structures that contain essential U 6+ are considered (of which 89 are minerals). They are arranged on the basis of the topological details of their structural units, which are formed by the polymerization of polyhedra containing higher-valence cations. Overarching structural categories correspond to those based upon isolated polyhedra (8), finite clusters (43), chains (57), sheets (204), and frameworks (56) of polyhedra. Within these categories, structures are organized and compared upon the basis of either their graphical representations, or in the case of sheets involving sharing of edges of polyhedra, upon the topological arrangement of anions within the sheets. (author)
Full Text Available Abstract Background A Southwest Asian origin and dispersal to North Africa in the Early Upper Palaeolithic era has been inferred in previous studies for mtDNA haplogroups M1 and U6. Both haplogroups have been proposed to show similar geographic patterns and shared demographic histories. Results We report here 24 M1 and 33 U6 new complete mtDNA sequences that allow us to refine the existing phylogeny of these haplogroups. The resulting phylogenetic information was used to genotype a further 131 M1 and 91 U6 samples to determine the geographic spread of their sub-clades. No southwest Asian specific clades for M1 or U6 were discovered. U6 and M1 frequencies in North Africa, the Middle East and Europe do not follow similar patterns, and their sub-clade divisions do not appear to be compatible with their shared history reaching back to the Early Upper Palaeolithic. The Bayesian Skyline Plots testify to non-overlapping phases of expansion, and the haplogroups’ phylogenies suggest that there are U6 sub-clades that expanded earlier than those in M1. Some M1 and U6 sub-clades could be linked with certain events. For example, U6a1 and M1b, with their coalescent ages of ~20,000–22,000 years ago and earliest inferred expansion in northwest Africa, could coincide with the flourishing of the Iberomaurusian industry, whilst U6b and M1b1 appeared at the time of the Capsian culture. Conclusions Our high-resolution phylogenetic dissection of both haplogroups and coalescent time assessments suggest that the extant main branching pattern of both haplogroups arose and diversified in the mid-later Upper Palaeolithic, with some sub-clades concomitantly with the expansion of the Iberomaurusian industry. Carriers of these maternal lineages have been later absorbed into and diversified further during the spread of Afro-Asiatic languages in North and East Africa.
Niu, Jinzhi; Shen, Guangmao; Christiaens, Olivier; Smagghe, Guy; He, Lin; Wang, Jinjun
Mites comprise a group of key agricultural pests on a wide range of crops. They cause harm through feeding on the plant and transferring dangerous pathogens, and the rapid evolution of pesticide resistance in mites highlights the need for novel control methods. Currently, RNA interference (RNAi) shows a great potential for insect pest control. Here, we review the literature associated with RNAi in mite pests. We discuss different target genes and RNAi efficiency in various mite species, a promising Varroa control program through RNAi, the synergy of RNAi with plant defense mechanisms and microorganisms, and the current understandings of systemic movement of dsRNA. Based on this, we can conclude that there is a clear potential for an RNAi-based mite control application but further research on several aspects is needed, including: (i) the factors influencing the RNAi efficiency, (ii) the mechanism of environmental RNAi and cross-kingdom dsRNA trafficking, (iii) the mechanism of possible systemic and parental RNAi, and (iv) non-target effects, specifically in predatory mites, should be considered during the RNAi target selection. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Full Text Available Abstract: A financial management eficiently begin, primarily, with an accounting record kept in the best possible conditions, this being conditioned on the adoption of a uniform forms, rational, clear and simple accounting. Throughout history, there have been known two forms of accounting: the simple and double entry. Romanian society after 1990 underwent a substantial change in social structure, the sector on which put a great emphasis being private, that of small manufacturers, peddler, freelance, who work independently and authorized or as associative form (family enterprises, various associations (owners, tenants, etc., liberal professions, etc.. They are obliged to keep a simple bookkeeping, because they have no juridical personality. Companies with legal personality are required to keep double entry bookkeeping; therefore, knowledge and border demarcation between the two forms of organisation of accounting is an essential. The material used for this work is mainly represented by the financial and accounting documents, by the analysis of the economic, by legislative updated sources, and as the method was used the comparison method, using hypothetical data, in case of an authorized individual and a legal entity. Based on the chosen material, an authorized individual (who perform single entry accounting system and a juridical entity (who perform double entry accounting system were selected comparative case studies, using hypothetical data, were analysed advantages and disadvantages in term of fiscal, if using two accounting systems, then were highlighted some conclusion that result.
Sherman, James H; Munyikwa, Tichafa; Chan, Stephen Y; Petrick, Jay S; Witwer, Kenneth W; Choudhuri, Supratim
During the 40th Annual Meeting of The Toxicology Forum, the current and potential future science, regulations, and politics of agricultural biotechnology were presented and discussed. The meeting session described herein focused on the technology of RNA interference (RNAi) in agriculture. The general process by which RNAi works, currently registered RNAi-based plant traits, example RNAi-based traits in development, potential use of double stranded RNA (dsRNA) as topically applied pesticide active ingredients, research related to the safety of RNAi, biological barriers to ingested dsRNA, recent regulatory RNAi science reviews, and regulatory considerations related to the use of RNAi in agriculture were discussed. Participants generally agreed that the current regulatory framework is robust and appropriate for evaluating the safety of RNAi employed in agricultural biotechnology and were also supportive of the use of RNAi to develop improved crop traits. However, as with any emerging technology, the potential range of future products, potential future regulatory frameworks, and public acceptance of the technology will continue to evolve. As such, continuing dialogue was encouraged to promote education of consumers and science-based regulations. Copyright © 2015 Elsevier Inc. All rights reserved.
Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl
Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe
Pang, Shi-Feng; Li, Xiao-Kun; Zhang, Qiang; Yang, Fang; Xu, Peilin
Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.
Full Text Available Concerns over the possibility of resistance developing to praziquantel (PZQ, has stimulated efforts to develop new drugs for schistosomiasis. In addition to the development of improved whole organism screens, the success of RNA interference (RNAi in schistosomes offers great promise for the identification of potential drug targets to initiate drug discovery. In this study we set out to contribute to RNAi based validation of putative drug targets. Initially a list of 24 target candidates was compiled based on the identification of putative essential genes in schistosomes orthologous of C. elegans essential genes. Knockdown of Calmodulin (Smp_026560.2 (Sm-Calm, that topped this list, produced a phenotype characterised by waves of contraction in adult worms but no phenotype in schistosomula. Knockdown of the atypical Protein Kinase C (Smp_096310 (Sm-aPKC resulted in loss of viability in both schistosomula and adults and led us to focus our attention on other kinase genes that were identified in the above list and through whole organism screening of known kinase inhibitor sets followed by chemogenomic evaluation. RNAi knockdown of these kinase genes failed to affect adult worm viability but, like Sm-aPKC, knockdown of Polo-like kinase 1, Sm-PLK1 (Smp_009600 and p38-MAPK, Sm-MAPK p38 (Smp_133020 resulted in an increased mortality of schistosomula after 2-3 weeks, an effect more marked in the presence of human red blood cells (hRBC. For Sm-PLK-1 the same effects were seen with the specific inhibitor, BI2536, which also affected viable egg production in adult worms. For Sm-PLK-1 and Sm-aPKC the in vitro effects were reflected in lower recoveries in vivo. We conclude that the use of RNAi combined with culture with hRBC is a reliable method for evaluating genes important for larval development. However, in view of the slow manifestation of the effects of Sm-aPKC knockdown in adults and the lack of effects of Sm-PLK-1 and Sm-MAPK p38 on adult viability
Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.
Abstract: A financial management eficiently begin, primarily, with an accounting record kept in the best possible conditions, this being conditioned on the adoption of a uniform forms, rational, clear and simple accounting. Throughout history, there have been known two forms of accounting: the simple and double entry. Romanian society after 1990 underwent a substantial change in social structure, the sector on which put a great emphasis being private, that of small manufacturers, ped...
Department of Transportation — The Bureau of Transportation Statistics (BTS) Border Crossing/Entry Data provides summary statistics for inbound crossings at the U.S.-Canadian and the U.S.-Mexican...
1. Introduction R Analyses of industrial competition have attained a new vigor with the application of game -theoretic methods. The process of... competition is represented in models that reflect genuine struggles for entry, market power, and continuing survival. Dynamics and informational effects are...presents a few of the models developed recently to study competitive processes that affect a firm’s entry into a market , and the decision to exit. The
Brutscher, Laura M.; Flenniken, Michelle L.
Honey bees play an important agricultural and ecological role as pollinators of numerous agricultural crops and other plant species. Therefore, investigating the factors associated with high annual losses of honey bee colonies in the US is an important and active area of research. Pathogen incidence and abundance correlate with Colony Collapse Disorder- (CCD-) affected colonies in the US and colony losses in the US and in some European countries. Honey bees are readily infected by single-stranded positive sense RNA viruses. Largely dependent on the host immune response, virus infections can either remain asymptomatic or result in deformities, paralysis, or death of adults or larvae. RNA interference (RNAi) is an important antiviral defense mechanism in insects, including honey bees. Herein, we review the role of RNAi in honey bee antiviral defense and highlight some parallels between insect and mammalian immune systems. A more thorough understanding of the role of pathogens on honey bee health and the immune mechanisms bees utilize to combat infectious agents may lead to the development of strategies that enhance honey bee health and result in the discovery of additional mechanisms of immunity in metazoans. PMID:26798663
Laura M. Brutscher
Full Text Available Honey bees play an important agricultural and ecological role as pollinators of numerous agricultural crops and other plant species. Therefore, investigating the factors associated with high annual losses of honey bee colonies in the US is an important and active area of research. Pathogen incidence and abundance correlate with Colony Collapse Disorder- (CCD- affected colonies in the US and colony losses in the US and in some European countries. Honey bees are readily infected by single-stranded positive sense RNA viruses. Largely dependent on the host immune response, virus infections can either remain asymptomatic or result in deformities, paralysis, or death of adults or larvae. RNA interference (RNAi is an important antiviral defense mechanism in insects, including honey bees. Herein, we review the role of RNAi in honey bee antiviral defense and highlight some parallels between insect and mammalian immune systems. A more thorough understanding of the role of pathogens on honey bee health and the immune mechanisms bees utilize to combat infectious agents may lead to the development of strategies that enhance honey bee health and result in the discovery of additional mechanisms of immunity in metazoans.
Zhang, Chi; Ruvkun, Gary
In the nematode Caenorhabditis elegans (C. elegans), gene inactivation by RNA interference can achieve remarkable potency due to the amplification of initial silencing triggers by RNA-dependent RNA polymerases (RdRPs). RdRPs catalyze the biogenesis of an abundant species of secondary small interfering RNAs (siRNAs) using the target mRNA as template. The interaction between primary siRNAs derived from the exogenous double-stranded RNA (dsRNA) trigger and the target mRNA is required for the recruitment of RdRPs. Other genetic requirements for RdRP activities have not been characterized. Recent studies have identified the RDE-10/RDE-11 complex which interacts with the primary siRNA bound target mRNA and acts upstream of the RdRPs. rde-10 and rde-11 mutants show an RNAi defective phenotype because the biogenesis of secondary siRNAs is completely abolished. In addition, the RDE-10/RDE-11 complex plays a similar role in the endogenous RNAi pathway for the biogenesis of a subset of siRNAs targeting recently acquired, duplicated genes.
de Pinto, Roberta; Strandberg, Morten Tune; Kostov, Kaloyan
both how to study fate and effects of the RNAi molecules. If COI or another gene apt for silencing will be successful in the earthworm L. terrestris, this could become a suggested positive control agent for future non-target studies of RNAi. The testing of environmental effects of NBT require...
Full Text Available The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR and the CRISPR-associated protein 9 (Cas9 (CRISPR/Cas9 system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. The conclusion drawn from this analysis is that RNAi will still occupy specific domains of biomedical research and clinical applications, under the current state of development of these technologies. However, further improvements in CRISPR/Cas9 based technology may ultimately enable it to dominate RNAi in the long term.
Seinen, Erwin; Burgerhof, Johannes G. M.; Jansen, Ritsert C.; Sibon, Ody C. M.; Polymenis, Michael
Background: RNAi technology is widely used to downregulate specific gene products. Investigating the phenotype induced by downregulation of gene products provides essential information about the function of the specific gene of interest. When RNAi is applied in Drosophila melanogaster or
Yang, K.N.; Maple, M.B.; DeLong, L.E.; Huber, J.G.; Junod, A.
Measurements of the superconducting- and normal-state heat capacity of U 6 X (XequivalentMn, Fe, Co, Ni) compounds have been performed over a temperature range 1 Kapprox. 6 X compounds have strong renormalizations of the free-carrier effective mass m/sup */ in the range 10m/sub e/approx. 6 X heat capacities suggest the presence of high densities of low-energy excitations of undetermined nature. The results are analyzed in terms of models appropriate to heavy-fermion liquids, and anisotropic or strong-coupled superconductors. The U 6 X compounds form a link between relatively low-m/sup */, high-transition-temperature A15 compounds and the more extreme examples of heavy-fermion superconductors such as UBe/sub 13/, UPt 3 , and CeCuSi 2 for which m/sup */∼10 2 m/sub e/. .AE
Perotti, E.; Vorage, M.
We model a setting in which citizens form coalitions to seek preferential entry to a given market. The lower entry the higher firm profits and political contributions, but the lower social welfare. Politicians choose to either control entry directly and be illegally bribed, or regulate entry using a
Hardin, John W.; Warnasooriya, Chandani; Kondo, Yasushi; Nagai, Kiyoshi; Rueda, David
In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31. PMID:26503251
Full Text Available BACKGROUND: Overcoming spaceflight-induced (pathophysiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown. METHODS: Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at -80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC on Earth were simultaneously grown under identical conditions. RESULTS: After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05. The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05. In spaceflight, RNAi against green fluorescent protein (gfp reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions. CONCLUSIONS: Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.
Dang, Tuong Vi T; Windelinckx, Saskia; Henry, Isabelle M; De Coninck, Barbara; Cammue, Bruno P A; Swennen, Rony; Remy, Serge
In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana. Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target
Full Text Available Clericuzio-type poikiloderma with neutropenia (PN is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3′ end polyuridine tails that are longer than those in normal cells and lack a terminal 2′,3′ cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis.
Lees, Jonathan G; Hériché, Jean-Karim; Morilla, Ian; Fernández, José M; Adler, Priit; Krallinger, Martin; Vilo, Jaak; Valencia, Alfonso; Ellenberg, Jan; Ranea, Juan A; Orengo, Christine
Most biological processes remain only partially characterized with many components still to be identified. Given that a whole genome can usually not be tested in a functional assay, identifying the genes most likely to be of interest is of critical importance to avoid wasting resources. Given a set of known functionally related genes and using a state-of-the-art approach to data integration and mining, our Functional Lists (FUN-L) method provides a ranked list of candidate genes for testing. Validation of predictions from FUN-L with independent RNAi screens confirms that FUN-L-produced lists are enriched in genes with the expected phenotypes. In this article, we describe a website front end to FUN-L. The website is freely available to use at http://funl.org © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: email@example.com.
This paper examines the use of exclusive dealing agreements to prevent the entry of rival firms. An exclusive dealing agreement is a contract between a buyer and a seller where the buyer commits to buy a good exclusively from the seller. One main concern of the literature is to explain how an incumbent seller is able to persuade the buyers to sign an exclusive dealing agreement that deters the entry of a more efficient rival seller. We propose a new explanation when the buyers are downstream ...
Perotti, E.C.; Volpin, P.
We develop a model of endogenous lobby formation in which wealth inequality and political accountability undermine entry and financial development. Incumbents seek a low level of effective investor protection to prevent potential entrants from raising capital. They succeed because they can promise
Yeon, Jeongho; Smith, Mark D.; Tapp, Joshua; Möller, Angela; zur Loye, Hans-Conrad
Two new uranium(IV) fluorides, Na3.13Mg1.43U6F30 (1) and Na2.50Mn1.75U6F30 (2), were synthesized through an in situ mild hydrothermal route, and were structurally characterized by single crystal X-ray diffraction. The compounds exhibit complex crystal structures composed of corner- or edge-shared UF9 and MF6 (M=Mg, Mn) polyhedra, forming hexagonal channels in the three-dimensional framework, in which ordered or disordered divalent metal and sodium atoms reside. The large hexagonal voids contain the nearly regular M(II)F6 octahedra and sodium ions, whereas the small hexagonal cavities include M(II) and sodium ions on a mixed-occupied site. Magnetic susceptibility measurements yielded effective magnetic moments of 8.36 and 11.6 μB for 1 and 2, respectively, confirming the presence and oxidation states of U(IV) and Mn(II). The large negative Weiss constants indicate the spin gap between a triplet and a singlet state in the U(IV). Magnetization data as a function of applied fields revealed that 2 exhibits paramagnetic behavior due to the nonmagnetic singlet ground state of U(IV) at low temperature. UV-vis diffuse reflectance and X-ray photoelectron spectroscopy data were also analyzed.
Agrawal, P.; Allen, G. A.; Hwang, H. H.; Marley, M. S.; McGuire, M. K.; Garcia, J. A.; Sklyanskiy, E.; Huynh, L. C.; Moses, R. W.
To better understand the technology requirements for Uranus atmospheric entry probe, Entry Vehicle Technology project funded an internal study with a multidisciplinary team from NASA Ames, Langley and JPL. The results of this study are communicated.
Department of Transportation — The dataset is known as “Border Crossing/Entry Data.” The Bureau of Transportation Statistics (BTS) Border Crossing/Entry Data provides summary statistics to the...
In the past few years metallurgical studies have been carried out to develop fuel dispersions with U-densities up to 7.0 Mg U m -3 . Uranium silicides have been considered to be the prime candidates as dispersants; U 6 Fe being a potential alternative on account of its higher U-density. The objective of this paper is to compare the metallurgical behaviour of these two material combinations with regard to the following aspects: (1) preparation of the compounds U 3 Si, U 3 Si 2 and U 6 Fe; (2) powder metallurgical processing to miniature fuel element plates; (3) reaction behaviour under equilibrium conditions in the relevant portions of the ternary U-Si-Al and U-Fe-Al systems; (4) dimensional stability of the fuel plates after prolonged thermal treatment; (5) thermochemical behaviour of fuel plates at temperatures near the melting point of the cladding. Based on this data, the possible advantages of each fuel combination are discussed. (author)
For some time now, efforts are being made to develop fuel dispersions that would permit the use of low (approx. 20% 235-U) enriched uranium (LEU) instead of the currently used highly (approx. 93% 235-U) enriched uranium (HEU) in research and test reactors. Since penalties in the performance of the reactor have to be avoided, the 235-U content in the dispersion has at least to be retained at current levels. On account of their high U-densities, the major development effort has been focussed on the uranium silicides (U 3 Si, U 3 Si(Al), and U 3 Si 2 -based dispersions). With silicides as dispersants, it is possible to fabricate fuel element plates with U-densities in the dispersion of about 6.0 gU/cm 3 . In comparison to the silicides, the U 6 Fe-phase offers several advantages namely: higher U-density (approx. 17.0 gU/cm 3 ); relative ease of formation compared to U 3 Si; possible advantages with regard to reprocessing of the spent fuel due to the absence of silicon. The studies outlined here were performed with a view to investigating the preparation, reaction behavior and dimensional stability after heat treatment of U 6 Fe-Al dispersions
For some time now, efforts are being made to develop fuel dispersions that would permit the use of low (∼ 20% 235-U) enriched uranium (LEU) instead of the currently used highly (∼ 93% 235-U) enriched uranium (HEU) in research and test reactors. Since penalties in the performance of the reactor have to be avoided, the 235-U content in the dispersion has at least to be retained at current levels. On account of their high U-densities, the major development effort has been focussed on the uranium silicides [U 3 Si, U 3 Si(Al), and U 3 Si 2 - based dispersions. With silicides as dispersants, it is possible to fabricate fuel element plates with U-densities in the dispersion of about 6.0 g U/cm 3 . In comparison to the silicides, the U 6 Fe-phase offers several advantages namely: - higher U-density (∼ 17.0 g U/cm 3 ); - relative ease of formation compared to U 3 Si; - possible advantages with regard to reprocessing of the spent fuel due to the absence of silicon. The studies outlined here were therefore performed with a view to investigating the preparation, reaction behaviour and dimensional stability after heat treatment of U 6 Fe-Al dispersions
RNAi is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-strand RNA molecules. In the 18 years since the initial report, RNAi has now been demonstrated to function in mammalian cells to alter gene expression and has been used as a means for genetic discovery as well as a possible strategy for genetic correction and genetic therapy in cancer and other disease. The aim of this review is to provide a general overview of how RNAi suppresses gene expression and to examine some published RNAi approaches that have resulted in changes in stem cell function and suggest the possible clinical relevance of this work in cancer therapy through targeting cancer stem cells.
Mon, Hiroaki; Li, Zhiqing; Kobayashi, Isao; Tomita, Shuichiro; Lee, JaeMan; Sezutsu, Hideki; Tamura, Toshiki; Kusakabe, Takahiro
RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Previously, the BmN4-SID1 cell expressing Caenorhabditis ele gans SID-1 was established, in which soaking RNAi could induce effective gene silencing. To establish its utility, 6 cell cycle progression related cDNAs, CDK1, MYC, MYB, RNRS, CDT1, and GEMININ, were isolated from the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), and their expressions were further silenced by soaking RNAi in the BmN4-SID1 cells. The cell cycle progression analysis using flow cytometer demonstrated that the small amount of double stranded RNA was enough to arrest cell cycle progression at the specific cell phases. These data suggest that RNAi in the BmN4-SID1 cells can be used as a powerful tool for loss-of-function analysis of B. mori genes. PMID:24773378
Liu, Ying; Ye, Xuecheng; Jiang, Feng; Liang, Chunyang; Chen, Dongmei; Peng, Junmin; Kinch, Lisa N; Grishin, Nick V; Liu, Qinghua
The catalytic engine of RNA interference (RNAi) is the RNA-induced silencing complex (RISC), wherein the endoribonuclease Argonaute and single-stranded small interfering RNA (siRNA) direct target mRNA cleavage. We reconstituted long double-stranded RNA- and duplex siRNA-initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We used this core reconstitution system to purify an RNAi regulator that we term C3PO (component 3 promoter of RISC), a complex of Translin and Trax. C3PO is a Mg2+-dependent endoribonuclease that promotes RISC activation by removing siRNA passenger strand cleavage products. These studies establish an in vitro RNAi reconstitution system and identify C3PO as a key activator of the core RNAi machinery.
Nicolás, Francisco E; Vila, Ana; Moxon, Simon; Cascales, María D; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M; Garre, Victoriano
RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants. Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway. This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential
Yu, Rong; Xu, Xinping; Liang, Yongkang; Tian, Honggang; Pan, Zhanqing; Jin, Shouheng; Wang, Na; Zhang, Wenqing
RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.
Carol D. Blair
Full Text Available RNA interference (RNAi was shown over 18 years ago to be a mechanism by which arbovirus replication and transmission could be controlled in arthropod vectors. During the intervening period, research on RNAi has defined many of the components and mechanisms of this antiviral pathway in arthropods, yet a number of unexplored questions remain. RNAi refers to RNA-mediated regulation of gene expression. Originally, the term described silencing of endogenous genes by introduction of exogenous double-stranded (dsRNA with the same sequence as the gene to be silenced. Further research has shown that RNAi comprises three gene regulation pathways that are mediated by small RNAs: the small interfering (siRNA, micro (miRNA, and Piwi-interacting (piRNA pathways. The exogenous (exo-siRNA pathway is now recognized as a major antiviral innate immune response of arthropods. More recent studies suggest that the piRNA and miRNA pathways might also have important roles in arbovirus-vector interactions. This review will focus on current knowledge of the role of the exo-siRNA pathway as an arthropod vector antiviral response and on emerging research into vector piRNA and miRNA pathway modulation of arbovirus-vector interactions. Although it is assumed that arboviruses must evade the vector’s antiviral RNAi response in order to maintain their natural transmission cycles, the strategies by which this is accomplished are not well defined. RNAi is also an important tool for arthropod gene knock-down in functional genomics studies and in development of arbovirus-resistant mosquito populations. Possible arbovirus strategies for evasion of RNAi and applications of RNAi in functional genomics analysis and arbovirus transmission control will also be reviewed.
Tokar, Derek R; Veleta, Katherine A; Canzano, Joseph; Hahn, Daniel A; Hatle, John D
Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph
report Impact on other disciplines: Nothing to report Impact on technology transfer: Nothing to report Impact on society : Nothing to report 5. CHANGES...AWARD NUMBER: W81XWH-15-1-0569 TITLE: Development of a Novel Targeted RNAi Delivery Technology in Therapies for Metabolic Diseases PRINCIPAL...COVERED 30Sep2016 - 29Sep2017 4. TITLE AND SUBTITLE Development of a Novel Targeted RNAi Delivery Technology in Therapies for Metabolic Diseases 5a
Ramon, Matthew; Devos, Yann; Lanzoni, Anna; Liu, Yi; Gomes, Ana; Gennaro, Andrea; Waigmann, Elisabeth
RNA interference (RNAi) is an emerging technology that offers new opportunities for the generation of new traits in genetically modified (GM) plants. Potential risks associated with RNAi-based GM plants and issues specific to their risk assessment were discussed during an international scientific workshop (June 2014) organized by the European Food Safety Authority (EFSA). Selected key outcomes of the workshop are reported here. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
Liang, Chunyang; Wang, Yibing; Murota, Yukiko; Liu, Xiang; Smith, Dean; Siomi, Mikiko C.; Liu, Qinghua
SUMMARY Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains Dicer-2(Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here, we identified the missing factor of RLC as TATA-binding protein associated factor 11 (TAF11) by genetic screen. Although an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11−/− ovary extract, we reconstituted the RLC in vitro using recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of Drosophila RLC and elucidate a novel cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency. PMID:26257286
Tushar Kanti Dutta
Full Text Available With the understanding of nematode-plant interactions at the molecular level, new avenues for engineering resistance have opened up, with RNA interference being one of them. Induction of RNAi by delivering double-stranded RNA (dsRNA has been very successful in the model non-parasitic nematode, Caenorhabditis elegans, while in plant nematodes, dsRNA delivery has been accomplished by soaking nematodes with dsRNA solution mixed with synthetic neurostimulants. The success of in vitro RNAi of target genes has inspired the use of in planta delivery of dsRNA to feeding nematodes. The most convincing success of host-delivered RNAi has been achieved against root-knot nematodes. Plant-mediated RNAi has been shown to lead to the specific down-regulation of target genes in invading nematodes, which had a profound effect on nematode development. RNAi-based transgenics are advantageous as they do not produce any functional foreign proteins and target organisms in a sequence-specific manner. Although the development of RNAi-based transgenics against plant nematodes is still in the preliminary stage, they offer novel management strategy for the future.
Schøler, Lone Vedel; Møller, Tine Hørning; Nørgaard, Steffen
The soil nematode Caenorhabditis elegans has become a popular genetic model organism used to study a broad range of complex biological processes, including development, aging, apoptosis, and DNA damage responses. Many genetic tools and tricks have been developed in C. elegans including knock down...... of gene expression via RNA interference (RNAi). In C. elegans RNAi can effectively be administrated via feeding the nematodes bacteria expressing double-stranded RNA targeting the gene of interest. Several commercial C. elegans RNAi libraries are available and hence gene inactivation using RNAi can...
The effect of purely persuasive advertising on generic market entry and social welfare is analysed. An incumbent has the possibility to invest in advertising which affects the prescribing physician's perceived relative qualities of the brand-name and the generic version of the drug. Advertising creates product differentiation and can induce generic market entry which is deterred without differentiation due to strong Bertrand competition. However, over-investment in advertising can deter generic market entry under certain conditions and reduces welfare as compared to accommodated market entry.
The function of an entry-control system in a total Physical Protection System is to allow the movement of authorized personnel and material through normal access routes, yet detect and delay unauthorized movement of personnel and material from uncontrolled areas. The ten chapters of this handbook cover: introduction, credentials, personnel identity verification systems, special nuclear materials monitors, metal detectors, explosives sensors, package search systems, criteria for selection of entry-control equipment, machine-aided manual entry-control systems, and automated entry-control systems. A system example and its cost are included as an appendix
Lemgo, Godwin Nana Yaw; Sabbadini, Silvia; Pandolfini, Tiziana; Mezzetti, Bruno
A major application of RNA interference (RNAi) is envisaged for the production of virus-resistant transgenic plants. For fruit trees, this remains the most, if not the only, viable option for the control of plant viral disease outbreaks in cultivated orchards, due to the difficulties associated with the use of traditional and conventional disease-control measures. The use of RNAi might provide an additional benefit for woody crops if silenced rootstock can efficiently transmit the silencing signal to non-transformed scions, as has already been demonstrated in herbaceous plants. This would provide a great opportunity to produce non-transgenic fruit from transgenic rootstock. In this review, we scrutinise some of the concerns that might arise with the use of RNAi for engineering virus-resistant plants, and we speculate that this virus resistance has fewer biosafety concerns. This is mainly because RNAi-eliciting constructs only express small RNA molecules rather than proteins, and because this technology can be applied using plant rootstock that can confer virus resistance to the scion, leaving the scion untransformed. We discuss the main biosafety concerns related to the release of new types of virus-resistant plants and the risk assessment approaches in the application of existing regulatory systems (in particular, those of the European Union, the USA, and Canada) for the evaluation and approval of RNAi-mediated virus-resistant plants, either as transgenic varieties or as plant virus resistance induced by transgenic rootstock.
Panfilio, Kristen A
Many insects undergo katatrepsis, essential reorganization by the extraembryonic membranes that repositions the embryo. Knockdown of the zen gene by RNA interference (RNAi) prevents katatrepsis in the milkweed bug Oncopeltus fasciatus. However, the precise morphogenetic defect has been uncertain, and katatrepsis itself has not been characterized in detail. The dynamics of wild type and zen(RNAi) eggs were analyzed from time-lapse movies, supplemented by analysis of fixed specimens. These investigations identify three zen(RNAi) defects. First, a reduced degree of tissue contraction implies a role for zen in baseline compression prior to katatrepsis. Subsequently, a characteristic 'bouncing' activity commences, leading to the initiation of katatrepsis in wild type eggs. The second zen(RNAi) defect is a delay in this activity, suggesting that a temporal window of opportunity is missed after zen knockdown. Ultimately, the extraembryonic membranes fail to rupture in zen(RNAi) eggs: the third defect. Nevertheless, the outer serosal membrane manages to contract, albeit in an aberrant fashion with additional phenotypic consequences for the embryo. These data identify a novel epithelial morphogenetic event - rupture of the 'serosal window' structure - as the ultimate site of defect. Overall, Oncopeltus zen seems to have a role in coordinating a number of pre-katatreptic events during mid embryogenesis.
Marker, Simone; Carradec, Quentin; Tanty, Véronique; Arnaiz, Olivier; Meyer, Eric
In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia. PMID:24860163
Perkins, Lizabeth A; Holderbaum, Laura; Tao, Rong; Hu, Yanhui; Sopko, Richelle; McCall, Kim; Yang-Zhou, Donghui; Flockhart, Ian; Binari, Richard; Shim, Hye-Seok; Miller, Audrey; Housden, Amy; Foos, Marianna; Randkelv, Sakara; Kelley, Colleen; Namgyal, Pema; Villalta, Christians; Liu, Lu-Ping; Jiang, Xia; Huan-Huan, Qiao; Wang, Xia; Fujiyama, Asao; Toyoda, Atsushi; Ayers, Kathleen; Blum, Allison; Czech, Benjamin; Neumuller, Ralph; Yan, Dong; Cavallaro, Amanda; Hibbard, Karen; Hall, Don; Cooley, Lynn; Hannon, Gregory J; Lehmann, Ruth; Parks, Annette; Mohr, Stephanie E; Ueda, Ryu; Kondo, Shu; Ni, Jian-Quan; Perrimon, Norbert
To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China). Copyright © 2015 by the Genetics Society of America.
Perkins, Lizabeth A.; Holderbaum, Laura; Tao, Rong; Hu, Yanhui; Sopko, Richelle; McCall, Kim; Yang-Zhou, Donghui; Flockhart, Ian; Binari, Richard; Shim, Hye-Seok; Miller, Audrey; Housden, Amy; Foos, Marianna; Randkelv, Sakara; Kelley, Colleen; Namgyal, Pema; Villalta, Christians; Liu, Lu-Ping; Jiang, Xia; Huan-Huan, Qiao; Wang, Xia; Fujiyama, Asao; Toyoda, Atsushi; Ayers, Kathleen; Blum, Allison; Czech, Benjamin; Neumuller, Ralph; Yan, Dong; Cavallaro, Amanda; Hibbard, Karen; Hall, Don; Cooley, Lynn; Hannon, Gregory J.; Lehmann, Ruth; Parks, Annette; Mohr, Stephanie E.; Ueda, Ryu; Kondo, Shu; Ni, Jian-Quan; Perrimon, Norbert
To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT–qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT–qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China). PMID:26320097
Guo, C Y; Chen, P; Zhang, M M; Ning, J J; Wang, С L; Wang, D L; Zhao, Y L
In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.
Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F
High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.
Ruiz-Vázquez, Rosa M; Nicolás, Francisco E; Torres-Martínez, Santiago; Garre, Victoriano
The basal fungus Mucor circinelloides has become, in recent years, a valuable model to study RNA-mediated gene silencing or RNA interference (RNAi). Serendipitously discovered in the late 1900s, the gene silencing in M. circinelloides is a landscape of consensus and dissents. Although similar to other classical fungal models in the basic design of the essential machinery that is responsible for silencing of gene expression, the existence of small RNA molecules of different sizes generated during this process and the presence of a mechanism that amplifies the silencing signal, give it a unique identity. In addition, M. circinelloides combines the components of RNAi machinery to carry out functions that not only limit themselves to the defense against foreign genetic material, but it uses some of these elements to regulate the expression of its own genes. Thus, different combinations of RNAi elements produce distinct classes of endogenous small RNAs (esRNAs) that regulate different physiological and developmental processes in response to environmental signals. The recent discovery of a new RNAi pathway involved in the specific degradation of endogenous mRNAs, using a novel RNase protein, adds one more element to the exciting puzzle of the gene silencing in M. circinelloides, in addition to providing hints about the evolutionary origin of the RNAi mechanism. Copyright © 2015 Elsevier Inc. All rights reserved.
Zhao, Chaoyang; Alvarez Gonzales, Miguel A; Poland, Therese M; Mittapalli, Omprakash
The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis. Copyright © 2015 Elsevier Ltd. All rights reserved.
Tetlak, Alicia G; Burnett, Jacob B; Hahn, Daniel A; Hatle, John D
Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.
Olesen, Morten Tobias Jarlstad; Gonzalez, Borja Ballarin; Howard, Ken
in which small interfering RNA (siRNA) mediates specific downregulation of key molecular targets of the IBD inflammatory process may offer a precise, potent and safer alternative to conventional treatments. This review describes the aetiology of Crohn’s disease and ulcerative colitis and the cellular...... and molecular basis for current treatments to highlight target candidates for an RNAi-based approach. Promising preclinical studies support an RNAi application; however, optimal siRNA designs that maximise potency and development of enabling technologies for site- and cellular-specific delivery......Inflammatory bowel disease (IBD) is a chronic, relapsing, idiopathic inflammation of the gastrointestinal tract with no permanent cure. Present immunosuppressive and anti-inflammatory therapies are often ineffective and associated with severe side effects. An RNA interference (RNAi)-based approach...
Roberts, Andrew F.; Devos, Yann; Lemgo, Godwin N. Y.; Zhou, Xuguo
RNA interference, or RNAi, refers to a set of biological processes that make use of conserved cellular machinery to silence genes. Although there are several variations in the source and mechanism, they are all triggered by double stranded RNA (dsRNA) which is processed by a protein complex into small, single stranded RNA, referred to as small interfering RNAs (siRNA) with complementarity to sequences in genes targeted for silencing. The use of the RNAi mechanism to develop new traits in plants has fueled a discussion about the environmental safety of the technology for these applications, and this was the subject of a symposium session at the 13th ISBGMO in Cape Town, South Africa. This paper continues that discussion by proposing research areas that may be beneficial for future environmental risk assessments of RNAi-based genetically modified plants, with a particular focus on non-target organism assessment. PMID:26594220
Xu, Ning; Gkountela, Sofia; Saeed, Khalid; Akusjärvi, Göran
Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.
Full Text Available Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (dsRNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain, validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL and B (FheCatB cysteine proteases, and a σ-class glutathione transferase (FheσGST.Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200-320 nt dsRNAs or 27 nt short interfering (siRNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control
Chen, Qing; Rehman, S; Smant, G; Jones, John T
RNA interference (RNAi) has been used widely as a tool for examining gene function and a method that allows its use with plant-parasitic nematodes recently has been described. Here, we use a modified method to analyze the function of secreted beta-1,4, endoglucanases of the potato cyst nematode Globodera rostochiensis, the first in vivo functional analysis of a pathogenicity protein of a plant-parasitic nematode. Knockout of the beta-1,4, endoglucanases reduced the ability of the nematodes to invade roots. We also use RNAi to show that gr-ams-1, a secreted protein of the main sense organs (the amphids), is essential for host location.
Fassnacht, Christina; Tocchini, Cristina; Kumari, Pooja; Gaidatzis, Dimos; Stadler, Michael B; Ciosk, Rafal
Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition.
Zhang, Chi; Montgomery, Taiowa A; Fischer, Sylvia E J; Garcia, Susana M D A; Riedel, Christian G; Fahlgren, Noah; Sullivan, Christopher M; Carrington, James C; Ruvkun, Gary
In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood. From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10, and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and other dosage-sensitive RNAi-defective genes rsd-2, rsd-6, and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation. Copyright © 2012 Elsevier Ltd. All rights reserved.
Mo Xiao; Peter F. Orazem
Past empirical literature provides strong evidence that competition increases when new firms enter a market. However, rarely have economists been able to examine how competition changes with the threat of entry. This paper uses the evolution of the zip code level market structure of facilities-based broadband providers from 1999 to 2004 to investigate how a firm adjusts its entry strategy when facing the threat of additional entrants. We identify the potential entrant into a local market as t...
Pačes, Jan; Nic, M.; Novotný, T.; Svoboda, Petr
Roč. 14, č. 6 (2017), č. článku 1246E. ISSN 2397-8325 Institutional support: RVO:68378050 Keywords : miRNA, , ,, , , * RNAi * siRNA * Dicer * Argonaute * dsRNA * off-targeting Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology
Garcia-Martin, Juan Antonio; Clote, Peter; Dotu, Ivan
Synthetic biology and nanotechnology are poised to make revolutionary contributions to the 21st century. In this article, we describe a new web server to support in silico RNA molecular design. Given an input target RNA secondary structure, together with optional constraints, such as requiring GC-content to lie within a certain range, requiring the number of strong (GC), weak (AU) and wobble (GU) base pairs to lie in a certain range, the RNAiFold web server determines one or more RNA sequences, whose minimum free-energy secondary structure is the target structure. RNAiFold provides access to two servers: RNA-CPdesign, which applies constraint programming, and RNA-LNSdesign, which applies the large neighborhood search heuristic; hence, it is suitable for larger input structures. Both servers can also solve the RNA inverse hybridization problem, i.e. given a representation of the desired hybridization structure, RNAiFold returns two sequences, whose minimum free-energy hybridization is the input target structure. The web server is publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold, which provides access to two specialized servers: RNA-CPdesign and RNA-LNSdesign. Source code for the underlying algorithms, implemented in COMET and supported on linux, can be downloaded at the server website.
Paul, Petra; van den Hoorn, Tineke; Jongsma, Marlieke L. M.; Bakker, Mark J.; Hengeveld, Rutger; Janssen, Lennert; Cresswell, Peter; Egan, David A.; van Ham, Marieke; ten Brinke, Anja; Ovaa, Huib; Beijersbergen, Roderick L.; Kuijl, Coenraad; Neefjes, Jacques
MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and
Hameed, Muhammad Salman; Wang, Zhengbing; Vasseur, Liette; Yang, Guang
Argonaute (Ago) protein family plays a key role in the RNA interference (RNAi) process in different insects including Lepidopteran. However, the role of Ago proteins in the RNAi pathway of Plutella xylostella is still unknown. We cloned an Argonaute3 gene in P. xylostella ( PxAgo3 ) with the complete coding sequence of 2832 bp. The encoded protein had 935 amino acids with an expected molecular weight of 108.9 kDa and an isoelectric point of 9.29. It contained a PAZ (PIWI/Argonaute/Zwile) domain and PIWI (P-element-induced whimpy testes) domain. PxAgo3 was classified into the Piwi subfamily of Ago proteins with a high similarity of 93.0% with Bombyx mori Ago3 (BmAgo3). The suppression of PxAgo3 by dsPxAgo3 was observed 3 h after treatment and was maintained until 24 h. Knockdown of PxAgo3 decreased the suppression level of PxActin by dsPxActin in P. xylostella cells, while overexpression of PxAgo3 increased the RNAi efficiency. Our results suggest that PxAgo3 play a key role in the double stranded RNA (dsRNA)-regulated RNAi pathway in P. xylostella .
Muhammad Salman Hameed
Full Text Available Argonaute (Ago protein family plays a key role in the RNA interference (RNAi process in different insects including Lepidopteran. However, the role of Ago proteins in the RNAi pathway of Plutella xylostella is still unknown. We cloned an Argonaute3 gene in P. xylostella (PxAgo3 with the complete coding sequence of 2832 bp. The encoded protein had 935 amino acids with an expected molecular weight of 108.9 kDa and an isoelectric point of 9.29. It contained a PAZ (PIWI/Argonaute/Zwile domain and PIWI (P-element-induced whimpy testes domain. PxAgo3 was classified into the Piwi subfamily of Ago proteins with a high similarity of 93.0% with Bombyx mori Ago3 (BmAgo3. The suppression of PxAgo3 by dsPxAgo3 was observed 3 h after treatment and was maintained until 24 h. Knockdown of PxAgo3 decreased the suppression level of PxActin by dsPxActin in P. xylostella cells, while overexpression of PxAgo3 increased the RNAi efficiency. Our results suggest that PxAgo3 play a key role in the double stranded RNA (dsRNA-regulated RNAi pathway in P. xylostella.
To develop low level of erucic acid in rapeseeds by intron-spliced hairpin RNA, an inverted repeat unit of a partial BnFAE1.1 gene interrupted by a spliceable intron ... In conclusion, the expression of endogenous BnFAE1.1 was efficiently silenced by the designed RNAi silencer, causing a significant down-regulation in the ...
Zhong, Rui; Dong, Xiaonan; Levine, Beth; Xie, Yang; Xiao, Guanghua
High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document. © 2014 Society for Laboratory Automation and Screening.
Broemer, Meike; Tenev, Tencho; Rigbolt, Kristoffer T G
-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1...
The Sheffield RNAi Screening Facility (SRSF) (www.rnai.group.shef.ac.uk) was established in 2008 with Wellcome Trust and University of Sheffield funding, with the task to provide the first UK RNAi screening resource for academic groups interested in identifying genes required in a diverse range of biological processes using Drosophila cell culture. The SRSF has carried out a wide range of screens varying in sizes from bespoke small-scale libraries, targeting a few hundred genes, to high-throughput, genome-wide studies. The SRSF has grown and improved with a dedicated partnership of its academic customers based mainly in the UK. We are part of the UK Academics Functional Genomics Network, participating in organizing an annual meeting in London and are part of the University of Sheffield's D3N (www.d3n.org.uk), connecting academics, biotech and pharmaceutical companies with a multidisciplinary network in Drug Discovery and Development. Recently, the SRSF has been funded by the Yorkshire Cancer Research Fund to perform genome-wide RNAi screens using human cells as part of a core facility for regional Yorkshire Universities and screens are now underway. Overall the SRSF has carried out more than 40 screens from Drosophila and human cell culture experiments.
Full Text Available Abstract Background RNA interference (RNAi is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly gene (corresponding to a putative gene CG5652/GM06434, that we named beltless based on an embryonic loss-of-function phenotype. Results Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. Conclusions We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should
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Calzada, Joan; Valletti, Tommaso
We develop a model of logit demand that extends to a multi-firm industry the traditional duopoly framework of network competition with access charges. Firstly, we show that, when incumbents do not face the threat of entry and compete in prices, they inefficiently establish the reciprocal access charge below cost. This inefficiency disappears if incumbents compete in utilities instead of prices. Secondly, we study how incumbents change their choices under the threat of entry when they determin...
Recent research suggests that adopting a common currency increases bilateral trade. In this paper, I explore experiences of currency union entry in the post-war period and find no effect on trade. Previous results derived from a large panel data set (covering more than 200 countries from 1948 through 1997) appear to depend crucially on the assumption of symmetry between currency union exits and entries: While countries leaving a currency union experience significant declines in trade, currenc...
Full Text Available The retinoblastoma (Rb tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.
Zotti, M J; Smagghe, G
The time has passed for us to wonder whether RNA interference (RNAi) effectively controls pest insects or protects beneficial insects from diseases. The RNAi era in insect science began with studies of gene function and genetics that paved the way for the development of novel and highly specific approaches for the management of pest insects and, more recently, for the treatment and prevention of diseases in beneficial insects. The slight differences in components of RNAi pathways are sufficient to provide a high degree of variation in responsiveness among insects. The current framework to assess the negative effects of genetically modified (GM) plants on human health is adequate for RNAi-based GM plants. Because of the mode of action of RNAi and the lack of genomic data for most exposed non-target organisms, it becomes difficult to determine the environmental risks posed by RNAi-based technologies and the benefits provided for the protection of crops. A better understanding of the mechanisms that determine the variability in the sensitivity of insects would accelerate the worldwide release of commercial RNAi-based approaches.
Full Text Available RNAi is a ubiquitous pathway that serves central functions throughout eukaryotes, including maintenance of genome stability and repression of transposon expression and movement. However, a number of organisms have lost their RNAi pathways, including the model yeast Saccharomyces cerevisiae, the maize pathogen Ustilago maydis, the human pathogen Cryptococcus deuterogattii, and some human parasite pathogens, suggesting there may be adaptive benefits associated with both retention and loss of RNAi. By comparing the RNAi-deficient genome of the Pacific Northwest Outbreak C. deuterogattii strain R265 with the RNAi-proficient genomes of the Cryptococcus pathogenic species complex, we identified a set of conserved genes that were lost in R265 and all other C. deuterogattii isolates examined. Genetic and molecular analyses reveal several of these lost genes play roles in RNAi pathways. Four novel components were examined further. Znf3 (a zinc finger protein and Qip1 (a homolog of N. crassa Qip were found to be essential for RNAi, while Cpr2 (a constitutive pheromone receptor and Fzc28 (a transcription factor are involved in sex-induced but not mitosis-induced silencing. Our results demonstrate that the mitotic and sex-induced RNAi pathways rely on the same core components, but sex-induced silencing may be a more specific, highly induced variant that involves additional specialized or regulatory components. Our studies further illustrate how gene network polymorphisms involving known components of key cellular pathways can inform identification of novel elements and suggest that RNAi loss may have been a core event in the speciation of C. deuterogattii and possibly contributed to its pathogenic trajectory.
Full Text Available Abstract Background The Ahringer C. elegans RNAi feeding library prepared by cloning genomic DNA fragments has been widely used in genome-wide analysis of gene function. However, the library has not been thoroughly validated by direct sequencing, and there are potential errors, including: 1 mis-annotation (the clone with the retired gene name should be remapped to the actual target gene; 2 nonspecific PCR amplification; 3 cross-RNAi; 4 mis-operation such as sample loading error, etc. Results Here we performed a reliability analysis on the Ahringer C. elegans RNAi feeding library, which contains 16,256 bacterial strains, using a bioinformatics approach. Results demonstrated that most (98.3% of the bacterial strains in the library are reliable. However, we also found that 2,851 (17.54% bacterial strains need to be re-annotated even they are reliable. Most of these bacterial strains are the clones having the retired gene names. Besides, 28 strains are grouped into unreliable category and 226 strains are marginal because of probably expressing unrelated double-stranded RNAs (dsRNAs. The accuracy of the prediction was further confirmed by direct sequencing analysis of 496 bacterial strains. Finally, a freely accessible database named CelRNAi (http://biocompute.bmi.ac.cn/CelRNAi/ was developed as a valuable complement resource for the feeding RNAi library by providing the predicted information on all bacterial strains. Moreover, submission of the direct sequencing result or any other annotations for the bacterial strains to the database are allowed and will be integrated into the CelRNAi database to improve the accuracy of the library. In addition, we provide five candidate primer sets for each of the unreliable and marginal bacterial strains for users to construct an alternative vector for their own RNAi studies. Conclusions Because of the potential unreliability of the Ahringer C. elegans RNAi feeding library, we strongly suggest the user examine
Full Text Available Lepidoptera comprise some of the most devastating herbivorous pest insects worldwide. One of the most promising novel pest control strategies is exploiting the RNA interference (RNAi mechanism to target essential genes for knockdown and incite toxic effects in the target species without harming other organisms in the ecosystem. However, many insects are refractory to oral RNAi, often due to rapid degradation of ingested dsRNA in their digestive system. This is the case for many lepidopteran insects, including the beet armyworm Spodoptera exigua, which is characterized by a very alkaline gut environment (pH > 9.0 and a strong intestinal nucleolytic activity. In this research, guanidine-containing polymers were developed to protect dsRNA against nucleolytic degradation, specifically in high pH environments. First, their ability to protect dsRNA against nucleolytic degradation in gut juice of the beet armyworm S. exigua was investigated ex vivo. Polymers with high guanidine content provided a strong protection against nucleolytic degradation at pH 11, protecting the dsRNA for up to 30 h. Next, cellular uptake of the dsRNA and the polyplexes in lepidopteran CF203 midgut cells was investigated by confocal microscopy, showing that the polymer also enhanced cellular uptake of the dsRNA. Finally, in vivo feeding RNAi bioassays demonstrated that using these guanidine-containing polymer nanoparticles led to an increased RNAi efficiency in S. exigua. Targeting the essential gene chitin synthase B, we observed that the mortality increased to 53% in the polymer-protected dsRNA treatment compared to only 16% with the naked dsRNA and found that polymer-protected dsRNA completely halted the development of the caterpillars. These results show that using guanylated polymers as a formulation strategy can prevent degradation of dsRNA in the alkaline and strongly nucleolytic gut of lepidopteran insects. Furthermore, the polymer also enhances cellular uptake in
Full Text Available Zhenghui Wang,1 Xiaoli Li,2 Xi-Jing He,3 Xianghong Zhang,1 Zhuangqun Yang,4 Min Xu,1 Baojun Wu,1 Junbo Tu,5 Huanan Luo,1 Jing Yan11Department of Otolaryngology – Head and Neck Surgery, 2Department of Dermatology, 3Department of Orthopedics, The Second Hospital, Xi’an Jiaotong University, 4Department of Plastic and Burns Surgery, The First Hospital, Xi’an Jiaotong University, 5Department of Oral and Maxillofacial Plastic Surgery, The Stomatological Hospital, Xi’an Jiaotong University, Xi’an, People’s Republic of ChinaPurpose: To determine the effects of RNA interference (RNAi on chondrocyte proliferation, function, and immunological rejection after allogenic tissue-engineered cartilage transplantation within bone matrix gelatin scaffolds.Methods: Seven million rat normal and RNAi chondrocytes were harvested and separately composited with fibrin glue to make the cell suspension, and then transplanted subcutaneously into the back of Sprague Dawley rats after being cultured for 10 days in vitro. Untransplanted animals served as the control group. The allograft and immunological response were examined at 1, 2, 4, 8, and 12 months postoperatively with hematoxylin and eosin histochemical staining, immunohistochemical staining (aggrecan, type II collagen, class I and II major histocompatibility complex, and flow cytometry for peripheral blood cluster of differentiation 4+ (CD4+ and CD8+ T-cells.Results: There was no infection or death in the rats except one, which died in the first week. Compared to the control group, the RNAi group had fewer eukomonocytes infiltrated, which were only distributed around the graft. The ratio of CD4+/CD8+ T-cells in the RNAi group was significantly lower than the normal one (P<0.05. There were many more positively stained chondrocytes and positively stained areas around the cells in the RNAi group, which were not found in the control group.Conclusion: The aggrecanase-1 and aggrecanase-2 RNAi for chondrocytes
Putzbach, William; Gao, Quan Q; Patel, Monal; Haluck-Kangas, Ashley; Murmann, Andrea E; Peter, Marcus E
Off-target effects (OTEs) represent a significant caveat for RNAi caused by substantial complementarity between siRNAs and unintended mRNAs. We now discuss the existence of three types of seed-dependent OTEs (sOTEs). Type I involves unintended targeting through the guide strand seed of an siRNA. Type II is caused by the activity of the seed on the designated siRNA passenger strand when loaded into the RNA-induced silencing complex (RISC). Both type I and II sOTEs will elicit unpredictable cellular responses. By contrast, in sOTE type III the guide strand seed preferentially targets essential survival genes resulting in death induced by survival gene elimination (DISE). In this Opinion article, we discuss DISE as a consequence of RNAi that may preferentially affect cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.
Lo Thi Mai Thu
Full Text Available Soybean mosaic virus (SMV and bean yellow mosaic virus (BYMV are two typical types of viruses that cause mosaic in soybean plants. Multiple viral infections at the same site can lead to 66% to 80% yield reduction. We have aimed to improve SMV and BYMV resistance in Vietnamese soybeans using gene transfer techniques under the mechanism of RNAi. In this study, we present newly generated transgenic tobacco plants carrying RNAi [CPi (SMV-BYMV] resistance to the two types of viruses; 73.08% of transgenic tobacco lines proved to be fully resistant to SMV and BYMV. In addition, the number of virus copies in transgenic tobacco plants was reduced on average by more than 51% compared to the control plants (wild type. This promising result shows the potential of transerring the CPi (SMV-BYMV structure in soybean to increase resistance of soybean to SMV and BYMV and advance the aims of antiviral soybean breeding in Vietnam.
Shekhar Kumar; Bijendra Kumar; Gelatar, J.K.; Pranay Kumar Sinha; Alok Kumar Mishra; Kamachi Mudali, U.
A study on direct dissolution of g-level metallic U and U-6 % Zr alloy bits by TBP-nitric acid adduct followed by in situ extraction at ambient pressures under batch and dynamic conditions was performed. The product organic solution was adjusted to 30 % TBP in dodecane and from it, U(VI) could be stripped quantitatively with 0.01 N nitric acid. Experimental results of the study are presented in this paper. (author)
Ahmed, Mohamed M S; Bian, Shiquan; Wang, Muyue; Zhao, Jing; Zhang, Bingwei; Liu, Qiaoquan; Zhang, Changquan; Tang, Shuzhu; Gu, Minghong; Yu, Hengxiu
Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, causes significant economic losses in rice production in China and many other Asian countries. Development of resistant varieties by using conventional breeding methods is limited, as germplasm with high level of resistance to RBSDV have not yet been found. One of the most promising methods to confer resistance against RBSDV is the use of RNA interference (RNAi) technology. RBSDV non-structural protein P7-2, encoded by S7-2 gene, is a potential F-box protein and involved in the plant-virus interaction through the ubiquitination pathway. P8, encoded by S8 gene, is the minor core protein that possesses potent active transcriptional repression activity. In this study, we transformed rice calli using a mini-twin T-DNA vector harboring RNAi constructs of the RBSDV genes S7-2 or S8, and obtained plants harboring the target gene constructs and the selectable marker gene, hygromycin phosphotransferase (HPT). From the offspring of these transgenic plants, we obtained selectable marker (HPT gene)-free plants. Homozygous T 5 transgenic lines which harbored either S7-2-RNAi or S8-RNAi exhibited high level resistance against RBSDV under field infection pressure from indigenous viruliferous small brown planthoppers. Thus, our results showed that RNA interference with the expression of S7-2 or S8 genes seemed an effective way to induce high level resistance in rice against RBSD disease.
Ahuja, Richa; Kumar, Vijay
RNA polymerase III transcribes structurally diverse group of essential noncoding RNAs including 5S ribosomal RNA (5SrRNA) and U6 snRNA. These noncoding RNAs are involved in RNA processing and ribosome biogenesis, thus, coupling Pol III activity to the rate of protein synthesis, cell growth, and proliferation. Even though a few Pol II-associated transcription factors have been reported to participate in Pol III-dependent transcription, its activation by activator protein 1 (AP-1) factors, c-Fos and c-Jun, has remained unexplored. Here, we show that c-Fos and c-Jun bind to specific sites in the regulatory regions of 5S rRNA (type I) and U6 snRNA (type III) gene promoters and stimulate their transcription. Our chromatin immunoprecipitation studies suggested that endogenous AP-1 factors bind to their cognate promoter elements during the G1/S transition of cell cycle apparently synchronous with Pol III transcriptional activity. Furthermore, the interaction of c-Jun with histone acetyltransferase p300 promoted the recruitment of p300/CBP complex on the promoters and facilitated the occupancy of Pol III transcriptional machinery via histone acetylation and chromatin remodeling. The findings of our study, together, suggest that AP-1 factors are novel regulators of Pol III-driven 5S rRNA and U6 snRNA expression with a potential role in cell proliferation. © 2017 Federation of European Biochemical Societies.
Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P; Mak, Ho Yi
RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments. Copyright © 2014 Elsevier Ltd. All rights reserved.
Arora, Shilpi; Gonzales, Irma M; Hagelstrom, R Tanner; Beaudry, Christian; Choudhary, Ashish; Sima, Chao; Tibes, Raoul; Mousses, Spyro; Azorsa, David O
Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.
Mark G Sterken
Full Text Available Orsay virus (OrV is the first virus known to be able to complete a full infection cycle in the model nematode species Caenorhabditis elegans. OrV is transmitted horizontally and its infection is limited by antiviral RNA interference (RNAi. However, we have no insight into the kinetics of OrV replication in C. elegans. We developed an assay that infects worms in liquid, allowing precise monitoring of the infection. The assay revealed a dual role for the RNAi response in limiting Orsay virus infection in C. elegans. Firstly, it limits the progression of the initial infection at the step of recognition of dsRNA. Secondly, it provides an inherited protection against infection in the offspring. This establishes the heritable RNAi response as anti-viral mechanism during OrV infections in C. elegans. Our results further illustrate that the inheritance of the anti-viral response is important in controlling the infection in the canonical wild type Bristol N2. The OrV replication kinetics were established throughout the worm life-cycle, setting a standard for further quantitative assays with the OrV-C. elegans infection model.
Knorr, Eileen; Fishilevich, Elane; Tenbusch, Linda; Frey, Meghan L F; Rangasamy, Murugesan; Billion, Andre; Worden, Sarah E; Gandra, Premchand; Arora, Kanika; Lo, Wendy; Schulenberg, Greg; Valverde-Garcia, Pablo; Vilcinskas, Andreas; Narva, Kenneth E
RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T 0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.
Garcia-Martin, Juan Antonio; Dotu, Ivan; Clote, Peter
Several algorithms for RNA inverse folding have been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been experimentally validated. The RNAiFold software is unique among approaches for inverse folding in that (exhaustive) constraint programming is used instead of heuristic methods. For that reason, RNAiFold can generate all sequences that fold into the target structure or determine that there is no solution. RNAiFold 2.0 is a complete overhaul of RNAiFold 1.0, rewritten from the now defunct COMET language to C++. The new code properly extends the capabilities of its predecessor by providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapping coding sequences; moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules. the web server, source code and linux binaries are publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold2.0. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
van der Made, Allard; Schoonbeek, Lambert
We consider a model of vertical product differentiation where consumers care about the environmental damage their consumption causes. An environmental group is capable of increasing consumers' environmental concern via a costly campaign. We show that the prospect of such a campaign can induce entry
Ebola virus is an enveloped virus with filamentous structure and causes a severe hemorrhagic fever in human and nonhuman primates. Host cell entry is the first essential step in the viral life cycle, which has been extensively studied as one of the therapeutic targets. A virus factor of cell entry is a surface glycoprotein (GP), which is an only essential viral protein in the step, as well as the unique particle structure. The virus also interacts with a lot of host factors to successfully enter host cells. Ebola virus at first binds to cell surface proteins and internalizes into cells, followed by trafficking through endosomal vesicles to intracellular acidic compartments. There, host proteases process GPs, which can interact with an intracellular receptor. Then, under an appropriate circumstance, viral and endosomal membranes are fused, which is enhanced by major structural changes of GPs, to complete host cell entry. Recently the basic research of Ebola virus infection mechanism has markedly progressed, largely contributed by identification of host factors and detailed structural analyses of GPs. This article highlights the mechanism of Ebola virus host cell entry, including recent findings.
Full Text Available Flaviviruses enter host cells by endocytosis initiated when the virus particles interact with cell surface receptors. The current model suggests that flaviviruses use at least two different sets of molecules for infectious entry: attachment factors that concentrate and/or recruit viruses on the cell surface and primary receptor(s that bind to virions and direct them to the endocytic pathway. Here, we present the currently available knowledge regarding the flavivirus receptors described so far with specific attention to C-type lectin receptors and the phosphatidylserine receptors, T-cell immunoglobulin and mucin domain (TIM and TYRO3, AXL and MER (TAM. Their role in flavivirus attachment and entry as well as their implication in the virus biology will be discussed in depth.
Taylor, M.J.; Keeney, J.A.; Wendel, M.W.; Demint, A.L.
The Oak Ridge Y-12 Plant (Y-12) is conducting highly coupled experimental and numerical studies to develop the technology needed to produce near-net-shape (NNS)-cast uranium-6 wt% niobium (U-6Nb) components which have a controlled carbon content. Current activities are focused on defining mechanical and metallurgical properties of cast material; experimental studies to define NNS casting, carbide particle flotation, and immersion-quench physics; and developing the numerical models needed to support the optimized design of NNS components. This paper summarizes the material characterization, experiment design, and model development activities
Stefanova, A.; Grudev, P.; Gencheva, R.
This paper presents the results from sensitivity calculations made with MEDICIS(ASTECv2) for investigation of the crust formation during the Molten Corium-Concrete Interaction(MCCI) in VB-U5 and VB-U6 VULCANO tests. All calculations are made with MEDICIS computer code. The main goal of these analyses is to assess how the assumptions for crust formation or not formation influence over the concrete ablation. Three calculations have been done for each one of the experiments with different crust thickness and lock of crust formation at the bottom, side and upper surface. (authors)
Wang, Shaowei; Wei, Xiaochun; Sun, Xiaojuan; Chen, Chongwei; Zhou, Jingming; Zhang, Ge; Wu, Heng; Guo, Baosheng
Background Cartilage degeneration affects millions of people but preventing its degeneration is a big challenge. Although RNA interference (RNAi) has been used in human trials via silencing specific genes, the cartilage RNAi has not been possible to date because the cartilage is an avascular and very dense tissue with very low permeability. Purpose The objective of this study was to develop and validate a novel lipid nanoparticle (LNP)-siRNA delivery system that can prevent cartilage degeneration by knocking down specific genes. Methods LNP transfection efficiency was evaluated in vitro and ex vivo. Indian Hedgehog (Ihh) has been correlated with cartilage degeneration. The in vivo effects of LNP-Ihh siRNA complexes on cartilage degeneration were evaluated in a rat model of surgery-induced osteoarthritis (OA). Results In vitro, 100% of chondrocytes were transfected with siRNA in the LNP-siRNA group. In accordance with the cell culture results, red positive signals could be detected even in the deep layer of cartilage tissue cultures treated by LNP-beacon. In vivo data showed that LNP is specific for cartilage, since positive signals were detected by fluorescence molecular tomography and confocal microscopy in joint cartilage injected with LNP-beacon, but not on the surface of the synovium. In the rat model of OA, intraarticular injection of LNP-Ihh siRNA attenuated OA progression, and PCR results showed LNP-Ihh siRNA exerted a positive impact on anabolic metabolism and negative impact on catabolic metabolism. Conclusion This study demonstrates that our LNP-RNAi delivery system has a significantly chondroprotective effect that attenuates cartilage degeneration and holds great promise as a powerful tool for treatment of cartilage diseases by knocking down specific genes. PMID:29440889
Wang, Shaowei; Wei, Xiaochun; Sun, Xiaojuan; Chen, Chongwei; Zhou, Jingming; Zhang, Ge; Wu, Heng; Guo, Baosheng; Wei, Lei
Cartilage degeneration affects millions of people but preventing its degeneration is a big challenge. Although RNA interference (RNAi) has been used in human trials via silencing specific genes, the cartilage RNAi has not been possible to date because the cartilage is an avascular and very dense tissue with very low permeability. The objective of this study was to develop and validate a novel lipid nanoparticle (LNP)-siRNA delivery system that can prevent cartilage degeneration by knocking down specific genes. LNP transfection efficiency was evaluated in vitro and ex vivo. Indian Hedgehog ( Ihh ) has been correlated with cartilage degeneration. The in vivo effects of LNP-Ihh siRNA complexes on cartilage degeneration were evaluated in a rat model of surgery-induced osteoarthritis (OA). In vitro, 100% of chondrocytes were transfected with siRNA in the LNP-siRNA group. In accordance with the cell culture results, red positive signals could be detected even in the deep layer of cartilage tissue cultures treated by LNP-beacon. In vivo data showed that LNP is specific for cartilage, since positive signals were detected by fluorescence molecular tomography and confocal microscopy in joint cartilage injected with LNP-beacon, but not on the surface of the synovium. In the rat model of OA, intraarticular injection of LNP-Ihh siRNA attenuated OA progression, and PCR results showed LNP-Ihh siRNA exerted a positive impact on anabolic metabolism and negative impact on catabolic metabolism. This study demonstrates that our LNP-RNAi delivery system has a significantly chondroprotective effect that attenuates cartilage degeneration and holds great promise as a powerful tool for treatment of cartilage diseases by knocking down specific genes.
Victor L Jensen
Full Text Available The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366; sdf-9(m708], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c. Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85, sdf-9(m708, and the wild-type N2 (at 27°C were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130 background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.
Kumar, Sanjeev; Tanti, Bhaben; Patil, Basavaprabhu L; Mukherjee, Sunil Kumar; Sahoo, Lingaraj
Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea.
Zanotto-Filho, Alfeu; Dashnamoorthy, Ravi; Loranc, Eva; de Souza, Luis H T; Moreira, José C F; Suresh, Uthra; Chen, Yidong; Bishop, Alexander J R
Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.
Full Text Available Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair, DNA-mRNA-protein metabolism (transcription/translation and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress/Unfolded Protein Responses (UPR in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.
Reich, Daniel P; Tyc, Katarzyna M; Bass, Brenda L
Cellular dsRNAs are edited by adenosine deaminases that act on RNA (ADARs). While editing can alter mRNA-coding potential, most editing occurs in noncoding sequences, the function of which is poorly understood. Using dsRNA immunoprecipitation (dsRIP) and RNA sequencing (RNA-seq), we identified 1523 regions of clustered A-to-I editing, termed editing-enriched regions (EERs), in four stages of Caenorhabditis elegans development, often with highest expression in embryos. Analyses of small RNA-seq data revealed 22- to 23-nucleotide (nt) siRNAs, reminiscent of viral siRNAs, that mapped to EERs and were abundant in adr-1;adr-2 mutant animals. Consistent with roles for these siRNAs in silencing, EER-associated genes (EAGs) were down-regulated in adr-1;adr-2 embryos, and this was dependent on associated EERs and the RNAi factor RDE-4. We observed that ADARs genetically interact with the 26G endogenous siRNA (endo-siRNA) pathway, which likely competes for RNAi components; deletion of factors required for this pathway ( rrf-3 or ergo-1 ) in adr-1;adr-2 mutant strains caused a synthetic phenotype that was rescued by deleting antiviral RNAi factors. Poly(A) + RNA-seq revealed EAG down-regulation and antiviral gene induction in adr-1;adr-2;rrf-3 embryos, and these expression changes were dependent on rde-1 and rde-4 Our data suggest that ADARs restrict antiviral silencing of cellular dsRNAs. © 2018 Reich et al.; Published by Cold Spring Harbor Laboratory Press.
Full Text Available Recent progress suggests gene therapy may one day be an option for treating some forms of limb girdle muscular dystrophy (LGMD. Nevertheless, approaches targeting LGMD have so far focused on gene replacement strategies for recessive forms of the disease. In contrast, no attempts have been made to develop molecular therapies for any of the eight dominantly inherited forms of LGMD. Importantly, the emergence of RNA interference (RNAi therapeutics in the last decade provided new tools to combat dominantly inherited LGMDs with molecular therapy. In this study, we describe the first RNAi-based, preclinical gene therapy approach for silencing a gene associated with dominant LGMD. To do this, we developed adeno-associated viral vectors (AAV6 carrying designed therapeutic microRNAs targeting mutant myotilin (MYOT, which is the underlying cause of LGMD type 1A (LGMD1A. Our best MYOT-targeted microRNA vector (called miMYOT significantly reduced mutant myotilin mRNA and soluble protein expression in muscles of LGMD1A mice (the TgT57I model both 3 and 9 months after delivery, demonstrating short- and long-term silencing effects. This MYOT gene silencing subsequently decreased deposition of MYOT-seeded intramuscular protein aggregates, which is the hallmark feature of LGMD1A. Histological improvements were accompanied by significant functional correction, as miMYOT-treated animals showed increased muscle weight and improved specific force in the gastrocnemius, which is one of the most severely affected muscles in TgT57I mice and patients with dominant myotilin mutations. These promising results in a preclinical model of LGMD1A support the further development of RNAi-based molecular therapy as a prospective treatment for LGMD1A. Furthermore, this study sets a foundation that may be refined and adapted to treat other dominant LGMD and related disorders.
Hashai, Niron; Geisler Asmussen, Christian; Benito, Gabriel
diversity across value chain activities and host markets. Analyzing a sample of Israeli based firms we show that larger firms exhibit a higher degree of entry mode diversity both across value chain activities and across host markets. Higher levels of knowledge intensity are also associated with more......This paper expands entry mode literature by referring to multiple modes exerted in different value chain activities within and across host markets, rather than to a single entry mode at the host market level. Scale of operations and knowledge intensity are argued to affect firms' entry mode...... diversity in firms' entry modes across both dimensions....
Morton, F M
Data on all generic drug entries in the period 1984-1994 are used to estimate which markets heterogeneous potential entrants will decide to enter. I find that organizational experience predicts entry. Firms tend to enter markets with supply and demand characteristics similar to the firm's existing drugs. Larger revenue markets, markets with more hospital sales, and products that treat chronic conditions attract more entry. The simultaneous nature of entry leads to an additional interpretation: specialization is profitable because of the severe risk to profits when a market is "overentered." However, I am unable to make any conclusions about the efficiency of entry decisions.
Wu, Ke; Hoy, Marjorie A.
Clathrin heavy chain has been shown to be important for viability, embryogenesis, and RNA interference (RNAi) in arthropods such as Drosophila melanogaster. However, the functional roles of clathrin heavy chain in chelicerate arthropods, such as the predatory mite Metaseiulus occidentalis, remain unknown. We previously showed that dsRNA ingestion, followed by feeding on spider mites, induced systemic and robust RNAi in M. occidentalis females. In the current study, we performed a loss-of-function analysis of the clathrin heavy chain gene in M. occidentalis using RNAi. We showed that ingestion of clathrin heavy chain dsRNA by M. occidentalis females resulted in gene knockdown and reduced longevity. In addition, clathrin heavy chain dsRNA treatment almost completely abolished oviposition by M. occidentalis females and the few eggs produced did not hatch. Finally, we demonstrated that clathrin heavy chain gene knockdown in M. occidentalis females significantly reduced a subsequent RNAi response induced by ingestion of cathepsin L dsRNA. The last finding suggests that clathrin heavy chain may be involved in systemic RNAi responses mediated by orally delivered dsRNAs in M. occidentalis. PMID:25329675
Månsson, Mats David Joakim
in a process termed ER-associated degradation (ERAD). This mechanism proceeds through four steps involving recognition, dislocation, ubiquitination and proteasomal degradation. This report describes a high-throughput screening method for identification of hitherto unknown pathways for degradation. We present...... fluorescence-based RNAi screens in mammalian cells on TCR-α-GFP and HANSκLC, for identification of ERAD pathways. By validating the obtained screening hits we concluded that UBE2J2 is involved in TCR-α-GFP degradation, possibly by ubiquitination of C-terminal serine residues in TCR-α-GFP. Additionally, we also...
Auweter-Kurtz, M.; Endlich, P.; Herdrich, G.; Kurtz, H.; Laux, T.; Löhle, S.; Nazina, N.; Pidan, S.
Entering the atmosphere of celestial bodies, spacecrafts encounter gases at velocities of several km/s, thereby being subjected to great heat loads. The thermal protection systems and the environment (plasma) have to be investigated by means of computational and ground facility based simulations. For more than a decade, plasma wind tunnels at IRS have been used for the investigation of TPS materials. Nevertheless, ground tests and computer simulations cannot re- place space flights completely. Particularly, entry mission phases encounter challenging problems, such as hypersonic aerothermodynamics. Concerning the TPS, radiation-cooled materials used for reuseable spacecrafts and ablator tech- nologies are of importance. Besides the mentioned technologies, there is the goal to manage guidance navigation, con- trol, landing technology and inflatable technologies such as ballutes that aim to keep vehicles in the atmosphere without landing. The requirement to save mass and energy for planned interplanetary missions such as Mars Society Balloon Mission, Mars Sample Return Mission, Mars Express or Venus Sample Return mission led to the need for manoeuvres like aerocapture, aero-breaking and hyperbolic entries. All three are characterized by very high kinetic vehicle energies to be dissipated by the manoeuvre. In this field flight data are rare. The importance of these manoeuvres and the need to increase the knowledge of required TPS designs and behavior during such mission phases point out the need of flight experiments. As result of the experience within the plasma diagnostic tool development and the plasma wind tunnel data base, flight experiments like the PYrometric RE-entry EXperiment PYREX were developed, fully qualified and successfully flown. Flight experiments such as the entry spectrometer RESPECT and PYREX on HOPE-X are in the conceptual phase. To increase knowledge in the scope of atmospheric manoeuvres and entries, data bases have to be created combining both
Chin, Wei-Xin; Ang, Swee Kim; Chu, Justin Jang Hann
In invertebrate eukaryotes and prokaryotes, respectively, the RNAi and clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) pathways are highly specific and efficient RNA and DNA interference systems, and are well characterised as potent antiviral systems. It has become possible to recruit or reconstitute these pathways in mammalian cells, where they can be directed against desired host or viral targets. The RNAi and CRISPR-Cas systems can therefore yield ideal antiviral therapeutics, capable of specific and efficient viral inhibition with minimal off-target effects, but development of such therapeutics can be slow. This review covers recent advances made towards developing RNAi or CRISPR-Cas strategies for clinical use. These studies address the delivery, toxicity or target design issues that typically plague the in vivo or clinical use of these technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.
Steiner, Florian A; Okihara, Kristy L; Hoogstrate, Suzanne W; Sijen, Titia; Ketting, René F
RNA interference (RNAi) is a process in which double-stranded RNA is cleaved into small interfering RNAs (siRNAs) that induce the destruction of homologous single-stranded mRNAs. Argonaute proteins are essential components of this silencing process; they bind siRNAs directly and can cleave RNA targets using a conserved RNase H motif. In Caenorhabditis elegans, the Argonaute protein RDE-1 has a central role in RNAi. In animals lacking RDE-1, the introduction of double-stranded RNA does not trigger any detectable level of RNAi. Here we show that RNase H activity of RDE-1 is required only for efficient removal of the passenger strand of the siRNA duplex and not for triggering the silencing response at the target-mRNA level. These results uncouple the role of the RDE-1 RNase H activity in small RNA maturation from its role in target-mRNA silencing in vivo.
Sindhu, Anoop S; Maier, Tom R; Mitchum, Melissa G; Hussey, Richard S; Davis, Eric L; Baum, Thomas J
Cyst nematodes are highly evolved sedentary plant endoparasites that use parasitism proteins injected through the stylet into host tissues to successfully parasitize plants. These secretory proteins likely are essential for parasitism as they are involved in a variety of parasitic events leading to the establishment of specialized feeding cells required by the nematode to obtain nourishment. With the advent of RNA interference (RNAi) technology and the demonstration of host-induced gene silencing in parasites, a new strategy to control pests and pathogens has become available, particularly in root-knot nematodes. Plant host-induced silencing of cyst nematode genes so far has had only limited success but similarly should disrupt the parasitic cycle and render the host plant resistant. Additional in planta RNAi data for cyst nematodes are being provided by targeting four parasitism genes through host-induced RNAi gene silencing in transgenic Arabidopsis thaliana, which is a host for the sugar beet cyst nematode Heterodera schachtii. Here it is reported that mRNA abundances of targeted nematode genes were specifically reduced in nematodes feeding on plants expressing corresponding RNAi constructs. Furthermore, this host-induced RNAi of all four nematode parasitism genes led to a reduction in the number of mature nematode females. Although no complete resistance was observed, the reduction of developing females ranged from 23% to 64% in different RNAi lines. These observations demonstrate the relevance of the targeted parasitism genes during the nematode life cycle and, potentially more importantly, suggest that a viable level of resistance in crop plants may be accomplished in the future using this technology against cyst nematodes.
Full Text Available Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV and BM-40 from hemocytes and fat cells.
Dönitz, Jürgen; Schmitt-Engel, Christian; Grossmann, Daniela; Gerischer, Lizzy; Tech, Maike; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor
The iBeetle-Base (http://ibeetle-base.uni-goettingen.de) makes available annotations of RNAi phenotypes, which were gathered in a large scale RNAi screen in the red flour beetle Tribolium castaneum (iBeetle screen). In addition, it provides access to sequence information and links for all Tribolium castaneum genes. The iBeetle-Base contains the annotations of phenotypes of several thousands of genes knocked down during embryonic and metamorphic epidermis and muscle development in addition to phenotypes linked to oogenesis and stink gland biology. The phenotypes are described according to the EQM (entity, quality, modifier) system using controlled vocabularies and the Tribolium morphological ontology (TrOn). Furthermore, images linked to the respective annotations are provided. The data are searchable either for specific phenotypes using a complex 'search for morphological defects' or a 'quick search' for gene names and IDs. The red flour beetle Tribolium castaneum has become an important model system for insect functional genetics and is a representative of the most species rich taxon, the Coleoptera, which comprise several devastating pests. It is used for studying insect typical development, the evolution of development and for research on metabolism and pest control. Besides Drosophila, Tribolium is the first insect model organism where large scale unbiased screens have been performed. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
Vélez, Ana María; Jurzenski, Jessica; Matz, Natalie; Zhou, Xuguo; Wang, Haichuan; Ellis, Marion; Siegfried, Blair D
Maize plants expressing dsRNA for the management of Diabrotica virgifera virgifera are likely to be commercially available by the end of this decade. Honey bees, Apis mellifera, can potentially be exposed to pollen from transformed maize expressing dsRNA. Consequently, evaluation of the biological impacts of RNAi in honey bees is a fundamental component for ecological risk assessment. The insecticidal activity of a known lethal dsRNA target for D. v. virgifera, the vATPase subunit A, was evaluated in larval and adult honey bees. Activity of both D. v. virgifera (Dvv)- and A. mellifera (Am)-specific dsRNA was tested by dietary exposure to dsRNA. Larval development, survival, adult eclosion, adult life span and relative gene expression were evaluated. The results of these tests indicated that Dvv vATPase-A dsRNA has limited effects on larval and adult honey bee survival. Importantly, no effects were observed upon exposure of Am vATPase-A dsRNA suggesting that the lack of response involves factors other than sequence specificity. The results from this study provide guidance for future RNAi risk analyses and for the development of a risk assessment framework that incorporates similar hazard assessments. Copyright © 2015 Elsevier Ltd. All rights reserved.
Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed
Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.
Song, Guo-qing; Sink, Kenneth C; Walworth, Aaron E; Cook, Meridith A; Allison, Richard F; Lang, Gregory A
Prunus necrotic ringspot virus (PNRSV) is a major pollen-disseminated ilarvirus that adversely affects many Prunus species. In this study, an RNA interference (RNAi) vector pART27-PNRSV containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid (triploid) cherry rootstocks, 'Gisela 6' (GI 148-1) and 'Gisela 7'(GI 148-8)', which are tolerant and sensitive, respectively, to PNRSV infection. One year after inoculation with PNRSV plus Prune Dwarf Virus, nontransgenic 'Gisela 6' exhibited no symptoms but a significant PNRSV titre, while the transgenic 'Gisela 6' had no symptoms and minimal PNRSV titre. The nontransgenic 'Gisela 7' trees died, while the transgenic 'Gisela 7' trees survived. These results demonstrate the RNAi strategy is useful for developing viral resistance in fruit rootstocks, and such transgenic rootstocks may have potential to enhance production of standard, nongenetically modified fruit varieties while avoiding concerns about transgene flow and exogenous protein production that are inherent for transformed fruiting genotypes. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
Shukla, Jayendra Nath; Palli, Subba Reddy
Sex in insects is determined by a cascade of regulators ultimately controlling sex-specific splicing of a transcription factor, Doublesex (Dsx). We recently identified homolog of dsx in the red flour beetle, Tribolium castaneum (Tcdsx). Here, we report on the identification and characterization of a regulator of Tcdsx splicing in T. castaneum. Two male-specific and one female-specific isoforms of T. castaneum transformer (Tctra) were identified. RNA interference-aided knockdown of Tctra in pupa or adults caused a change in sex from females to males by diverting the splicing of Tcdsx pre-mRNA to male-specific isoform. All the pupa and adults developed from Tctra dsRNA injected final instar larvae showed male-specific sexually dimorphic structures. Tctra parental RNAi caused an elimination of females from the progeny resulting in production of all male progeny. Transformer parental RNAi could be used to produce all male population for use in pest control though sterile male release methods. PMID:22924109
Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M
The existence of an RNA-mediated silencing mechanism in the opportunistic fungal pathogen Mucor circinelloides was first described in the early 2000. Since then, Mucor has reached an outstanding position within the fungal kingdom as a model system to achieve a deeper understanding of regulation of endogenous functions by the RNA interference (RNAi) machinery. M. circinelloides combines diverse components of its RNAi machinery to carry out functions not only limited to the defense against invasive nucleic acids, but also to regulate expression of its own genes by producing different classes of endogenous small RNA molecules (esRNAs). The recent discovery of a novel RNase that participates in a new RNA degradation pathway adds more elements to the gene silencing-mediated regulation. This review focuses on esRNAs in M. circinelloides, the different pathways involved in their biogenesis, and their roles in regulating specific physiological and developmental processes in response to environmental signals, highlighting the complexity of silencing-mediated regulation in fungi. Copyright © 2015 Elsevier Inc. All rights reserved.
Rodrigues, Thais B; Rieske, Lynne K; J Duan, Jian; Mogilicherla, Kanakachari; Palli, Subba R
The ingestion of double-strand RNAs (dsRNA) targeting essential genes in an insect could cause mortality. Based on this principle, a new generation of insect control methods using RNA interference (RNAi) are being developed. In this work, we developed a bioassay for oral delivery of dsRNA to an invasive forest and urban tree pest, the emerald ash borer (EAB, Agrilus planipennis). EAB feeds and develops beneath the bark, killing trees rapidly. This behavior, coupled with the lack of a reliable artificial diet for rearing larvae and adults, make them difficult to study. We found that dsRNA is transported and processed to siRNAs by EAB larvae within 72 h after ingestion. Also, feeding neonate larvae with IAP (inhibitor of apoptosis) or COP (COPI coatomer, β subunit) dsRNA silenced their target genes and caused mortality. Both an increase in the concentration of dsRNA fed and sequential feeding of two different dsRNAs increased mortality. Here we provide evidence for successful RNAi in EAB, and demonstrate the development of a rapid and effective bioassay for oral delivery of dsRNA to screen additional genes.
Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C
Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.
Ibrahim, Abdulrazak B; Monteiro, Tatiane R; Cabral, Glaucia B; Aragão, Francisco J L
RNA interference (RNAi)-based transgenic technologies have evolved as potent biochemical tools for silencing specific genes of plant pathogens and pests. The approach has been demonstrated to be useful in silencing genes in insect species. Here, we report on the successful construction of RNAi-based plasmid containing an interfering cassette designed to generate dsRNAs that target a novel v-ATPase transcript in whitefly (Bemisia tabaci), an important agricultural pest in tropical and sub-tropical regions. The presence of the transgene was confirmed in T 0 and T 1 generations of transgenic lettuce lines, segregating in a Mendelian fashion. Seven lines were infested with whiteflies and monitored over a period of 32 days. Analysis of mortality showed that within five days of feeding, insects on transgenic plants showed a mortality rate of 83.8-98.1%. In addition, a reduced number of eggs (95 fold less) was observed in flies feeding on transgenic lettuce plants than insects on control lines. Quantitative reverse transcription PCR showed decreased expression level of endogenous v-ATPase gene in whiteflies feeding on transgenic plants. This technology is a foundation for the production of whitefly-resistant commercial crops, improving agricultural sustainability and food security, reducing the use of more environmentally aggressive methods of pest control.
Firnhaber Christopher B
Full Text Available Abstract Background The Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in Drosophila cell culture to identify genes that modify Notch-dependent transcription. Results Employing complementary data analyses, we found 399 putative modifiers: 189 promoting and 210 antagonizing Notch activated transcription. These modifiers included several known Notch interactors, validating the robustness of the assay. Many novel modifiers were also identified, covering a range of cellular localizations from the extracellular matrix to the nucleus, as well as a large number of proteins with unknown function. Chromatin-modifying proteins represent a major class of genes identified, including histone deacetylase and demethylase complex components and other chromatin modifying, remodeling and replacement factors. A protein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number of the identified proteins interact physically with these core chromatin components. Conclusions The genome-wide RNAi screen identified many genes that can modulate Notch transcriptional output. A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymes and remodelers that regulate Notch transcription. Our results open new avenues to explore the mechanisms of Notch signal regulation and the integration of this pathway into diverse cellular processes.
Castanotto, Daniela; Sakurai, Kumi; Lingeman, Robert; Li, Haitang; Shively, Louise; Aagaard, Lars; Soifer, Harris; Gatignol, Anne; Riggs, Arthur; Rossi, John J.
Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs. PMID:17660190
Familial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low density lipoprotein cholesterol (LDL-C) and increasing the risk of cardio vascular diseases. FH and many other liver diseases can possibly be treated with RNA interference (RNAi). RNAi is a natural process
Bai Hongbo; Zhang Jinfu; Zhou Xianrong
We study the energy level statistics of the SO(5) limit of super-symmetry U(6/4) in odd-A nucleus using the interacting boson-fermion model. The nearest neighbor spacing distribution (NSD) and the spectral rigidity (Δ 3 ) are investigated, and the factors that affect the properties of level statistics are also discussed. The results show that the boson number N is a dominant factor. If N is small, both the interaction strengths of subgroups SO B (5) and SO BF (5) and the spin play important roles in the energy level statistics, however, along with the increase of N, the statistics distribution would tend to be in Poisson form.
Ream, W.K.; Espinoza, J.
An entry control system to automatically control access to nuclear facilities is described. The design uses a centrally located console, integrated into the regular security system, to monitor the computer-controlled passage into and out of sensitive areas. Four types of entry control points are used: an unmanned enclosed portal with metal and SNM detectors for contraband detection with positive personnel identification, a bypass portal for contraband search after a contraband alarm in a regular portal also with positive personnel identification, a single door entry point with positive personnel identification, and a single door entry point with only a magnetic card-type identification. Security force action is required only as a response to an alarm. The integration of the entry control function into the security system computer is also described. The interface between the entry control system and the monitoring security personnel utilizing a color graphics display with touch screen input is emphasized. 2 refs., 7 figs
Gundersen, Anne Kathrine Navestad
As the mature markets of developed economies have become increasingly saturated, firms are turning their attention towards emerging markets for further enterprise growth. However, these countries often present significant challenges for foreign entrants, forcing firms to adapt their strategies to the new context. While MNEs? entry mode choice is an extensively studied field, there is a deficit in the entry mode research on SMEs, and even more so when it comes to entry into emerging markets in...
Kort, Peter M.; Wrzaczek, Stefan
The paper studies the incumbent-entrant problem in a fully dynamic setting. We find that under an open-loop information structure the incumbent anticipates entry by overinvesting, whereas in the Markov perfect equilibrium the incumbent slightly underinvests in the period before the entry. The entry cost level where entry accommodation passes into entry deterrence is lower in the Markov perfect equilibrium. Further we find that the incumbent’s capital stock level needed to deter entry is hump shaped as a function of the entry time, whereas the corresponding entry cost, where the entrant is indifferent between entry and non-entry, is U-shaped. PMID:26435573
D. A. Seliverstov
The prime focus in this article is on key findings concerning theoretical aspects of strategic behavior by incumbents to deter market entry of new firms. The author summarizes main lines of scientific research in the topic which give an insight into the patterns of the incumbent’s impact on the behavior of the entrants, the entry deterrence instruments and the consequences of these actions. Today the free entry markets are considered to be a rare phenomenon. The market entry of new firms is a...
One of the controls for information entered into the data bases created and maintained by the DOE Technical Information Center is the standardized name for the corporate entity or the corporate author. The purpose of Energy Information Data Base: Corporate Author Entries is to provide a means for the consistent citing of the names of organizations in bibliographic records. These entries serve as guides for users of the DOE/RECON computerized data bases who want to locate information originating in particular organizations. The entries in this revision include the corporate entries used in report bibliographic citations since 1973 and list approximately 28,000 corporate sources
Peng, S.S. [West Virginia University, Morgantown, WV (USA). Mining Engineering Dept.
The paper explains the applications of open entries in mining and different roof techniques to relieve longwall abutment pressures. The primary support for the open (or cut-through) entries and recovery rooms are normally similar to other development entries in the same mines. The supplementary supports installed can be divided into the following three types: complete backfill of open entries, supplemental roof and/or rib-bolt reinforcement only - no standing support and rows of standing support with or without supplemental roof and/or rib-bolt reinforcement. 3 figs.
Full Text Available Despite the unquestionable success of antiretroviral therapy (ART in the treatment of HIV infection, the cost, need for daily adherence, and HIV-associated morbidities that persist despite ART all underscore the need to develop a cure for HIV. The cure achieved following an allogeneic hematopoietic stem cell transplant (HSCT using HIV-resistant cells, and more recently, the report of short-term but sustained, ART-free control of HIV replication following allogeneic HSCT, using HIV susceptible cells, have served to both reignite interest in HIV cure research, and suggest potential mechanisms for a cure. In this review, we highlight some of the obstacles facing HIV cure research today, and explore the roles of gene therapy targeting HIV entry, and allogeneic stem cell transplantation in the development of strategies to cure HIV infection.
Foda, Bardees M.; Singh, Upinder
RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683
Foda, Bardees M; Singh, Upinder
RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Full Text Available Shaowei Wang,1 Xiaochun Wei,1 Xiaojuan Sun,1 Chongwei Chen,1 Jingming Zhou,2 Ge Zhang,3 Heng Wu,3 Baosheng Guo,3 Lei Wei1,2 1Department of Orthopaedics, The 2nd Hospital of Shanxi Medical University, Taiyuan, Shanxi, China; 2Department of Orthopaedics, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI, USA; 3Integrated Traditional Chinese and Western Medicine, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong Background: Cartilage degeneration affects millions of people but preventing its degeneration is a big challenge. Although RNA interference (RNAi has been used in human trials via silencing specific genes, the cartilage RNAi has not been possible to date because the cartilage is an avascular and very dense tissue with very low permeability. Purpose: The objective of this study was to develop and validate a novel lipid nanoparticle (LNP-siRNA delivery system that can prevent cartilage degeneration by knocking down specific genes. Methods: LNP transfection efficiency was evaluated in vitro and ex vivo. Indian Hedgehog (Ihh has been correlated with cartilage degeneration. The in vivo effects of LNP-Ihh siRNA complexes on cartilage degeneration were evaluated in a rat model of surgery-induced osteoarthritis (OA. Results: In vitro, 100% of chondrocytes were transfected with siRNA in the LNP-siRNA group. In accordance with the cell culture results, red positive signals could be detected even in the deep layer of cartilage tissue cultures treated by LNP-beacon. In vivo data showed that LNP is specific for cartilage, since positive signals were detected by fluorescence molecular tomography and confocal microscopy in joint cartilage injected with LNP-beacon, but not on the surface of the synovium. In the rat model of OA, intraarticular injection of LNP-Ihh siRNA attenuated OA progression, and PCR results showed LNP-Ihh siRNA exerted a positive impact on anabolic metabolism and negative
Department of Transportation — The dataset is known as “Border Crossing/Entry Data.” The Bureau of Transportation Statistics (BTS) Border Crossing/Entry Data provides summary statistics to the...
Steiner, F.A.; Okihara, K.L.; Hoogstrate, S.W.; Sijen, T.; Ketting, R.F.
RNA interference (RNAi) is a process in which double-stranded RNA is cleaved into small interfering RNAs (siRNAs) that induce the destruction of homologous single-stranded mRNAs. Argonaute proteins are essential components of this silencing process; they bind siRNAs directly and can cleave RNA
Wallace, Lindsay M; Saad, Nizar Y; Pyne, Nettie K; Fowler, Allison M; Eidahl, Jocelyn O; Domire, Jacqueline S; Griffin, Danielle A; Herman, Adam C; Sahenk, Zarife; Rodino-Klapac, Louise R; Harper, Scott Q
RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement, with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal-dominant disorder facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene DUX4 using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s). We previously demonstrated proof of concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 overexpression and sequence-specific off-target silencing. In this study, we describe improvements in vector design and expansion of our miDUX4 sequence repertoire and report differential toxicity elicited by two miDUX4 sequences, of which one was toxic and the other was not. This study provides important data to help advance our goal of translating RNAi gene therapy for facioscapulohumeral muscular dystrophy.
Kennedy, Lisa M; Grishok, Alla
Endogenous short RNAs and the conserved plant homeodomain (PHD) zinc-finger protein ZFP-1/AF10 regulate overlapping sets of genes in Caenorhabditis elegans, which suggests that they control common biological pathways. We have shown recently that the RNAi factor RDE-4 and ZFP-1 negatively modulate transcription of the insulin/PI3 signaling-dependent kinase PDK-1 to promote C. elegans fitness. Moreover, we have demonstrated that the insulin/IGF-1-PI3K-signaling pathway regulates the activity of the DAF-16/FOXO transcription factor in the hypodermis to nonautonomously promote the anterior migrations of the hermaphrodite-specific neurons (HSNs) during embryogenesis of C. elegans. In this study, we implicate the PHD-containing isoform of ZFP-1 and endogenous RNAi in the regulation of HSN migration. ZFP-1 affects HSN migration in part through its negative effect on pdk-1 transcription and modulation of downstream DAF-16 activity. We also identify a novel role for ZFP-1 and RNAi pathway components, including RDE-4, in the regulation of HSN migration in parallel with DAF-16. Therefore, the coordinated activities of DAF-16, ZFP-1, and endogenous RNAi contribute to gene regulation during development to ensure proper neuronal positioning.
Danielsen, Erik Thomas
and duration required for juvenile-adult transition. This PhD project demonstrates the power of Drosophila genetics by taking an in vivo genome-wide RNAi screening approach to uncover genes required for the function of steroid producing tissue and developmental maturation. In total, 1909 genes were found...... to be required for the prothoracic gland function and affected the developmental timing for the juvenile-adult transition. Among the screen hits, we focused on an uncharacterized gene, sit (CG5278), which is highly expressed in the gland and is required for ecdysone production. Sit is a homolog of mammalian very...... flux of cholesterol uptake in the gland cells and affected the endosomal trafficking. Therefore this gene was suggested to be named stuck in traffic (sit). Sit’s role in cholesterol uptake was also supported by the observation that the developmental delayed phenotype from loss of sit expression...
Sun, Hongyan; Chen, Zhong-Hua; Chen, Fei
Background Understanding the mechanism of low Cd accumulation in crops is crucial for sustainable safe food production in Cd-contaminated soils. Results Confocal microscopy, atomic absorption spectrometry, gas exchange and chlorophyll fluorescence analyses revealed a distinct difference in Cd...... with a substantial difference between the two genotypes. Cd stress led to higher expression of genes involved in transport, carbohydrate metabolism and signal transduction in the low-grain-Cd-accumulating genotype. Novel transporter genes such as zinc transporter genes were identified as being associated with low Cd...... accumulation. Quantitative RT-PCR confirmed our microarray data. Furthermore, suppression of the zinc transporter genes HvZIP3 and HvZIP8 by RNAi silencing showed increased Cd accumulation and reduced Zn and Mn concentrations in barley grains. Thus, HvZIP3 and HvZIP8 could be candidate genes related to low...
Gengyo-Ando, Keiko; Yoshina, Sawako; Inoue, Hideshi; Mitani, Shohei
In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusion gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode
O'Donnell, Christopher D.; Kovacs, Maria; Akhtar, Jihan; Valyi-Nagy, Tibor; Shukla, Deepak
Heparan sulfate (HS) proteoglycans are commonly exploited by multiple viruses for initial attachment to host cells. Herpes simplex virus-1 (HSV-1) is unique because it can use HS for both attachment and penetration, provided specific binding sites for HSV-1 envelope glycoprotein gD are present. The interaction with gD is mediated by specific HS moieties or 3-O sulfated HS (3-OS HS), which are generated by all but one of the seven isoforms of 3-O sulfotransferases (3-OSTs). Here we demonstrate that several common experimental cell lines express unique sets of 3-OST isoforms. While the isoforms 3-OST-3, -5 and -6 were most commonly expressed, isoforms 3-OST-2 and -4 were undetectable in the cell lines examined. Since most cell lines expressed multiple 3-OST isoforms, we addressed the significance of 3-OS HS in HSV-1 entry by down-regulating 2-O-sulfation, a prerequisite for 3-OS HS formation, by knocking down 2-OST expression by RNA interference (RNAi). 2-OST knockdown was verified by reverse-transcriptase PCR and Western blot analysis, while 3-OS HS knockdown was verified by immunofluorescence. Cells showed a significant decrease in viral entry, suggesting an important role for 3-OS HS. Implicating 3-OS HS further, cells knocked down for 2-OST expression also demonstrated decreased cell-cell fusion when cocultivated with effector cells transfected with HSV-1 glycoproteins. Our findings suggest that 3-OS HS may play an important role in HSV-1 entry into many different cell lines.
Wang, Ying; Baker, Nicholas; Amdam, Gro V
This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception. RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species. The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.
Al-Ayedh, Hassan; Rizwan-Ul-Haq, Muhammad; Hussain, Abid; Aljabr, Ahmed M
Palm trees around the world are prone to notorious Rhynchophorus ferrugineus, which causes heavy losses of palm plantations. In Middle Eastern countries, this pest is a major threat to date palm orchards. Conventional pest control measures with the major share of synthetic insecticides have resulted in insect resistance and environmental issues. Therefore, in order to explore better alternatives, the RNAi approach was employed to knock down the catalase gene in fifth and tenth larval instars with different dsRNA application methods, and their insecticidal potency was studied. dsRNA of 444 bp was prepared to knock down catalase in R. ferrugineus. Out of the three dsRNA application methods, dsRNA injection into larvae was the most effective, followed by dsRNA application by artificial feeding. Both methods resulted in significant catalase knockdown in various tissues, especially the midgut. As a result, the highest growth inhibition of 123.49 and 103.47% and larval mortality of 80 and 40% were observed in fifth-instar larvae, whereas larval growth inhibition remained at 86.83 and 69.08% with larval mortality at 30 and 10% in tenth-instar larvae after dsRNA injection and artificial diet treatment. The topical application method was the least efficient, with the lowest larval growth inhibition of 57.23 and 45.61% and 0% mortality in fifth- and tenth-instar larvae. Generally, better results were noted at the high dsRNA dose of 5 µL. Catalase enzyme is found in most insect body tissues, and thus its dsRNA can cause broad-scale gene knockdown within the insect body, depending upon the application method. Significant larval mortality and growth inhibition after catalase knockdown in R. ferrugineus confirms its insecticidal potency and suggests a bright future for RNAi-based bioinsecticides in pest control. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.
Falkenberg, K J; Newbold, A; Gould, C M; Luu, J; Trapani, J A; Matthews, G M; Simpson, K J; Johnstone, R W
Vorinostat is an FDA-approved histone deacetylase inhibitor (HDACi) that has proven clinical success in some patients; however, it remains unclear why certain patients remain unresponsive to this agent and other HDACis. Constitutive STAT (signal transducer and activator of transcription) activation, overexpression of prosurvival Bcl-2 proteins and loss of HR23B have been identified as potential biomarkers of HDACi resistance; however, none have yet been used to aid the clinical utility of HDACi. Herein, we aimed to further elucidate vorinostat-resistance mechanisms through a functional genomics screen to identify novel genes that when knocked down by RNA interference (RNAi) sensitized cells to vorinostat-induced apoptosis. A synthetic lethal functional screen using a whole-genome protein-coding RNAi library was used to identify genes that when knocked down cooperated with vorinostat to induce tumor cell apoptosis in otherwise resistant cells. Through iterative screening, we identified 10 vorinostat-resistance candidate genes that sensitized specifically to vorinostat. One of these vorinostat-resistance genes was GLI1, an oncogene not previously known to regulate the activity of HDACi. Treatment of vorinostat-resistant cells with the GLI1 small-molecule inhibitor, GANT61, phenocopied the effect of GLI1 knockdown. The mechanism by which GLI1 loss of function sensitized tumor cells to vorinostat-induced apoptosis is at least in part through interactions with vorinostat to alter gene expression in a manner that favored apoptosis. Upon GLI1 knockdown and vorinostat treatment, BCL2L1 expression was repressed and overexpression of BCL2L1 inhibited GLI1-knockdown-mediated vorinostat sensitization. Taken together, we present the identification and characterization of GLI1 as a new HDACi resistance gene, providing a strong rationale for development of GLI1 inhibitors for clinical use in combination with HDACi therapy.
Corporate author entries provide a means for consistent citing of the names of organizations in bibliographic records in the data bases of the DOE Technical Information Center. These entries serve as guides for users of the DOE/RECON computerized data bases who want to locate information originating in particular organizations
D. A. Seliverstov
Full Text Available The prime focus in this article is on key findings concerning theoretical aspects of strategic behavior by incumbents to deter market entry of new firms. The author summarizes main lines of scientific research in the topic which give an insight into the patterns of the incumbent’s impact on the behavior of the entrants, the entry deterrence instruments and the consequences of these actions. Today the free entry markets are considered to be a rare phenomenon. The market entry of new firms is associated with significant entry costs, which allow the incumbents to take advantage of their dominant position and derive positive economic profits. In case of entry threat by potential competitors the incumbents take strategic actions aimed at deterring entry and preserving their dominant position. Among the most efficient strategic actions one can emphasize the erection of additional barriers to entry for the newcomers through producing the limit output and price, investments in sunk assets, capacity expansion and product differentiation. Meanwhile by taking strategic actions the incumbents are not always trying to affect the entrant’s costs and profit directly, they often aim at changing the entrant’s expectations regarding future intentions of the incumbents to preserve dominant position.
...-entry interval applies, including, but not limited to, soil, water, air, or surfaces of plants; and (2...-entry activity, the agricultural employer shall provide a decontamination site in accordance with § 170... running water for routine and emergency decontamination and mechanical devices that would reduce the...
Schaumans, C.B.C.; Verboven, F.L.
We propose a methodology for estimating the competition effects from entry when firms sell differentiated products. We first derive precise conditions under which Bres- nahan and Reiss'entry threshold ratios (ETRs) can be used to test for the presence and to measure the magnitude of competition
We analyse a simple entry-deterrence game, where a `Potential Intruder' only imperfectly observes the decision of an `Incumbent' to commit or to not commit to fight any entry by the Potential Intruder. Our game generalises the one studied in Bonanno (1992) by allowing for a richer information tec...
Nicolás, Francisco E.; Moxon, Simon; de Haro, Juan P.; Dalmay, Tamas; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M
The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1 − M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi. PMID:23935973
Background Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. Results Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. Conclusions Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens. PMID:23006893
Deng, Paul Duo; Jefferson, Gary H.
We adopt the framework of Schumpeterian creative destruction formalized by Aghion et al. (2009) to analyze the impact of foreign entry on the productivity growth of domestic firms. In the face of foreign entry, domestic firms exhibit heterogeneous patterns of growth depending on their technologic...... manufacturing. Our empirical results confirm that foreign entry indeed generates strong heterogeneous growth patterns among domestic firms.......We adopt the framework of Schumpeterian creative destruction formalized by Aghion et al. (2009) to analyze the impact of foreign entry on the productivity growth of domestic firms. In the face of foreign entry, domestic firms exhibit heterogeneous patterns of growth depending on their technological...... distance from foreign firms. Domestic firms with smaller technological distance from their foreign counterparts tend to experience faster productivity growth, while firms with larger technological distance tend to lag further behind. We test this hypothesis using a unique firm-level data of Chinese...
Proschowsky, Morten Smidt
The reduced size of many mobile devices makes it difficult to enter text with them. The text entry methods are often slow or complicated to use. This affects the performance and user experience of all applications and services on the device. This work introduces new easy-to-use text entry methods...... for mobile devices and a framework for adaptive context-aware language models. Based on analysis of current text entry methods, the requirements to the new text entry methods are established. Transparent User guided Prediction (TUP) is a text entry method for devices with one dimensional touch input. It can...... be touch sensitive wheels, sliders or similar input devices. The interaction design of TUP is done with a combination of high level task models and low level models of human motor behaviour. Three prototypes of TUP are designed and evaluated by more than 30 users. Observations from the evaluations are used...
Zhao, Shu-Hua; Zhao, Fan; Zheng, Jing-Ying; Gao, Li-Fang; Zhao, Xue-Jian; Cui, Man-Hua
The aim of the study was to investigate the suppressive effects of pSilencer2.1-U6-siRNA-stat3 recombinant plasmids on the growth of ovarian cancer in vitro. Three pairs of DNA template (stat3-1, stat3-2, stat3-3) specific for different target sites on stat3 mRNA were synthesized to reconstruct pSilencer2.1-U6-siRNA-stat3s, which were transfected into SKOV3 cells. The expressions of STAT3, BcL-2, cyclin D1 and C-myc in these cells were detected by Western blot and Northern blot. The cell cycle and the growth were determined by flow cytometry (FCM) and MTT assay, respectively. Cell apoptosis was determined by TUNEL staining. Of the three siRNAs, only siRNA targeting stat3-3 markedly suppressed the protein expression of stat3 in SKOV3 cells; MTT assay and FCM showed that transfection of stat3-3 siRNA could significantly suppress the growth of SKOV3 cells and arrest the cell cycle in vitro. TUNEL staining also showed massive apoptosis in SKOV3 cells transfected with stat3-3 siRNA. pSilencer2.1-U6-siRNA-stat3-3 can significantly inhibit the STAT3 expression in human ovarian cancer cells resulting in the inhibition of the cancer growth and the increase of apoptosis of cancer cells
Pareek, Manish; Rajam, Manchikatla Venkat
Fusarium oxysporum is a soil-borne plant fungal pathogen, and causes colossal losses in several crop plants including tomato. Effective control measures include the use of harmful fungicides and resistant cultivars, but these methods have shown limited success. Conventional methods to validate fungal pathogenic genes are labour intensive. Therefore, an alternative strategy is required to efficiently characterize unknown pathogenic genes. RNA interference (RNAi) has emerged as a potential tool to functionally characterize novel fungal pathogenic genes and also to control fungal diseases. Here, we report an efficient method to produce stable RNAi transformants of F. oxysporum using Agrobacterium-mediated transformation (AMT). We have transformed F. oxysporum spores using RNAi constructs of Fmk1, Hog1, and Pbs2 MAP kinase signalling genes. Fmk1 RNAi fungal transformants showed loss of surface hydrophobicity, reduced invasive growth on tomato fruits and hypo-virulence on tomato seedlings. Hog1 and Pbs2 RNAi transformants showed altered conidial size, and reduced invasive growth and pathogenesis. These results showed that AMT using RNAi constructs is an effective approach for dissecting the role of genes involved in pathogenesis in F. oxysporum and this could be extended for other fungal systems. The obtained knowledge can be easily translated for developing fungal resistant crops by RNAi. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Computerized provider order entry (CPOE) systems are designed to replace a hospital's paper-based ordering system. They allow users to electronically write the full range of orders, maintain an online medication administration record, and review changes made to an order by successive personnel. They also offer safety alerts that are triggered when an unsafe order (such as for a duplicate drug therapy) is entered, as well as clinical decision support to guide caregivers to less expensive alternatives or to choices that better fit established hospital protocols. CPOE systems can, when correctly configured, markedly increase efficiency and improve patient safety and patient care. However, facilities need to recognize that currently available CPOE systems require a tremendous amount of time and effort to be spent in customization before their safety and clinical support features can be effectively implemented. What's more, even after they've been customized, the systems may still allow certain unsafe orders to be entered. Thus, CPOE systems are not currently a quick or easy remedy for medical errors. ECRI's Evaluation of CPOE systems--conducted in collaboration with the Institute for Safe Medication Practices (ISMP)--discusses these and other related issues. It also examines and compares CPOE systems from three suppliers: Eclipsys Corp., IDX Systems Corp., and Siemens Medical Solutions Health Services Corp. Our testing focuses primarily on the systems' interfacing capabilities, patient safeguards, and ease of use.
Full Text Available RNAi-based genetically engineered (GE crops for the management of insect pests are likely to be commercialized by the end of this decade. Without a workable framework for conducting the ecological risk assessment (ERA and a standardized ERA protocol, however, the utility of RNAi transgenic crops in pest management remains uncertain. The overall goal of this study is to assess the risks of RNAi-based GE crops on a non-target soil micro-arthropod, Sinella curviseta, which could be exposed to plant-protected dsRNAs deposited in crop residues. Based on the preliminary research, we hypothesized that insecticidal dsRNAs targeting at the western corn rootworm, Diabrotica virgifera virgifera, a billion-dollar insect pest, has no adverse impacts on S. curviseta, a soil decomposer. Following a tiered approach, we tested this risk hypothesis using a well-designed dietary RNAi toxicity assay. To create the worst-case scenario, the full-length cDNA of v-ATPase subunit A from S. curviseta were cloned and a 400 bp fragment representing the highest sequence similarity between target pest and non-target arthropods was selected as the template to synthesize insecticidal dsRNAs. Specifically, 10-day old S. curviseta larvae were subjected to artificial diets containing v-ATPase A dsRNAs from both D. v. virgifera (dsDVV and S. curviseta (dsSC, respectively, a dsRNA control, β-glucuronidase, from plant (dsGUS, and a vehicle control, H2O. The endpoint measurements included gene expression profiles, survival, and life history traits, such as developmental time, fecundity, hatching rate, and body length. Although S. curviseta larvae developed significantly faster under the treatments of dsDVV and dsSC than the vehicle control, the combined results from both temporal RNAi effect study and dietary RNAi toxicity assay support the risk hypothesis, suggesting that the impacts of ingested arthropod-active dsRNAs on this representative soil decomposer are negligible.
The future exploration of the Solar System will require innovations in transportation and the use of entry, descent, and landing (EDL) systems at many planetary landing sites. The cost of space missions has always been prohibitive, and using the natural planetary and planet s moons atmospheres for entry, descent, and landing can reduce the cost, mass, and complexity of these missions. This paper will describe some of the EDL ideas for planetary entry and survey the overall technologies for EDL that may be attractive for future Solar System missions.
Beaumont, A.; Brundige, E.; DesJardin, R.; Rivera, R.
The exchange badge system which was used at the Plutonium Facility located in Technical Area 55 was replaced on a trial basis with an automated Entry Control System. As a result of the success of the trial system, a new system incorporating expanded features and increased reliability is being implemented. The new Entry Control System incorporates several features not previously available in relatively inexpensive entry systems. The reliability of the system is enhanced by redundant microprocessors incorporating bubble memory for nonvolatile storage of the system data base. The badge readers incorporate dual communication lines to two different controllers to further increase the total system reliability
Entry control hardware has been used extensively in the past to assist security forces in separating the authorized from the unauthorized at the plant perimeter. As more attention is being focused on the insider threat, these entry control elements are being used to extend the security inspectors' presence into the plant by compartmentalizing access and monitoring vital components. This paper summarizes the experiences expressed by the participants at the March 16 to 19, 1982 INMM Physical Protection Workshop in utilizing access control and contraband detection hardware for plant wide entry control applications
He, J; Chaparro, A; Nguyen, B; Burge, R J; Crandall, J; Chaparro, B; Ni, R; Cao, S
Research indicates that using a cell phone to talk or text while maneuvering a vehicle impairs driving performance. However, few published studies directly compare the distracting effects of texting using a hands-free (i.e., speech-based interface) versus handheld cell phone, which is an important issue for legislation, automotive interface design and driving safety training. This study compared the effect of speech-based versus handheld text entries on simulated driving performance by asking participants to perform a car following task while controlling the duration of a secondary text-entry task. Results showed that both speech-based and handheld text entries impaired driving performance relative to the drive-only condition by causing more variation in speed and lane position. Handheld text entry also increased the brake response time and increased variation in headway distance. Text entry using a speech-based cell phone was less detrimental to driving performance than handheld text entry. Nevertheless, the speech-based text entry task still significantly impaired driving compared to the drive-only condition. These results suggest that speech-based text entry disrupts driving, but reduces the level of performance interference compared to text entry with a handheld device. In addition, the difference in the distraction effect caused by speech-based and handheld text entry is not simply due to the difference in task duration. Copyright © 2014 Elsevier Ltd. All rights reserved.
including the proposed moderating effect, on average, yield higher post-entry performance. This study sheds light on inconsistent results found in previous research investigating the impact of international experience and has practical implications for managerial decision-making.......This study investigates whether the relationship between mode of international market entry and non-location bound international experience is weaker for firms that are large or have a high foreign to total sales ratio, labeled multinational experience. Empirical evidence based on 250 foreign...... market entries made by Norwegian, Danish and Swedish firms suggests that the association between equity mode choice and non-location bound international experience diminishes in the presence of higher levels of multinational experience. Furthermore, firms whose entry mode choice is predicted by the model...
Department of Transportation — Border Crossing/Entry Data provides summary statistics for incoming crossings at the U.S.-Canadian and the U.S.-Mexican border at the port level. Data are available...
Boyd, Britta; Dyhr Ulrich, Anna Marie
Based on a sample of 177 exporting SMEs, this study investigates what market entry strategy is used by Danish family and non-family businesses. From a resource-based view, three critical internal factors (risk, flexibility and control) affecting the entry mode choice into the BRIC markets...... are analysed. The effective management of firms’ resources and capabilities is influenced by the perception of these internal factors when expanding into foreign markets. Our results confirmed that family firms build up longer lasting relationships in the host country by choosing high commitment entry modes...... when entering the BRIC markets. In contrast, family firms choose high commitment entry modes which involve high risk and low flexibility when entering the BRIC markets. Further implications discuss the suitability of export strategies to BRIC markets for managers of Danish family and non-family firms....
One of the controls for information entered into the data bases created and maintained by the DOE Technical Information Center is the standardized name for the corporate entity or the corporate author. The purpose of Energy Information Data Base: Corporate Author Entries (TID-4585-R1) and this supplemental list of authorized or standardized corporate entries is to provide a means for the consistent citing of the names of organizations in bibliographic records. In general, an entry in Corporate Author Entries consists of the seven-digit code number assigned to the particular corporate entity, the two-letter country code, the largest element of the corporate name, the location of the corporate entity, and the smallest element of the corporate name (if provided). This supplement [DOE/TIC-4585-R1(Suppl.5)] contains additions to the base document (TID-4585-R1) and is intended to be used with that publication
National Aeronautics and Space Administration — Building on our successful Phase I Tactile Data Entry program, Barron Associates proposes development of a Glove-Enabled Computer Operations (GECO) system to permit...
Tabara, Hiroaki; Yigit, Erbay; Siomi, Haruhiko; Mello, Craig C
Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.
Noriyuki Yanagawa; Ryoko Oki
This paper examines the effects of exclusive dealing contracts in a simple model with manufacturers-distributors relations. We consider entrants in both manufacturing and distribution sectors. It is well-known that a potential entry threat is welfare increasing under homogenous price competition, even though the potential entrant is less productive. This paper reexamines this intuition by employing the above model. We show that the entry threat of a less-productive manufacturer is welfare dec...
Kongsted, Hans Christian
This paper establishes the general deterministic limit that corresponds to Dixit's model of entry and exit decisions under uncertainty. The interlinked nature of decisions is shown to be essential also in the deterministic limit. A numerical example illustrates the result......This paper establishes the general deterministic limit that corresponds to Dixit's model of entry and exit decisions under uncertainty. The interlinked nature of decisions is shown to be essential also in the deterministic limit. A numerical example illustrates the result...
Kim, Kevin; Lee, Young Sik; Carthew, Richard W.
In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA. PMID:17123955
Pernet, Olivier; Pohl, Christine; Ainouze, Michelle; Kweder, Hasan; Buckland, Robin
Nipah virus (NiV) is a zoonotic biosafety level 4 paramyxovirus that emerged recently in Asia with high mortality in man. NiV is a member, with Hendra virus (HeV), of the Henipavirus genus in the Paramyxoviridae family. Although NiV entry, like that of other paramyxoviruses, is believed to occur via pH-independent fusion with the host cell's plasma membrane we present evidence that entry can occur by an endocytic pathway. The NiV receptor ephrinB2 has receptor kinase activity and we find that ephrinB2's cytoplasmic domain is required for entry but is dispensable for post-entry viral spread. The mutation of a single tyrosine residue (Y304F) in ephrinB2's cytoplasmic tail abrogates NiV entry. Moreover, our results show that NiV entry is inhibited by constructions and drugs specific for the endocytic pathway of macropinocytosis. Our findings could potentially permit the rapid development of novel low-cost antiviral treatments not only for NiV but also HeV.
This paper reviews existing models of radon entry into houses. The primary mechanism of radon entry in houses with high indoor concentrations is, in most cases, convective entry of radon bearing soil-gas from the surrounding soil. The driving force for this convective entry is the small indoor-outdoor pressure difference arising from the stack effect and other causes. Entry points for the soil-gas generally are the cracks or gaps in the building substructure, or though other parts of the building shell in direct contact with the soil, although entry may also occur by flow though permeable concrete or cinder block walls of the substructure. Models using analytical solutions to idealized geometrical configurations with simplified boundary conditions obtain analytical tractability of equations to be solved at the cost of severe approximations; their strength is in the insights they offer with their solutions. Models based on lumped parameters attempt to characterize the significant physical behavioral characteristics of the soil-gas and radon flow. When realistic approximations are desired for the boundary conditions and terms in the governing equations, numerical models must be used; these are usually based on finite difference or finite element solutions to the governing equations. Limited data are now available for experimental verification of model predictions. The models are briefly reviewed and their strengths and limitations are discussed
Wei, Xudong; He, Jian; Wang, Jingyu; Wang, Wei
Previous studies have confirmed that CD133+ cells in laryngeal tumor tissue have the characteristics of cancer stem cells. Bmi-1 gene expression is central to the tumorigenicity of CD133+ cells. In this study, we tried to develop a new siRNA carrier system using chitosan-methoxypolyethylene nanoparticles (CS-mPEG-NPs) that exhibit higher tumor-targeting ability and enhanced gene silencing efficacy in CD133+ tumor stem cells. It is hoped to block the self-renewal and kill the stem cells of laryngeal carcinoma. The mPEG-CS-Bmi-1RNAi-NPs were synthesized and their characters were checked. The changes in invasion ability and sensitivity to radiotherapy and chemotherapy of CD133+Hep-2 tumor cells were observed after Bmi-1 gene silencing. The mPEG-CS-Bmi-1RNAi-NPs synthesized in this experiment have a regular spherical form, a mean size of 139.70 ±6.40 nm, an encapsulation efficiency of 85.21 ± 1.94%, with drug loading capacity of 18.47 ± 1.83%, as well as low cytotoxicity, providing good protection to the loaded gene, strong resistance to nuclease degradation and high gene transfection efficiency. After Bmi-1 gene silencing, the invasion ability of CD133+ cells was weakened. Co-cultured with paclitaxel, the survival rates of CD133+Bmi-1RNAi cells were lower. After radiotherapy, the mean growth inhibition rate of CD133+/Bmi-1RNAi cells was significantly lower than CD133+ cells. In conclusion, the mPEG-CS nano-carrier is an ideal vector in gene therapy, while silencing the Bmi-1 gene can enhance the sensitivity of CD133+ tumor stem cells to chemoradiotherapy and abate their invasion ability.
Calo, Silvia; Nicolás, Francisco E; Lee, Soo Chan; Vila, Ana; Cervantes, Maria; Torres-Martinez, Santiago; Ruiz-Vazquez, Rosa M; Cardenas, Maria E; Heitman, Joseph
Mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. Recent studies revealed that these pathogens activate an RNAi-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug FK506. To elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in Mucor circinelloides that revealed an inhibitory role for the non-canonical RdRP-dependent Dicer-independent silencing pathway, which is an RNAi-based mechanism involved in mRNA degradation that was recently identified. Thus, mutations that specifically block the mRNA degradation pathway, such as those in the genes r3b2 and rdrp3, enhance the production of drug resistant epimutants, similar to the phenotype previously described for mutation of the gene rdrp1. Our genetic analysis also revealed two new specific components of the epimutation pathway related to the quelling induced protein (qip) and a Sad-3-like helicase (rnhA), as mutations in these genes prevented formation of drug-resistant epimutants. Remarkably, drug-resistant epimutant production was notably increased in M. circinelloides f. circinelloides isolates from humans or other animal hosts. The host-pathogen interaction could be a stressful environment in which the phenotypic plasticity provided by the epimutant pathway might provide an advantage for these strains. These results evoke a model whereby balanced regulation of two different RNAi pathways is determined by the activation of the RNAi-dependent epimutant pathway under stress conditions, or its repression when the regular maintenance of the mRNA degradation pathway operates under non-stress conditions.
Full Text Available Mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. Recent studies revealed that these pathogens activate an RNAi-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug FK506. To elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in Mucor circinelloides that revealed an inhibitory role for the non-canonical RdRP-dependent Dicer-independent silencing pathway, which is an RNAi-based mechanism involved in mRNA degradation that was recently identified. Thus, mutations that specifically block the mRNA degradation pathway, such as those in the genes r3b2 and rdrp3, enhance the production of drug resistant epimutants, similar to the phenotype previously described for mutation of the gene rdrp1. Our genetic analysis also revealed two new specific components of the epimutation pathway related to the quelling induced protein (qip and a Sad-3-like helicase (rnhA, as mutations in these genes prevented formation of drug-resistant epimutants. Remarkably, drug-resistant epimutant production was notably increased in M. circinelloides f. circinelloides isolates from humans or other animal hosts. The host-pathogen interaction could be a stressful environment in which the phenotypic plasticity provided by the epimutant pathway might provide an advantage for these strains. These results evoke a model whereby balanced regulation of two different RNAi pathways is determined by the activation of the RNAi-dependent epimutant pathway under stress conditions, or its repression when the regular maintenance of the mRNA degradation pathway operates under non-stress conditions.
Fuentes, Alejandro; Carlos, Natacha; Ruiz, Yoslaine; Callard, Danay; Sánchez, Yadira; Ochagavía, María Elena; Seguin, Jonathan; Malpica-López, Nachelli; Hohn, Thomas; Lecca, Maria Rita; Pérez, Rosabel; Doreste, Vivian; Rehrauer, Hubert; Farinelli, Laurent; Pujol, Merardo; Pooggin, Mikhail M
RNA interference (RNAi) is a widely used approach to generate virus-resistant transgenic crops. However, issues of agricultural importance like the long-term durability of RNAi-mediated resistance under field conditions and the potential side effects provoked in the plant by the stable RNAi expression remain poorly investigated. Here, we performed field trials and molecular characterization studies of two homozygous transgenic tomato lines, with different selection markers, expressing an intron-hairpin RNA cognate to the Tomato yellow leaf curl virus (TYLCV) C1 gene. The tested F6 and F4 progenies of the respective kanamycin- and basta-resistant plants exhibited unchanged field resistance to TYLCV and stably expressed the transgene-derived short interfering RNA (siRNAs) to represent 6 to 8% of the total plant small RNAs. This value outnumbered the average percentage of viral siRNAs in the nontransformed plants exposed to TYLCV-infested whiteflies. As a result of the RNAi transgene expression, a common set of up- and downregulated genes was revealed in the transcriptome profile of the plants selected from either of the two transgenic events. A previously unidentified geminivirus causing no symptoms of viral disease was detected in some of the transgenic plants. The novel virus acquired V1 and V2 genes from TYLCV and C1, C2, C3, and C4 genes from a distantly related geminivirus and, thereby, it could evade the repressive sequence-specific action of transgene-derived siRNAs. Our findings shed light on the mechanisms of siRNA-directed antiviral silencing in transgenic plants and highlight the applicability limitations of this technology as it may alter the transcriptional pattern of nontarget genes.
Wiles, Amy M.; Ravi, Dashnamoorthy; Bhavani, Selvaraj; Bishop, Alexander J.R.
Genome-wide RNAi screening is a powerful, yet relatively immature technology that allows investigation into the role of individual genes in a process of choice. Most RNAi screens identify a large number of genes with a continuous gradient in the assessed phenotype. Screeners must then decide whether to examine just those genes with the most robust phenotype or to examine the full gradient of genes that cause an effect and how to identify the candidate genes to be validated. We have used RNAi in Drosophila cells to examine viability in a 384-well plate format and compare two screens, untreated control and treatment. We compare multiple normalization methods, which take advantage of different features within the data, including quantile normalization, background subtraction, scaling, cellHTS2 1, and interquartile range measurement. Considering the false-positive potential that arises from RNAi technology, a robust validation method was designed for the purpose of gene selection for future investigations. In a retrospective analysis, we describe the use of validation data to evaluate each normalization method. While no normalization method worked ideally, we found that a combination of two methods, background subtraction followed by quantile normalization and cellHTS2, at different thresholds, captures the most dependable and diverse candidate genes. Thresholds are suggested depending on whether a few candidate genes are desired or a more extensive systems level analysis is sought. In summary, our normalization approaches and experimental design to perform validation experiments are likely to apply to those high-throughput screening systems attempting to identify genes for systems level analysis. PMID:18753689
Xiao, Han; Zhao, Huimin
Background Furfural is a major growth inhibitor in lignocellulosic hydrolysates and improving furfural tolerance of microorganisms is critical for rapid and efficient fermentation of lignocellulosic biomass. In this study, we used the RNAi-Assisted Genome Evolution (RAGE) method to select for furfural resistant mutants of Saccharomyces cerevisiae, and identified a new determinant of furfural tolerance. Results By using a genome-wide RNAi (RNA-interference) screen in S. cerevisiae for genes in...
Xie, Zhongcong; Dong, Yuanlin; Maeda, Uta; Xia, Weiming; Tanzi, Rudolph E
Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.
Full Text Available Abstract Amyloid-β-protein (Aβ, the key component of senile plaques in Alzheimer's disease (AD brain, is produced from amyloid precursor protein (APP by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB domains, including Dab (gene: DAB and Numb (gene: NUMB, can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells or APP-C99 (H4-APP-C99 cells increased levels of APP-C-terminal fragments (APP-CTFs and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.
Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided. PMID:23211096
Adams, Felix F; Heckl, Dirk; Hoffmann, Thomas; Talbot, Steven R; Kloos, Arnold; Thol, Felicitas; Heuser, Michael; Zuber, Johannes; Schambach, Axel; Schwarzer, Adrian
RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system. Copyright © 2017 Elsevier Ltd. All rights reserved.
Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen
RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.
Full Text Available Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila. miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.
Judge, Adam D; Robbins, Marjorie; Tavakoli, Iran; Levi, Jasna; Hu, Lina; Fronda, Anna; Ambegia, Ellen; McClintock, Kevin; MacLachlan, Ian
siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.
Rodrigues, Thais B; Duan, Jian J; Palli, Subba R; Rieske, Lynne K
Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), caused up to 90% mortality of larvae and adults. In EAB eggs, larvae, and adults, the hsp is expressed at higher levels when compared to that of shi. Ingestion of dsHSP and dsSHI caused mortality in both neonate larvae and adults. Administration of a mixture of both dsRNAs worked better than either dsRNA by itself. In contrast, injection of EAB.dsHSP and EAB.dsSHI did not cause mortality in T. castaneum. Thus, the two genes identified cause high mortality in the EAB with no apparent phenotype effects in a non-target organism, the red flour beetle, and could be used in RNAi-mediated control of this invasive pest.
Raman, Pravrutha; Zaghab, Soriayah M; Traver, Edward C; Jose, Antony M
Long double-stranded RNA (dsRNA) can silence genes of matching sequence upon ingestion in many invertebrates and is therefore being developed as a pesticide. Such feeding RNA interference (RNAi) is best understood in the worm Caenorhabditis elegans, where the dsRNA-binding protein RDE-4 initiates silencing by recruiting an endonuclease to process long dsRNA into short dsRNA. These short dsRNAs are thought to move between cells because muscle-specific rescue of rde-4 using repetitive transgenes enables silencing in other tissues. Here, we extend this observation using additional promoters, report an inhibitory effect of repetitive transgenes, and discover conditions for cell-autonomous silencing in animals with tissue-specific rescue of rde-4. While expression of rde-4(+) in intestine, hypodermis, or neurons using a repetitive transgene can enable silencing also in unrescued tissues, silencing can be inhibited wihin tissues that express a repetitive transgene. Single-copy transgenes that express rde-4(+) in body-wall muscles or hypodermis, however, enable silencing selectively in the rescued tissue but not in other tissues. These results suggest that silencing by the movement of short dsRNA between cells is not an obligatory feature of feeding RNAi in C. elegans. We speculate that similar control of dsRNA movement could modulate tissue-specific silencing by feeding RNAi in other invertebrates. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Habig, Jeffrey W; Aruscavage, P Joseph; Bass, Brenda L
C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.
Shirayama, Masaki; Stanney, William; Gu, Weifeng; Seth, Meetu; Mello, Craig C
Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing. Copyright © 2014 Elsevier Ltd. All rights reserved.
Liu, Ying; Tan, Huiling; Tian, Hui; Liang, Chunyang; Chen, She; Liu, Qinghua
The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading the Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct the Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used the human Ago2 minimal RISC system to purify Sjögren's syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. Copyright Â© 2011 Elsevier Inc. All rights reserved.
Wagner, Nicholas; Mroczka, Andrew; Roberts, Peter D; Schreckengost, William; Voelker, Toni
Suppression of the microsomal ω6 oleate desaturase during the seed development of soybean (Glycine max) with the 420-bp soybean FAD2-1A intron as RNAi trigger shifts the conventional fatty acid composition of soybean oil from 20% oleic and 60% polyunsaturates to one containing greater than 80% oleic acid and less than 10% polyunsaturates. To determine whether RNAi could be attenuated by reducing the trigger fragment length, transgenic plants were generated to express successively shorter 5' or 3' deletion derivatives of the FAD2-1A intron. We observed a gradual reduction in transcript suppression with shorter trigger fragments. Fatty acid composition was less affected with shorter triggers, and triggers less than 60 bp had no phenotypic effect. No trigger sequences conferring significantly higher or lower suppression efficiencies were found, and the primary determinant of suppression effect was sequence length. The observed relationship of transcript suppression with the induced fatty acid phenotype indicates that RNAi is a saturation process and not a step change between suppressed and nonsuppressed states and intermediate suppression states can be achieved. © 2010 Monsanto. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd.
Jeffrey W Habig
Full Text Available C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.
Suka, Intan Elya; Chew, Bee Lynn; Goh, Hoe-Han; Isa, Nurulhikma Md
PROTEOLYSIS 6 plays major role in the N-end rule pathway as N-recognin which functions as E3 ligase enzyme. It mediates ubiquitin processes that lead to degradation of unstable substrate protein. The aim of the current study is to transform the PRT6 gene into tomato (Solanum lycopersicum) from the cultivar Micro-Tom and to investigate its function in regulating ripening in tomato fruits. The PRT6_RNAi construct was successfully transformed into Agrobacterium C58 via heat shock method and transformed into seven days old cotyledon explants. Factors affecting transformation efficiency such as co-cultivation time and type of plant growth regulator combination were evaluated. Results from this study found that pre-cultured cotyledons from seven days old seedlings incubated for 2 days in co-cultivation medium increased shoot regeneration. Plant growth hormones zeatin combine with auxin produced a higher number of callus formation but lower shoot proliferation and transformation frequency compared to treatments of single plant hormone in the selection medium. Polymerase chain reaction (PCR) was performed on the regenerated shoots to confirm the integration of PRT6 fragment into the genome of transgenic plants. Based on PCR analysis, all putative shoots were positive transformants.
Full Text Available Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM. Here, we have performed a microscopy-based genome-wide RNA interference (RNAi screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1 nine are required for centriole duplication, (2 11 are required for centrosome maturation, (3 nine are required for both functions, and (4 three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.
Full Text Available Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C-Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila. Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development.
Full Text Available Glioblastoma Multiforme (GBM cells are highly invasive, infiltrating into the surrounding normal brain tissue, making it impossible to completely eradicate GBM tumors by surgery or radiation. Increasing evidence also shows that these migratory cells are highly resistant to cytotoxic reagents, but decreasing their migratory capability can re-sensitize them to chemotherapy. These evidences suggest that the migratory cell population may serve as a better therapeutic target for more effective treatment of GBM. In order to understand the regulatory mechanism underlying the motile phenotype, we carried out a genome-wide RNAi screen for genes inhibiting the migration of GBM cells. The screening identified a total of twenty-five primary hits; seven of them were confirmed by secondary screening. Further study showed that three of the genes, FLNA, KHSRP and HCFC1, also functioned in vivo, and knocking them down caused multifocal tumor in a mouse model. Interestingly, two genes, KHSRP and HCFC1, were also found to be correlated with the clinical outcome of GBM patients. These two genes have not been previously associated with cell migration.
Full Text Available Abstract Background Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. Methods Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. Results Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. Conclusions Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.
Habayeb, Mazen S; Ekström, Jens-Ola; Hultmark, Dan
Drosophila melanogaster is widely used to decipher the innate immune system in response to various pathogens. The innate immune response towards persistent virus infections is among the least studied in this model system. We recently discovered a picorna-like virus, the Nora virus which gives rise to persistent and essentially symptom-free infections in Drosophila melanogaster. Here, we have used this virus to study the interaction with its host and with some of the known Drosophila antiviral immune pathways. First, we find a striking variability in the course of the infection, even between flies of the same inbred stock. Some flies are able to clear the Nora virus but not others. This phenomenon seems to be threshold-dependent; flies with a high-titer infection establish stable persistent infections, whereas flies with a lower level of infection are able to clear the virus. Surprisingly, we find that both the clearance of low-level Nora virus infections and the stability of persistent infections are unaffected by mutations in the RNAi pathways. Nora virus infections are also unaffected by mutations in the Toll and Jak-Stat pathways. In these respects, the Nora virus differs from other studied Drosophila RNA viruses.
Full Text Available Hedgehog (Hh proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.
Cheng-Xu Delon Toh
Full Text Available Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries.
Wang, Xiaoyun; Chen, Wenjun; Tian, Yanli; Huang, Yan; Li, Xuerong; Yu, Xinbing
Clonorchis sinensis (C. sinensis) infection is still a common public health problem in freshwater fish consumption areas in Asian countries. More molecular evidence are required to speed up the prevention strategies to control this kind of infectious disease. In the present study, to confirm the biological importance of Csenolase followed by our previous observations of the key metabolic enzyme, we explored the RNA silence effect of the Csenolase-derived RNA interference (RNAi) in C. sinensis. The extramembranous region aa105-226 was selected as the target sequence of RNA silence. Csenolase-derived double strand RNA (dsRNA-Csenolase, 366 bp) was synthetized and delivered into C. sinensis by soaking approach. The penetration of dsRNA into adult worms and metacercariae was tracked using fluorescently labeled RNA. Western blotting and qRT-PCR experiments were performed to determine dsRNA-Csenolase-silencing effect. Our results showed that, after incubating for 120 h, dsRNA-Csenolase could effectively target and downregulate the expression of Csenolase in both adult worms (P sinensis adult worms (P sinensis, allowing further applications in identifying functional genes in C. sinensis.
Full Text Available Retinal neovascularization (NV due to retinal ischemia remains one of the principal causes of vision impairment in patients with ischemic retinal diseases. We recently reported that periostin (POSTN may play a role in the development of preretinal fibrovascular membranes, but its role in retinal NV has not been determined. The purpose of this study was to examine the expression of POSTN in the ischemic retinas of a mouse model of oxygen-induced retinal NV. We also studied the function of POSTN on retinal NV using Postn KO mice and human retinal endothelial cells (HRECs in culture. In addition, we used a novel RNAi agent, NK0144, which targets POSTN to determine its effect on the development of retinal NV. Our results showed that the expression of POSTN was increased in the vascular endothelial cells, pericytes, and M2 macrophages in ischemic retinas. POSTN promoted the ischemia-induced retinal NV by Akt phosphorylation through integrin αvβ3. NK0144 had a greater inhibitory effect than canonical double-stranded siRNA on preretinal pathological NV in vivo and in vitro. These findings suggest a causal relationship between POSTN and retinal NV, and indicate a potential therapeutic role of intravitreal injection of NK0144 for retinal neovascular diseases.
Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar
Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H.
Avgousti, Daphne C; Palani, Santhosh; Sherman, Yekaterina; Grishok, Alla
Endogenous small interfering RNAs (endo-siRNAs) have been discovered in many organisms, including mammals. In C. elegans, depletion of germline-enriched endo-siRNAs found in complex with the CSR-1 Argonaute protein causes sterility and defects in chromosome segregation in early embryos. We discovered that knockdown of either csr-1, the RNA-dependent RNA polymerase (RdRP) ego-1, or the dicer-related helicase drh-3, leads to defects in histone mRNA processing, resulting in severe depletion of core histone proteins. The maturation of replication-dependent histone mRNAs, unlike that of other mRNAs, requires processing of their 3'UTRs through an endonucleolytic cleavage guided by the U7 snRNA, which is lacking in C. elegans. We found that CSR-1-bound antisense endo-siRNAs match histone mRNAs and mRNA precursors. Consistently, we demonstrate that CSR-1 directly binds to histone mRNA in an ego-1-dependent manner using biotinylated 2'-O-methyl RNA oligonucleotides. Moreover, we demonstrate that increasing the dosage of histone genes rescues the lethality associated with depletion of CSR-1 and EGO-1. These results support a positive and direct effect of RNAi on histone gene expression.
Trung Anh Trieu
Full Text Available Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. Here, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of Mucor circinelloides silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of mcplD, which encodes a Phospholipase D, and mcmyo5, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of M. circinelloides. Knock-out mutants for those genes showed reduced virulence in both Galleria mellonella and Mus musculus models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in M. circinelloides that could represent promising targets for future antifungal therapies.
Mansisidor, Andres R; Cecere, Germano; Hoersch, Sebastian; Jensen, Morten B; Kawli, Trupti; Kennedy, Lisa M; Chavez, Violeta; Tan, Man-Wah; Lieb, Jason D; Grishok, Alla
Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16-dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.
Full Text Available Abstract Background Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. Results We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. Conclusion Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.
Full Text Available The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC lineage differentiation, and ISC-to-enteroendocrine (EE lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.
Kraehling, Jan R.; Chidlow, John H.; Rajagopal, Chitra; Sugiyama, Michael G.; Fowler, Joseph W.; Lee, Monica Y.; Zhang, Xinbo; Ramírez, Cristina M.; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W.; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L.; Fernández-Hernando, Carlos; Sessa, William C.
In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL. PMID:27869117
Full Text Available Hedgehog (Hh signaling is highly conserved in all metazoan animals and plays critical roles in many developmental processes. Dysregulation of the Hh signaling cascade has been implicated in many diseases, including cancer. Although key components of the Hh pathway have been identified, significant gaps remain in our understanding of the regulation of individual Hh signaling molecules. Here, we report the identification of novel regulators of the Hh pathway, obtained from an in vivo RNA interference (RNAi screen in Drosophila. By selectively targeting critical genes functioning in post-translational modification systems utilizing ubiquitin (Ub and Ub-like proteins, we identify two novel genes (dUba3 and dUbc12 that negatively regulate Hh signaling activity. We provide in vivo and in vitro evidence illustrating that dUba3 and dUbc12 are essential components of the neddylation pathway; they function in an enzyme cascade to conjugate the ubiquitin-like NEDD8 modifier to Cullin proteins. Neddylation activates the Cullin-containing ubiquitin ligase complex, which in turn promotes the degradation of Cubitus interruptus (Ci, the downstream transcription factor of the Hh pathway. Our study reveals a conserved molecular mechanism of the neddylation pathway in Drosophila and sheds light on the complex post-translational regulations in Hh signaling.
Liang, Chunyang; Wang, Yibing; Murota, Yukiko; Liu, Xiang; Smith, Dean; Siomi, Mikiko C; Liu, Qinghua
Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here we identified the missing factor of RLC as TATA-binding protein-associated factor 11 (TAF11) by genetic screen. Although it is an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11(-/-) ovary extract, we reconstituted the RLC in vitro using the recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of the Drosophila RLC and elucidate a cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency. Copyright © 2015 Elsevier Inc. All rights reserved.
Tatiana I Novobrantseva
Full Text Available Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP for durable and potent in vivo RNA interference (RNAi-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA. In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
Firnhaber, Christopher; Hammarlund, Marc
Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes. Using the GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism.
Hajeri, Subhas; Killiny, Nabil; El-Mohtar, Choaa; Dawson, William O; Gowda, Siddarame
A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D. citri. Coincident cohabitation in phloem tissue by CLas, D. citri and CTV was exploited to develop a novel method to mitigate HLB through RNA interference (RNAi). Since CTV has three RNA silencing suppressors, it was not known if CTV-based vector could induce RNAi in citrus. Yet, expression of sequences targeting citrus phytoene desaturase gene by CTV-RNAi resulted in photo-bleaching phenotype. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, induced altered Awd expression when silencing triggers ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. This impaired ability of D. citri to fly would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control. Copyright © 2014. Published by Elsevier B.V.
DeWitt, Dawn; Canny, Benedict J; Nitzberg, Michael; Choudri, Jennifer; Porter, Sarah
There is ongoing debate regarding the optimal length of medical training, with concern about the cost of prolonged training. Two simultaneous tracks currently exist in Australia: direct entry from high school and graduate entry for students with a bachelor degree. Medical schools are switching to graduate entry based on maturity, academic preparedness and career-choice surety. We tested the assumption that graduate entry is better by exploring student preferences, coping, burnout, empathy and alcohol use. From a potential pool of 2188 participants, enrolled at five Australian medical schools, a convenience sample of 688 (31%) first and second year students completed a survey in the middle of the academic year. Participants answered questions about demographics, satisfaction and coping and completed three validated instruments. Over 90% of students preferred their own entry-type, though more graduate-entry students were satisfied with their programme (82.4% versus 65.3%, p students in self-reported coping or in the proportion of students meeting criteria for burnout (50.7% versus 51.2%). Direct-entry students rated significantly higher for empathy (concern, p = 0.022; personal distress, p = 0.031). Graduate-entry students reported significantly more alcohol use and hazardous drinking (30.0% versus 22.8%; p = 0.017). Our multi-institution data confirm that students are generally satisfied with their choice of entry pathway and do not confirm significant psychosocial benefits of graduate entry. Overall, our data suggest that direct-entry students cope with the workload and psychosocial challenges of medical school, in the first 2 years, as well as graduate-entry students. Burnout and alcohol use should be addressed in both pathways. Despite studies showing similar academic outcomes, and higher total costs, more programmes in Australia are becoming graduate entry. Further research on non-cognitive issues and outcomes is needed so that universities, government
... 19 Customs Duties 2 2010-04-01 2010-04-01 false Information on entry summary. 151.41 Section 151... Products § 151.41 Information on entry summary. On the entry summary for petroleum or petroleum products in.... If the exact volumetric quantity cannot be determined in advance, the entry summary may be made for...
An Entry Control System has been developed which is appropriate for use at an installation with a large population requiring access over a large area. This is accomplished by centralizing the data base management and enrollment functions and decentralizing the guard-assisted, positive personnel identification and access functions. Current information pertaining to all enrollees is maintained through user-friendly enrollment stations. These stations may be used to enroll individuals, alter their area access authorizations, change expiration dates, and other similar functions. An audit trail of data base alterations is provided to the System Manager. Decentrailized systems exist at each area to which access is controlled. The central system provides these systems with the necessary entry control information to allow them to operate microprocessor-driven entry control devices. The system is comprised of commercially available entry control components and is structured such that it will be able to incorporate improved devices as technology porogresses. Currently, access is granted to individuals who possess a valid credential, have current access authorization, can supply a memorized personal identification number, and whose physical hand dimensions match their profile obtained during enrollment. The entry control devices report misuses as security violations to a Guard Alarm Display and Assessment System
Boyd, Britta; Dyhr Ulrich, Anna Marie; Hollensen, Svend
Based on a survey of 170 Danish SMEs the paper examines influences on entry mode choices and the financial outcome of these decisions. The main research objectives are divided into two steps: Step 1: To determine the factors influencing the choice of foreign entry modes by Danish companies. Step ...... and implications are provided for companies willing to invest more into foreign markets in order to achieve a higher degree of control and better financial results.......Based on a survey of 170 Danish SMEs the paper examines influences on entry mode choices and the financial outcome of these decisions. The main research objectives are divided into two steps: Step 1: To determine the factors influencing the choice of foreign entry modes by Danish companies. Step 2......: To determine the relationship between the choice of entry mode and export performance, measured in terms of financial outcome. Drawing from transaction cost theory the authors develop and test a model where different factors affect the level of control chosen by the parent company. This study contributes...
Prabhu, Dinesh K.; Spilker, Thomas R.; Allen, Gary A., Jr.; Hwang, Helen H.; Cappuccio, Gelsomina; Moses, Robert W.
The present study considers direct ballistic entries into the atmosphere of Venus using a 45deg sphere-cone rigid aeroshell, a legacy shape that has been used successfully in the past in the Pioneer Venus Multiprobe Mission. For a number of entry mass and heatshield diameter combinations (i.e., various ballistic coefficients) and entry velocities, the trajectory space in terms of entry flight path angles between skip out and -30deg is explored with a 3DoF trajectory code, TRAJ. From these trajectories, the viable entry flight path angle space is determined through the use of mechanical and thermal performance limits on the thermal protection material and science payload; the thermal protection material of choice is entry-grade carbon phenolic, for which a material thermal response model is available. For mechanical performance, a 200 g limit is placed on the peak deceleration load experienced by the science instruments, and 10 bar is assumed as the pressure limit for entry-grade carbon-phenolic material. For thermal performance, inflection points in the total heat load distribution are used as cut off criteria. Analysis of the results shows the existence of a range of critical ballistic coefficients beyond which the steepest possible entries are determined by the pressure limit of the material rather than the deceleration load limit.
Ulhøi, John Parm
(first-mover or follower). Invention is understood as the conversion of human creativity, time and financial resources into new ideas. Innovation in turn reflects the practical and financial return on such investments. While there is little disagreement about what an innovator strategy is, imitative......This paper focuses on the initial questions of how and when to enter a market from the perspective of a firm. By entry mode is meant a firm’s strategy (innovation or imitation) for entering the market in response to environmental changes. Entry order refers to the related issue of market timing...... strategies are more ambiguous. Based on a corporate technology and innovation strategy perspective, the paper reconceptualises and extends existing modes and orders of market entry, and in particular clarifies the ambiguity associated with imitative strategies. Four distinct imitator strategies...
Zuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R; Herrmann, Harald; Sison, Edward A; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J; Johns, Christopher; Chicas, Agustin; Mulloy, James C; Kogan, Scott C; Brown, Patrick; Valent, Peter; Bradner, James E; Lowe, Scott W; Vakoc, Christopher R
Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.
Felipe M Vigoder
Full Text Available Cold acclimation is a critical physiological adaptation for coping with seasonal cold. By increasing their cold tolerance individuals can remain active for longer at the onset of winter and can recover more quickly from a cold shock. In insects, despite many physiological studies, little is known about the genetic basis of cold acclimation. Recently, transcriptomic analyses in Drosophila virilis and D. montana revealed candidate genes for cold acclimation by identifying genes upregulated during exposure to cold. Here, we test the role of myo-inositol-1-phosphate synthase (Inos, in cold tolerance in D. montana using an RNAi approach. D. montana has a circumpolar distribution and overwinters as an adult in northern latitudes with extreme cold. We assessed cold tolerance of dsRNA knock-down flies using two metrics: chill-coma recovery time (CCRT and mortality rate after cold acclimation. Injection of dsRNAInos did not alter CCRT, either overall or in interaction with the cold treatment, however it did induced cold-specific mortality, with high levels of mortality observed in injected flies acclimated at 5°C but not at 19°C. Overall, injection with dsRNAInos induced a temperature-sensitive mortality rate of over 60% in this normally cold-tolerant species. qPCR analysis confirmed that dsRNA injection successfully reduced gene expression of Inos. Thus, our results demonstrate the involvement of Inos in increasing cold tolerance in D. montana. The potential mechanisms involved by which Inos increases cold tolerance are also discussed.
Pasi, K John; Rangarajan, Savita; Georgiev, Pencho; Mant, Tim; Creagh, Michael D; Lissitchkov, Toshko; Bevan, David; Austin, Steve; Hay, Charles R; Hegemann, Inga; Kazmi, Rashid; Chowdary, Pratima; Gercheva-Kyuchukova, Liana; Mamonov, Vasily; Timofeeva, Margarita; Soh, Chang-Heok; Garg, Pushkal; Vaishnaw, Akshay; Akinc, Akin; Sørensen, Benny; Ragni, Margaret V
Current hemophilia treatment involves frequent intravenous infusions of clotting factors, which is associated with variable hemostatic protection, a high treatment burden, and a risk of the development of inhibitory alloantibodies. Fitusiran, an investigational RNA interference (RNAi) therapy that targets antithrombin (encoded by SERPINC1), is in development to address these and other limitations. In this phase 1 dose-escalation study, we enrolled 4 healthy volunteers and 25 participants with moderate or severe hemophilia A or B who did not have inhibitory alloantibodies. Healthy volunteers received a single subcutaneous injection of fitusiran (at a dose of 0.03 mg per kilogram of body weight) or placebo. The participants with hemophilia received three injections of fitusiran administered either once weekly (at a dose of 0.015, 0.045, or 0.075 mg per kilogram) or once monthly (at a dose of 0.225, 0.45, 0.9, or 1.8 mg per kilogram or a fixed dose of 80 mg). The study objectives were to assess the pharmacokinetic and pharmacodynamic characteristics and safety of fitusiran. No thromboembolic events were observed during the study. The most common adverse events were mild injection-site reactions. Plasma levels of fitusiran increased in a dose-dependent manner and showed no accumulation with repeated administration. The monthly regimen induced a dose-dependent mean maximum antithrombin reduction of 70 to 89% from baseline. A reduction in the antithrombin level of more than 75% from baseline resulted in median peak thrombin values at the lower end of the range observed in healthy participants. Once-monthly subcutaneous administration of fitusiran resulted in dose-dependent lowering of the antithrombin level and increased thrombin generation in participants with hemophilia A or B who did not have inhibitory alloantibodies. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02035605 .).
Eglous, Najat Mohamed; Ali, Zainon Mohd; Hassan, Maizom; Zainal, Zamri
Tomato (Solanum Lycopersicum L.) is the second most cultivated vegetable in the world and widely used as a system for studying the role of ethylene during fruit ripening. Our objective was to study the oxidative stress and antioxidative metabolism during ripening of non transgenic tomato and transgenic line-21 tomato which reduced ethylene. The line-21 of transgenic tomato plants (RNAi ACO1) had lower ethylene production and longer shelf-life more than 32 days as compared to the wild-type fruits which have very short shelf-life. In this study, tomato fruit were divided into five different stages (MG: mature green 5%, B: breaker 25%, T: turning 50%, O: orange75%, RR: red ripe100%). The activity of lipoxygenase (LOX) and lipid peroxidation (MDA) were measured to assess changes in oxidative stress. The LOX activity and MDA content decreased significantly obtaining 2.6-fold and 1.2-fold, respectively, as compared to the wild type fruit. However, superoxide dismutase (SOD) and catalase (CAT) activities were increased to 1.9 and 1.2 folds from the mature green to the fully ripe stage in transgenic tomatoes. Furthermore, the wild type tomato increases 1.3 in SOD and 1.6 in CAT activities. The overall results indicate that the wild type tomato fruit showed a faster rate of ripening, parallel to decline in the rate of enzymatic antioxidative systems as compared to the transgenic line-21 tomato fruit. In addition, the results show that the antioxidant capacity is improved during the ripening process and is accompanied by an increase in the oxidative stress.
It is worth stressing that distinguishing between the form and its meaning in comparing the material 6 languages belonging to three different groups of Slavic languages (as is the case in the MONDILEX Project will allow us to avoid numeorus substantiva mistakes and erroneous conclusions. Hence dictionary entries should be verified and made uniform in that respect before they are “digitalized”... Distinction between the form and its meaning in a dictionary entry is fully possible, as shown by works of Z. Saloni (2002 and A.Przepiórkowski (2008.
Johnson, Wyatt R.; Lu, Ping; Stachowiak, Susan J.
This paper discusses the implementation and simulated performance of the FNPEG (Fully Numerical Predictor-corrector Entry Guidance) algorithm into GNC FSW (Guidance, Navigation, and Control Flight Software) for use in an autonomous re-entry vehicle. A few modifications to FNPEG are discussed that result in computational savings -- a change to the state propagator, and a modification to cross-range lateral logic. Finally, some Monte Carlo results are presented using a representative vehicle in both a high-fidelity 6-DOF (degree-of-freedom) sim as well as in a 3-DOF sim for independent validation.
Full Text Available Allele-specific gene silencing by RNA interference (RNAi is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi, the design and assessment of small interfering RNA (siRNA duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs against mutant alleles of the human Prion Protein (PRNP gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs, of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense
H.G. Barkema (Harry); J.H.J. Bell (John); J.M.E. Pennings
textabstractThis paper examines the longevity of foreign entries. Hypotheses are developed on the mode (start-ups vs. acquisitions) and ownership structure (wholly owned vs. joint ventures) in relation to cultural distance. The hypotheses are tested within a framework of organizational learning,
Full Text Available Purpose: The purpose of the paper is to explore the entry modes of EU firms setting up operations in Vietnam. Design/methodology/approach: we use a case study approach on Haymarket, Cadbury, Creative Education, Fairchild, Aventis and Artemisinin and Farming International using interviews from managerial professionals in Vietnam. Findings: Despite the fact that Vietnam has been opening up for more than 20 years, licensing is the preferred entry mode because of the risks involved in venturing with local firms; that preference signals a low level commitment and a high perception of risk and state interference. In line with Vietnam transition to state - rather than private market - capitalism, a foreign company opting for a joint-venture will do so with a state-owned rather than privately-owned company. The choice of a subsidiary can be explained by the lack of trust in partners and institutions, not by improvement in the socio-political environment. Limitations: In determining the entry mode strategy, the paper focuses on the Uppsala school’s “psychic distance” (e.g. cultural distance, lack of trust rather than on firm-specific advantages (Rugman, 1980; 2006. Key-words: international entry mode; emerging markets; subsidiary; joint-venture; India; Vietnam
In this study, the investigator examines the correlation between entry requirements and academic performance of undergraduate students at the University of Buea. The quality of performance on the Cameroon General Certificate Examination at the Advanced Level is the predictor while the criterion is the cumulative grade ...
Smit, Jolanda M.; Moesker, Bastiaan; Rodenhuis-Zybert, Izabela; Wilschut, Jan
Flaviviruses, such as dengue virus and West Nile virus, are enveloped viruses that infect cells through receptor-mediated endocytosis and fusion from within acidic endosomes. The cell entry process of flaviviruses is mediated by the viral E glycoprotein. This short review will address recent
Russell, Dorothy S.
The author reviews the entry of women into the teaching profession in nineteenth-century America, noting that, while the primary motivation for encouraging women to teach was that they could be paid less than men, this economic justification was obscured by sentimental pronouncements about women's superior moral and nurturant power. (SJL)
Lutz, C.H.M.; Kemp, R.G.M.; Dijkstra, S.G.
This article uses factor analysis to identify the underlying dimensions of strategic and structural entry barriers. We find that, in the perception of firms, both types of barriers are important and that the effectiveness of strategic barriers depends on attributes of the market structure. Based on
Lutz, Clemens H. M.; Kemp, Ron G. M.; Dijkstra, S. Gerhard
This article uses factor analysis to identify the underlying dimensions of strategic and structural entry barriers. We find that, in the perception of firms, both types of barriers are important and that the effectiveness of strategic barriers depends on attributes of the market structure. Based on
Full Text Available Enterovirus B species (EV-B are responsible for a vast number of mild and serious acute infections. They are also suspected of remaining in the body, where they cause persistent infections contributing to chronic diseases such as type I diabetes. Recent studies of the infectious entry pathway of these viruses revealed remarkable similarities, including non-clathrin entry of large endosomes originating from the plasma membrane invaginations. Many cellular factors regulating the efficient entry have recently been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1, actin, Na/H exchanger, phospholipace C (PLC and protein kinase Cα (PKCα. Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The biogenesis of neutral multivesicular bodies is crucial for their infection, at least for echovirus 1 (E1 and coxsackievirus A9 (CVA9. These pathways are triggered by the virus binding to their receptors on the plasma membrane, and they are not efficiently recycled like other cellular pathways used by circulating receptors. Therefore, the best “markers” of these pathways may be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of enterovirus uncoating and genome release from the endosomes to start efficient replication.
The DOE Energy Information Data Base has been created and is maintained by the DOE Technical Information Center. One of the controls for information entered into the base is the standardized name of the corporate entity or the corporate author. The purpose of this list of authorized or standardized corporate entries is to provide a means for the consistent citing of the names of organizations in bibliographic records. It also serves as a guide for users who retrieve information from a bibliographic data base and who want to locate information originating in particular organizations. This authority is a combination of entries established by the Technical Information Center and the International Atomic Energy Agency's International Nuclear Information System (INIS). The format calls, in general, for the name of the organization represented by the literature being cataloged to be cited as follows: the largest element, the place, the smallest element, e.g., Brigham Young Univ., Provo, Utah (USA), Dept. of Chemical Engineering. Code numbers are assigned to each entry to provide manipulation by computer. Cross references are used to reflect name changes and invalid entries
state which mainly determines its polarizability . ∆r2 = r2u− r2l is the difference between Radiation Database for Earth and Mars Entry RTO-EN-AVT...NO A← X (0,0) band in the presence of argon and nitrogen. Journal of Quantitative Spectroscopy and Radiative Transfer, 47:375–390, 1992. Radiation
... DEPARTMENT OF HOMELAND SECURITY Bureau of Customs and Border Protection DEPARTMENT OF THE TREASURY 19 CFR Part 141 Entry of Merchandise CFR Correction In Title 19 of the Code of Federal Regulations, Parts 141 to 199, revised as of April 1, 2010, on page 6, the second general authority citation for part...
Eiselein, E. B.; Topper, Martin
The article describes some of the roles open to anthropologists in radio and television. Entry strategies for occupying these roles include taking the first step in approaching the broadcast station, learning about broadcasting, and communicating anthropology to the broadcasters. (Author/NQ)
Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G S; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang
Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.
Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G. S.; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang
Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides. PMID:24837592
Pompey, Justine M; Foda, Bardees; Singh, Upinder
Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.
Justine M Pompey
Full Text Available Dicer enzymes process double-stranded RNA (dsRNA into small RNAs that target gene silencing through the RNA interference (RNAi pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.
Li, Xinxin; Zhang, Yonggen; Hannoufa, Abdelali; Yu, Peiqiang
Lignin, a phenylpropanoid polymer present in secondary cell walls, has a negative impact on feed digestibility. TT8 and HB12 genes were shown to have low expression levels in low-lignin tissues of alfalfa, but to date, there has been no study on the effect of down-regulation of these two genes in alfalfa on nutrient chemical profiles and availability in ruminant livestock systems. The objectives of this study were to investigate the effect of transformation of alfalfa with TT8 and HB12 RNAi constructs on carbohydrate (CHO) structure and CHO nutritive value in ruminant livestock systems. The results showed that transformation with TT8 and HB12 RNAi constructs reduced rumen, rapidly degraded CHO fractions (RDCA4, P = 0.06; RDCB1, P alfalfa with TT8 and HB12 RNAi constructs induced molecular structure changes. Different CHO functional groups had different sensitivities and different responses to the transformation. The CHO molecular structure changes induced by the transformation were associated with predicted CHO availability. Compared with HB12 RNAi, transformation with TT8 RNAi could improve forage quality by increasing the availability of both NDF and DM. Further study is needed on the relationship between the transformation-induced structure changes at a molecular level and nutrient utilization in ruminant livestock systems when lignification is much higher.
Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi
The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.
Culleton Bridget A
Full Text Available Abstract Background Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance. Results To identify such genes, a panel of expressed sequence tags (ESTs enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species. Conclusions This study has identified and characterised the
Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin
Levits'kij, R.R.; Lyisnij, B.M.
In order to study the dielectric, piezoelectric and elastic properties of ferroelectrics and antiferroelectrics of the KH 2 PO 4 family, we consider an extended proton tunneling model that takes into account the shear strain u 6 . In the four-particle cluster approximation for the short-range interactions and the mean field approximation for the long-range interaction we calculate the transverse components of the dielectric susceptibility tensor of the KH 2 PO 4 family ferroelectrics
An increasing number of European football clubs has developed global strategies with an emphasis on the Chinese market, which seems to be a shortcut to their global success. In order to achieve an effective expansion, the selection of appropriate entry modes should be conducted cautiously. This paper aims to select feasible entry modes for Tottenham Hotspur F.C., an emerging power in the Premier League, to enter into the Chinese market. A framework that combines different theories based ...
The thesis considers the entry strategy and development of the company McDonald's into international markets. The theoretical aspects of the entry strategy of the company into the international markets. Analyzes the key features of the development of McDonald's in Russia. Investigated the prospects of the company in international markets. In theoretic part there was regarded some important aspects of international strategic management, such as: strategic alternatives, elements and levels o...
Dogan, Mevlut; Ulu, Melike; Gennerakis, Giannis; Zouros, Theo J. M.
A new hemispherical deflector analyzer (HDA) which is designed for electron energy analysis in atomic collisions has been constructed and tested. Using the crossed beam technique at the electron spectrometer, test measurements were performed for electron beam (200 eV) - Helium atoms interactions. These first experimental results show that the paracentric entries give almost twice as good resolution as that for the conventional entry. Supporting simulations of the entire lens+HDA spectrometer are found in relatively good agreement with experiment.
Tiago Campos Pereira
Full Text Available The RNA interference (RNAi technique is a recent technology that uses double-stranded RNA molecules to promote potent and specific gene silencing. The application of this technique to molecular biology has increased considerably, from gene function identification to disease treatment. However, not all small interfering RNAs (siRNAs are equally efficient, making target selection an essential procedure. Here we present Strand Analysis (SA, a free online software tool able to identify and classify the best RNAi targets based on Gibbs free energy (deltaG. Furthermore, particular features of the software, such as the free energy landscape and deltaG gradient, may be used to shed light on RNA-induced silencing complex (RISC activity and RNAi mechanisms, which makes the SA software a distinct and innovative tool.
Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Yan; McGraw, John; Woods, Matthew; Murray, Stuart; Eckert, Amy; Liu, Wei; Ryan, Terence E
ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.
Abd El Halim, Hesham M; Alshukri, Baida M H; Ahmad, Munawar S; Nakasu, Erich Y T; Awwad, Mohammed H; Salama, Elham M; Gatehouse, Angharad M R; Edwards, Martin G
The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.
Bai, Hua; Palli, Subba R.
Bursicon is an insect neuropeptide hormone that is secreted from the central nervous system into the hemolymph and initiates cuticle tanning. The receptor for bursicon is encoded by the rickets (rk) gene and belongs to the G protein-coupled receptor (GPCR) superfamily. The bursicon and its receptor regulate cuticle tanning as well as wing expansion after adult eclosion. However, the molecular action of bursicon signaling remains unclear. We utilized RNA interference (RNAi) and microarray to study the function of the bursicon receptor (Tcrk) in the model insect, Tribolium castaneum. The data included here showed that in addition to cuticle tanning and wing expansion reported previously, Tcrk is also required for development and expansion of integumentary structures and adult eclosion. Using custom microarrays, we identified 24 genes that are differentially expressed between Tcrk RNAi and control insects. Knockdown in the expression of one of these genes, TC004091, resulted in the arrest of adult eclosion. Identification of genes that are involved in bursicon receptor mediated biological processes will provide tools for future studies on mechanisms of bursicon action. PMID:20457145
Full Text Available The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1 by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.
Ali, H. K.; Braun, R. D.
This work aims to study the suitability of multi-pass entry trajectories for harnessing of vehicle kinetic energy through magnetohydrodynamic power generation from the high temperature entry plasma. Potential mission configurations are analyzed.
... UNITED STATES TRADE REPRESENTATIVE ALLOCATION OF TARIFF-RATE QUOTA ON IMPORTED SUGARS, SYRUPS AND... present a certificate to the appropriate customs official at the date of entry of specialty sugars. Entry...
Barad, Shiri; Sela, Noa; Dubey, Amit K; Kumar, Dilip; Luria, Neta; Ment, Dana; Cohen, Shahar; Schaffer, Arthur A; Prusky, Dov
The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared. C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process. Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit
Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results The presence of RNA interference (RNAi mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1 gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90 as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM, an inhibitor of HSP90. Conclusions Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These
Kokotti, H.; Kalliokoski, P.
Radon levels, ventilation rate and pressure differences were monitored continuously in four apartment houses with different ventilation systems. Two of them were ventilated by mechanical exhaust, one by mechanical supply and exhaust, and one by natural ventilation. The two-storey houses were constructed from concrete elements on a slab and located on a gravel esker. It was surprising to find that increasing the ventilation rate increased levels of radon in the apartments. Increased ventilation caused increased outdoor-indoor pressure difference, which in turn increased the entry rate of radon and counteracted the diluting effect of ventilation. The increase was significant when the outdoor-indoor pressure difference exceeded 5 Pa. Especially in the houses with mechanical exhaust ventilation the pressure difference was the most important factor of radon entry rate, and contributed up to several hundred Bq m -3 h -1 . (Author)
Whitnah, A. M.; Howes, D. B.
The aerodynamic data that were derived in 1967 from the analysis of flight-generated data for the Gemini entry module are presented. These data represent the aerodynamic characteristics exhibited by the vehicle during the entry portion of Gemini 2, 3, 5, 8, 10, 11, and 12 missions. For the Gemini, 5, 8, 10, 11, and 12 missions, the flight-generated lift-to-drag ratios and corresponding angles of attack are compared with the wind tunnel data. These comparisons show that the flight generated lift-to-drag ratios are consistently lower than were anticipated from the tunnel data. Numerous data uncertainties are cited that provide an insight into the problems that are related to an analysis of flight data developed from instrumentation systems, the primary functions of which are other than the evaluation of flight aerodynamic performance.
Smith, Kelly M.; Rea, Jeremy
For Exploration Missions 1 and 2, the Orion capsules will be entering the Earth's atmosphere with speeds in excess of 11 km/s. In the event of a degraded Guidance, Navigation, and Control system, attempting the nominal guided entry may be inadvisable due to the potential for failures that result in a loss of vehicle (or crew, when crew are aboard). In such a case, a method of assuring Earth capture, water landing, and observence of trajectory constraints (heating, loads) is desired. Such a method should also be robust to large state uncertainty and variations in entry interface states. This document will explore four approaches evaluated and their performance in ensuring a safe return of the Orion capsule in the event of onboard system degradation.
Automated entry control has become an increasingly important issue at facilities where budget constraints are limiting options for manned entry control points. Ongoing work at Sandia National Laboratories is attempting to establish a data base for use by facility security managers working the problem of how to maintain security on a limited budget. Sandia National Laboratories conducted a performance test of the following biometric verifiers: (1) voice verifier by Alpha Microsystems of Santa Ana, California; (2) signature dynamics verifier by Autosig Systems of Irving, Texas; (3) voice verifier by Ecco Industries of Danvers, Massachusetts (now International Electronics); (4) retinal pattern verifier by EyeDentify of Portland, Oregon; (5) fingerprint verifier by Identix of Sunnyvale, California; and (6) hand geometry verifier by Recognition Systems of San Jose, California
Bartosch, Birke; Cosset, Francois-Loic
Hepatitis C virus (HCV), an important human pathogen, is an enveloped, positive-stranded RNA virus classified in the hepacivirus genus of the Flaviviridae family. Cell attachment of flaviviruses generally leads to endocytosis of bound virions. Systems that support HCV replication and particle formation in vitro are emerging only now, 16 years after the discovery of the virus. Albeit this limitation, the route of HCV cell entry as well as 'capture' molecules involved in low-affinity interactions for the initial contact of HCV with target cells and potential high-affinity receptor candidates that may mediate HCV trafficking and fusion has been described. The objective of this review is to summarize the contribution of different HCV model systems to our current knowledge about structure of the HCV GPs E1 and E2 and their roles in cell entry comprising cell attachment, interactions with cellular receptors, endocytosis, and fusion
Koskenniemi, Kimmo Matti
A practical method for interactive guessing of LEXC lexicon entries is presented. The method is based on describing groups of similarly inflected words using regular expressions. The patterns are compiled into a finite-state transducer (FST) which maps any word form into the possible LEXC lexicon entries which could generate it. The same FST can be used (1) for converting conventional headword lists into LEXC entries, (2) for interactive guessing of entries, (3) for corpus-assisted interactiv...
Heilimo, J.; Harri, A.-M.; Aleksashkin, S.; Koryanov, V.; Arruego, I.; Schmidt, W.; Haukka, H.; Finchenko, V.; Martynov, M.; Ostresko, B.; Ponomarenko, A.; Kazakovtsev, V.; Martin, S.; Siili, T.
A new generation of inflatable Entry, Descent and Landing System (EDLS) for Mars has been developed. It is used in both the initial atmospheric entry and atmospheric descent before the semi-hard impact of the penetrator into Martian surface. The EDLS applicability to Earth's atmosphere is studied by the EU/RITD  project. Project focuses on the analysis and tests of the transonic behaviour of this compact and light weight payload entry system at the Earth re-entry.
Maican, Florin; Orth, ´Matilda
We use a dynamic oligopoly model of entry and exit to evaluate how entry regulations affect profitability and market structure in retail. The model incorporates demand and store-level heterogeneity. Based on unique data for all retail food stores in Sweden, we find that the average entry costs for small and large stores are 10 and 18 percent lower, respectively, in markets with liberal compared with restrictive regulations. Counterfactual simulations show that lower entry costs in restrictive...
Maican, Florin; Orth, Matilda
We use a dynamic oligopoly model of entry and exit with store-type differentiation to evaluate how entry regulations affect profitability, market structure and welfare. Based on unique data for all retail food stores in Sweden, we estimate demand, recover variable profits, and estimate entry costs and fixed costs by store type. Counterfactual policy experiments show that welfare increases when competition is enhanced by lower entry costs. Protecting small stores by imposing licensing fees on ...
Falck, Oliver; Heblich, Stephan
This paper takes a different perspective toward the escape entry incentive of incumbent firms to innovate. New entrants spawned from incumbents are not necessarily a threat; they can complement incumbents' production by commercializing knowledge incumbents are not willing or able to exploit. Accordingly, incumbent innovation determines exploitable knowledge externalities for spinoffs while, at the same time, spinoffs are expected to influence incumbent innovation. To overcome this problem of ...
Crampes, Claude; Haritchabalet, Carole; Jullien, Bruno
This paper presents a model of media competition with free entry when media operators are financed both from advertisers and customers. The relation between advertising receipts and sales receipts, which are both complementary and antagonist, is different if media operators impose a price or a quantity to advertisers. When consumers dislike advertising, media operators are better off setting an advertising price than an advertising quantity. We establish a relationship between the equilibrium...
Kellogg, M.; Gallup, J.M.; Shlaer, S.; Spencer, N.
This manual describes the systems for producing histograms and dot plots that were designed for use in connection with the Q general-purpose data-acquisition system. These systems allow for the creation of histograms; the entry, retrieval, and plotting of data in the form of histograms; and the dynamic display of scatter plots as data are acquired. Although the systems are designed for use with Q, they can also be used as a part of other applications. 3 figures
Full Text Available In human and nonhuman primates, filoviruses (Ebola and Marburg viruses cause severe hemorrhagic fever．Recently, other animals such as pigs and some species of fruit bats have also been shown to be susceptible to these viruses. While having a preference for some cell types such as hepatocytes, endothelial cells, dendritic cells, monocytes, and macrophages, filoviruses are known to be pantropic in infection of primates. The envelope glycoprotein (GP is responsible for both receptor binding and fusion of the virus envelope with the host cell membrane. It has been demonstrated that filovirus GP interacts with multiple molecules for entry into host cells, whereas none of the cellular molecules so far identified as a receptor/coreceptor fully explains filovirus tissue tropism and host range. Available data suggest that the mucin-like region (MLR on GP plays an important role in attachment to the preferred target cells, whose infection is likely involved in filovirus pathogenesis, whereas the MLR is not essential for the fundamental function of the GP in viral entry into cells in vitro. Further studies elucidating the mechanisms of cellular entry of filoviruses may shed light on the development of strategies for prophylaxis and treatment of Ebola and Marburg hemorrhagic fevers.
... 31 Money and Finance: Treasury 2 2010-07-01 2010-07-01 false Conversions to book-entry. 337.6... HOUSING ADMINISTRATION DEBENTURES Certificated Debentures § 337.6 Conversions to book-entry. Upon implementation of the book-entry debenture system, to be announced in advance by separate public notice, all new...
... 32 National Defense 4 2010-07-01 2010-07-01 true Rights of entry. 643.11 Section 643.11 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE General § 643.11 Rights of entry. Pending the signing of the formal instrument, no right of entry will be...
... 19 Customs Duties 2 2010-04-01 2010-04-01 false Entry by nonresident corporation. 141.18 Section....18 Entry by nonresident corporation. A nonresident corporation (i.e., one which is not incorporated... entry is located who is authorized to accept service of process against that corporation or, in the case...
... 19 Customs Duties 2 2010-04-01 2010-04-01 false Information on entry summary. 151.63 Section 151.63 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF... Information on entry summary. Each entry summary covering wool or hair subject to duty at a rate per clean...
Prabu, Dinesh K.; Allen, Gary A., Jr.; Cappuccio, Gelsomina; Spilker, Thomas R.; Hwang, Helen H.; Moses, Robert W.
The present study considers ballistic entries into the atmosphere of Venus using a 45deg sphere-cone rigid aeroshell, a legacy shape that has been used successfully in the past in the Pioneer Venus Multiprobe Mission. For a number of entry mass and capsule diameter combinations (i.e., various ballistic coefficients) and entry velocities, the trajectory space in terms of entry flight path angles between skip out and -30 is explored with a 3DOF trajectory code, TRAJ. Assuming that the thermal protection material of choice is carbon phenolic of flight heritage, the entry flight path angle space is constrained a posteriori by the mechanical and thermal performance parameters of the material. For mechanical performance, a 200 g limit is placed on the peak deceleration load and 10 bar is assumed as the limit for heritage carbon-phenolic material. It is shown that both constraints cannot be active simultaneously. For thermal performance, a heat flux 2.5 kW/sq cm is utilized as a threshold below which the heritage carbon phenolic is considered mass inefficient. Using these constraints, viable entry flight path angle corridors are determined. Analysis of the results also hints at the existence of a range of "critical" ballistic coefficients beyond which the steepest possible entries are determined by the pressure limit of 10 bar. The results are verified against known performance of the various probes used in the Pioneer Venus mission. It is anticipated that the results presented here will serve as a baseline in the development of a new class of ablative materials for future Venus missions.
Tierney, P; Tobin, D P; Blake, C; Delahunt, E
Global Positioning System (GPS) technology is commonly utilized in team sports, including rugby union. It has been used to describe the average running demands of rugby union. This has afforded an enhanced understanding of the physical fitness requirements for players. However, research in team sports has suggested that training players relative to average demands may underprepare them for certain scenarios within the game. To date, no research has investigated the running demands of attacking 22 entries in rugby union. Additionally, no research has been undertaken to determine whether differences exist in the running intensity of successful and unsuccessful attacking 22 entries in rugby union. The first aim of this study was to describe the running intensity of attacking 22 entries. The second aim of this study was to investigate whether differences exist in the running intensity of successful and unsuccessful attacking 22 entries. Running intensity was measured using meters per minute (m min -1 ) for (a) total distance, (b) running distance, (c) high-speed running distance, and (d) very high-speed running distance. This study provides normative data for the running intensity of attacking 22 entries in rugby union. Forwards achieved greater high-speed running intensity in successful (3.6 m min -1 ) compared to unsuccessful (1.8 m min -1 ) attacking 22 entries. Forwards should try and achieve greater high-speed running intensity in attacking 22 entries to increase the likelihood of successful outcomes during this period of gameplay. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
When the product is digital, it will most often be distributed directly to the customer through the Internet, and therefore the entry modes, considered in this paper, are different flavors of the entry mode called direct export: Virtual export channel are generally understood as the entry mode fo...... for digital product providers. However other types of entry modes like what wee call direct digital export with F2F-sales, direct digital export with F2F-support and virtual sales subsidiary are entry modes that respond to a higher degree of pre- and after-sales complexity....
Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Chuan, Tjin Swee; Yong, Ken-Tye; Yoon, Ho Sup
RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications. (paper)
Full Text Available For sustainable utilization of limited coal resources, it is important to increase the coal recovery rate and reduce mine accidents, especially those occurring in the entry (gateroad. Entry stabilities are vital for ventilation, transportation and other essential services in underground coal mining. In the present study, a finite difference model was built to investigate stress evolutions around the entry, and true triaxial tests were carried out at the laboratory to explore entry wall stabilities under different mining conditions. The modeling and experimental results indicated that a wide coal pillar was favorable for entry stabilities, but oversize pillars caused a serious waste of coal resources. As the width of the entry wall decreased, the integrated vertical stress, induced by two adjacent mining panels, coupled with each other and experienced an increase on the entry wall, which inevitably weakened the stability of the entry. Therefore, mining with coal pillars always involves a tradeoff between economy and safety. To address this problem, an innovative non-pillar mining technique by optimizing the entry surrounding structures was proposed. Numerical simulation showed that the deformation of the entry roof decreased by approximately 66% after adopting the new approach, compared with that using the conventional mining method. Field monitoring indicated that the stress condition of the entry was signiﬁcantly improved and the average roof pressure decreased by appropriately 60.33% after adopting the new technique. This work provides an economical and effective approach to achieve sustainable exploitation of underground coal resources.
John W. UPSON
Full Text Available Market entry is the essence of strategy and is largely viewed as a dichotomous event: entry or no entry. What has not been acknowledged is the uniqueness of each market entry. Our study highlights the scale of market entry in the context of multipoint competition. We assert that entry scale varies based on the risk of market incumbent retaliation. Theory suggests that when risk associated with retaliation are low, firms enter with large scale and when associated risks are high, firms enter with low scale. Further, survival is viewed as dependent on following theory. We argue and find supporting evidence that firms behave in the opposite manner and do so to their own benefit, thereby revealing a unique discrepancy between theory and practice among 75 product market entries by 27 firms.
Ujino, Saneyuki; Nishitsuji, Hironori; Hishiki, Takayuki; Sugiyama, Kazuo; Takaku, Hiroshi; Shimotohno, Kunitada
Various host factors are involved in the cellular entry of hepatitis C virus (HCV). In addition to the factors previously reported, we discovered that the very-low-density lipoprotein receptor (VLDLR) mediates HCV entry independent of CD81. Culturing Huh7.5 cells under hypoxic conditions significantly increased HCV entry as a result of the expression of VLDLR, which was not expressed under normoxic conditions in this cell line. Ectopic VLDLR expression conferred susceptibility to HCV entry of CD81-deficient Huh7.5 cells. Additionally, VLDLR-mediated HCV entry was not affected by the knockdown of cellular factors known to act as HCV receptors or HCV entry factors. Because VLDLR is expressed in primary human hepatocytes, our results suggest that VLDLR functions in vivo as an HCV receptor independent of canonical CD81-mediated HCV entry.
Kretova, Olga V; Fedoseeva, Daria M; Gorbacheva, Maria A; Gashnikova, Natalya M; Gashnikova, Maria P; Melnikova, Nataliya V; Chechetkin, Vladimir R; Kravatsky, Yuri V; Tchurikov, Nickolai A
RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs) due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%-97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT), integrase, vpu, gp120, and p17. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G) in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Hansen, K. R.; Ibarra, P. T.; Thon, G.
. Mutations and conditions perturbing histone acetylation had similar effects further demonstrating that the tlh genes are normally repressed by heterochromatin. In contrast, mutations in the RNAi factors Dcr1, Ago1 or Rdp1 led only to a modest derepression of the tlh genes indicating an alternate pathway...
Gu, Keyu; Tian, Dongsheng; Mao, Huizhu; Wu, Lifang; Yin, Zhongchao
Jatropha curcas L. is a potential biofuel plant and its seed oil is suitable for biodiesel production. Despite this promising application, jatropha seeds contain two major toxic components, namely phorbol esters and curcins. These compounds would reduce commercial value of seed cake and raise safety and environment concerns on jatropha plantation and processing. Curcins are Type I ribosome inactivating proteins. Several curcin genes have been identified in the jatropha genome. Among which, the Curcin 1 (C1) gene is identified to be specifically expressed in endosperm, whereas the Curcin 2A (C2A) is mainly expressed in young leaves. A marker-free RNAi construct carrying a β-estradiol-regulated Cre/loxP system and a C1 promoter-driven RNAi cassette for C1 gene was made and used to generate marker-free transgenic RNAi plants to specifically silence the C1 gene in the endosperm of J. curcas. Plants of transgenic line L1, derived from T0-1, carry two copies of marker-free RNAi cassette, whereas plants of L35, derived from T0-35, harbored one copy of marker-free RNAi cassette and three copies of closely linked and yet truncated Hpt genes. The C1 protein content in endosperm of L1 and L35 seeds was greatly reduced or undetectable, while the C2A proteins in young leaves of T0-1 and T0-35 plants were unaffected. In addition, the C1 mRNA transcripts were undetectable in the endosperm of T3 seeds of L1 and L35. The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette. The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable. Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas. The marker-free transgenic plants and curcin-deficient seeds developed in this study provided a solution for the toxicity of curcins in jatropha seeds and
Hollis, Brian R.; Borrelli, Salvatore
In this chapter, the aerothermodynamic phenomena of blunt body entry vehicles are discussed. Four topics will be considered that present challenges to current computational modeling techniques for blunt body environments: turbulent flow, non-equilibrium flow, rarefied flow, and radiation transport. Examples of comparisons between computational tools to ground and flight-test data will be presented in order to illustrate the challenges existing in the numerical modeling of each of these phenomena and to provide test cases for evaluation of computational fluid dynamics (CFD) code predictions.
Laskowski, Roman A.; Jabłońska, Jagoda; Pravda, Lukáš; Vařeková, Radka Svobodová; Thornton, Janet M.
Debra J. Aron
Under what circumstances will a successful incumbent in a related market be the first to enter a new market? We present a model in which the order of entry into new markets has long run effects on the firms' profits. We assume that a firm that is successfully producing in a related market has valuable information about the demand in the new market. By his choice of location in product space in the new market the incumbent reveals information about the demand to the potential entrant. Thus, th...
Laskowski, Roman A; Jabłońska, Jagoda; Pravda, Lukáš; Vařeková, Radka Svobodová; Thornton, Janet M
Tegtmeier, Silke; Kurczewska, Agnieszka
with a positive influence, namely having an entrepreneurial role model and having attended seminars about starting a business. In our model, these factors support waiting longer before starting a business. In light of these results, we call for a more experimental approach in entrepreneurship education.......This paper explores the nascence period - the time between idea generation and business entry -among women entrepreneurs with a graduate degree. To address this research problem and to better understand the specifics of a window of opportunity, we combine selected theories of human and social...
Jensen, Njal Winther
Abstract In eukaryotes, secretory proteins are translocated into the endoplasmic reticulum (ER) for folding assistance, acquisition of posttranslational modifications and sorting. Proteins that do not obtain their native conformation are eliminated by ER-associated degradation (ERAD). ERAD...... is a sophisticated pathway that recognizes misfolded proteins and targets them for degradation by the 26S proteasome residing in the cytosol. More than 60 diseases including Alzheimer’s disease, Huntington’s disease and Parkinson’s disease have been linked to the ERAD pathway underscoring its crucial role...... for cellular homeostasis. The aim of this thesis has been to gain insight into ERAD. The experimental approach was RNAi screening, which is a fast and efficient method for initial evaluation of a large pool of genes. Since relatively few proteins routinely are used as ERAD substrates, the first goal...
Full Text Available Globalization causes high mobility of human and livestock, hence increase the transmission of infectious diseases, including avian influenza, severe acute respiratory syndrome (SARS, and swine influenza. Therefore, prevention of those diseases is required. Vaccines are effective to prevent infectious diseases; however, their development takes a long time and they cannot provide immediate protection in pandemic cases. This paper describes several gene silencing technologies including antisense oligonucleotide (ASO, RNA interference (RNAi and single strand-small interfering RNA (ss-siRNA for controlling diseases. The primary mechanism of these technologies is inhibition of gene expression, typically by causing the destruction of specific RNA molecule of the pathogen. The use of gene silencing technologies is expected to give new alternative that is more effective in eradication of infectious diseases in animals before threaten human being.
Schyth, Brian Dall; Hajiabadi, Seyed Amir Hossein Jalali; Kristensen, Lasse Bøgelund Juel
Small RNAs acting in the recently discovered gene regulatory mechanism called RNA interference has a potential as diagnostic signatures of disease and immunological state and when produced synthetically as prophylactic treatment of such diseases. In the RNAi mechanism the cell produces different....... The mechanism can be programmed with several types of small double stranded RNAs - the type of which defines the destiny of the target. One such class of regulatory RNAs called microRNAs are upregulated due to various physiological responses of the cell and they suppress many genes simultaneously believed...... small RNAs which inhibit gene expression through more or less specific interaction with messenger RNAs resulting in repression of translation to protein. In this way cells can turn of genes of specific pathways thereby leading to altered physiological stages of tissues and possibly of whole organisms...
Full Text Available The Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1, Pb03 (PS2 and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis.In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA from a Gateway-adapted cassette (cALf which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach.We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.
Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto
Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.
Full Text Available BACKGROUND: Pheromone biosynthesis activating neuropeptide (PBAN is a neurohormone that regulates sex pheromone synthesis in female moths. Bombyx mori is a model organism that has been used to explore the signal transduction pattern of PBAN, which is mediated by a G-protein coupled receptor (GPCR. Although significant progress has been made in elucidating PBAN-regulated lipolysis that releases the precursor of the sex pheromone, little is known about the molecular components involved in this step. To better elucidate the molecular mechanisms of PBAN-stimulated lipolysis of cytoplasmic lipid droplets (LDs, the associated lipase genes involved in PBAN- regulated sex pheromone biosynthesis were identified using digital gene expression (DGE and subsequent RNA interference (RNAi. RESULTS: Three DGE libraries were constructed from pheromone glands (PGs at different developed stages, namely, 72 hours before eclosion (-72 h, new emergence (0 h and 72 h after eclosion (72 h, to investigate the gene expression profiles during PG development. The DGE evaluated over 5.6 million clean tags in each PG sample and revealed numerous genes that were differentially expressed at these stages. Most importantly, seven lipases were found to be richly expressed during the key stage of sex pheromone synthesis and release (new emergence. RNAi-mediated knockdown confirmed for the first time that four of these seven lipases play important roles in sex pheromone synthesis. CONCLUSION: This study has identified four lipases directly involved in PBAN-stimulated sex pheromone biosynthesis, which improve our understanding of the lipases involved in releasing bombykol precursors from triacylglycerols (TAGs within the cytoplasmic LDs.
Moazeni, Maryam; Khoramizadeh, Mohammad Reza; Kordbacheh, Parivash; Sepehrizadeh, Zargham; Zeraati, Hojat; Noorbakhsh, Fatemeh; Teimoori-Toolabi, Ladan; Rezaie, Sassan
The introduction of RNA silencing machinery in fungi has led to the promising application of RNAi methodology to knock down essential vital factor or virulence factor genes in the microorganisms. Efg1p is required for development of a true hyphal growth form which is known to be essential for interactions with human host cells and for the yeast's pathogenesis. In this paper, we describe the development of a system for presenting and studying the RNAi function on the EFG1 gene in C. albicans. The 19-nucleotide siRNA was designed on the basis of the cDNA sequence of the EFG1 gene in C. albicans and transfection was performed by use of a modified-PEG/LiAc method. To investigate EFG1 gene silencing in siRNA-treated cells, the yeasts were grown in human serum; to induce germ tubes a solid medium was used with the serum. Quantitative changes in expression of the EFG1 gene were analyzed by measuring the cognate EFG1 mRNA level by use of a quantitative real-time RT-PCR assay. Compared with the positive control, true hyphae formation was significantly reduced by siRNA at concentrations of 1 μM, 500 nM, and 100 nM (P < 0.05). In addition, siRNA at a concentration of 1 μM was revealed to inhibit expression of the EFG1 gene effectively (P < 0.05). On the basis of the potential of post-transcriptional gene silencing to control the expression of specific genes, these techniques may be regarded as promising means of drug discovery, with applications in biomedicine and functional genomics analysis.
Full Text Available Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L. vacuolar H(+-ATPase subunit A (OsVHA-A gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+-ATPase activity and an enhancement of plasma membrane H(+-ATPase activity, thereby increasing the concentrations of extracellular H(+ and intracellular K(+ and Na(+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+-ATPase 3 and downregulation of CAM1 (calmodulin 1, CAM3 (calmodulin 3 and YDA1 (YODA, a MAPKK gene. Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.
Rawlings, Renata A.; Krishnan, Vishalakshi; Walter, Nils G.
RNA interference (RNAi) is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response against viruses and retrotransposons. During viral infection, the RNase III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs), 21–24 nucleotides in length, and helps load them into the RNA-induced silencing complex (RISC) to guide cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressor (RSS) proteins that tightly, and presumably quantitatively, bind siRNAs to thwart RNAi-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus (CIRV), as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding ((1.69 ± 0.07)×108 M−1s−1) and marked dissociation (koff = 0.062 ± 0.002 s−1). We also observe that p19 efficiently competes with recombinant Dicer and inhibits formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple-turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy. PMID:21354178
Olga V. Kretova
Full Text Available RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%–97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT, integrase, vpu, gp120, and p17. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS. Keywords: HIV-1, RNAi targets, gene therapy, ultra-deep sequencing, conserved HIV-1 sequences
Panwar, Vinay; Jordan, Mark; McCallum, Brent; Bakkeren, Guus
Leaf rust, caused by the pathogenic fungus Puccinia triticina (Pt), is one of the most serious biotic threats to sustainable wheat production worldwide. This obligate biotrophic pathogen is prevalent worldwide and is known for rapid adaptive evolution to overcome resistant wheat varieties. Novel disease control approaches are therefore required to minimize the yield losses caused by Pt. Having shown previously the potential of host-delivered RNA interference (HD-RNAi) in functional screening of Pt genes involved in pathogenesis, we here evaluated the use of this technology in transgenic wheat plants as a method to achieve protection against wheat leaf rust (WLR) infection. Stable expression of hairpin RNAi constructs with sequence homology to Pt MAP-kinase (PtMAPK1) or a cyclophilin (PtCYC1) encoding gene in susceptible wheat plants showed efficient silencing of the corresponding genes in the interacting fungus resulting in disease resistance throughout the T 2 generation. Inhibition of Pt proliferation in transgenic lines by in planta-induced RNAi was associated with significant reduction in target fungal transcript abundance and reduced fungal biomass accumulation in highly resistant plants. Disease protection was correlated with the presence of siRNA molecules specific to targeted fungal genes in the transgenic lines harbouring the complementary HD-RNAi construct. This work demonstrates that generating transgenic wheat plants expressing RNAi-inducing transgenes to silence essential genes in rust fungi can provide effective disease resistance, thus opening an alternative way for developing rust-resistant crops. © 2017 Her Majesty the Queen in Right of Canada. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Full Text Available Face Interface is a wearable prototype that combines the use of voluntary gaze direction and facial activations, for pointing and selecting objects on a computer screen, respectively. The aim was to investigate the functionality of the prototype for entering text. First, three on-screen keyboard layout designs were developed and tested (n=10 to find a layout that would be more suitable for text entry with the prototype than traditional QWERTY layout. The task was to enter one word ten times with each of the layouts by pointing letters with gaze and select them by smiling. Subjective ratings showed that a layout with large keys on the edge and small keys near the center of the keyboard was rated as the most enjoyable, clearest, and most functional. Second, using this layout, the aim of the second experiment (n=12 was to compare entering text with Face Interface to entering text with mouse. The results showed that text entry rate for Face Interface was 20 characters per minute (cpm and 27 cpm for the mouse. For Face Interface, keystrokes per character (KSPC value was 1.1 and minimum string distance (MSD error rate was 0.12. These values compare especially well with other similar techniques.
The US Department of Energy is one of three agencies funding the major portion of government-supported research. One of the ways to locate the results of this research is to find reports in the Energy Data Base (EDB), the comprehensive data base of the Office of Scientific and Technical Information, Technical Information Center, and in publications derived therefrom by referring to the corporate organization performing the research. This information field has been established as an index point retrievable in on-line searching and is included as an index in printed publications. To provide consistent citing of names in bibliographic entries, this authority has been created and maintained as a means of entry of corporate names into the EDB. To locate such information, one can (1) use the seven-digit code number assigned to the corporate entity of interest (enter, for example, IC=9506086) or (2) use one word at a time from the corporate name given (enter, for example, CS=Dominion)
... Entry Summary-- Declaration of Value AGENCY: Customs and Border Protection, Department of Homeland... manner by which the declared transaction value on imported merchandise was determined. This requirement... whether the transaction value of imported merchandise is determined on the basis of the price paid by the...
This note shows that a feasible reference drag profile for the first entry portion of a skip entry can be generated as a polynomial expression of the velocity. The coefficients of that polynomial are found through the resolution of a system composed of m + 1 equations, where m is the degree of the drag polynomial. It has been shown that a minimum of five equations (m = 4) are required to establish the range and the initial and final conditions on velocity and flight path angle. It has been shown that at least one constraint on the trajectory can be imposed through the addition of one extra equation in the system, which must be accompanied by the increase in the degree of the drag polynomial. In order to simplify the resolution of the system of equations, the drag was considered as being a probability density function of the velocity, with the velocity as a distribution function of the drag. Combining this notion with the introduction of empirically derived constants, it has been shown that the system of equations required to generate the drag profile can be successfully reduced to a system of linear algebraic equations. For completeness, the resulting drag profiles have been flown using the feedback linearization method of differential geometric control as a guidance law with the error dynamics of a second order homogeneous equation in the form of a damped oscillator. Satisfactory results were achieved when the gains in the error dynamics were changed at a certain point along the trajectory that is dependent on the velocity and the curvature of the drag as a function of the velocity. Future work should study the capacity to update the drag profile in flight when dispersions are introduced. Also, future studies should attempt to link the first entry, as presented and controlled in this note, with a more standard control concept for the second entry, such as the Apollo entry guidance, to try to assess the overall skip entry performance. A guidance law that includes
Lv, Ye-hui; Ma, Kai-jun; Zhang, Heng; He, Meng; Zhang, Ping; Shen, Yi-wen; Jiang, Nan; Ma, Duan; Chen, Long
Determining the postmortem interval (PMI) is important in criminal, civil, and forensic cases. We examined the feasibility of using the transcript abundances of mRNAs, 18S rRNA, U6 snRNA, and microRNAs as a means to estimate the PMI. We removed spleen tissues from rats at different PMIs under 4°C or 25°C and examined gene transcript abundances in these samples by RT-qPCR. Using the algorithm geNorm, we found that microRNAs to be appropriate control markers because they were less affected by PMI and temperature. We also characterized relationships between observed PMI and the transcript levels of the above-mentioned RNAs. GAPDH1 and ACTB1 fluctuated slightly like cubic curves, while GAPDH2 and ACTB2 decreased rapidly. 18S rRNA transcript level exhibited a parabolic-like trend at 25°C and exponential growth at 4°C, while U6 transcript level exhibited exponential decay at 25°C and a parabolic-like trend at 4°C. Following validation, we conclude that GAPDH2, ACTB2, and 18S rRNA are suitable makers in the accurate determination of PMI. © 2014 American Academy of Forensic Sciences.
Ream, W.K.; Espinoza, J.
An entry control system to automatically control access to nuclear facilities is described. The design uses a centrally located console, integrated into the regular security system, to monitor the computer-controlled passage into and out of sensitive areas. Four types of entry control points are used: an unmanned enclosed portal with metal and SNM detectors for contraband detection with positive personnel identification, a bypass portal for contraband search after a contraband alarm in a regular portal also with positive personnel identification, a single door entry point with positive personnel identification, and a single door entry point with only a magnetic card-type identification. Security force action is required only as a response to an alarm. The integration of the entry control function into the security system computer is also described. The interface between the entry control system and the monitoring security personnel utilizing a color graphics display with touch screen input is emphasized
Bengali, Zain; Satheshkumar, P.S. [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210 (United States); Moss, Bernard, E-mail: firstname.lastname@example.org [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210 (United States)
Vaccinia virus (VACV) enters cells by a low pH endosomal route or by direct fusion with the plasma membrane. We previously found differences in entry properties of several VACV strains: entry of WR was enhanced by low pH, reduced by bafilomycin A1 and relatively unaffected by heparin, whereas entry of IHD-J, Copenhagen and Elstree were oppositely affected. Since binding and entry modes may have been selected by specific conditions of in vitro propagation, we now examined the properties of three distinct, recently isolated cowpox viruses and a monkeypox virus as well as additional VACV and cowpox virus strains. The recent isolates were more similar to WR than to other VACV strains, underscoring the biological importance of endosomal entry by orthopoxviruses. Sequence comparisons, gene deletions and gene swapping experiments indicated that viral determinants, other than or in addition to the A26 and A25 'fusion-suppressor' proteins, impact entry properties.
This paper examines the impact of restricting foreign bank entry on bank net interest margins while controlling for (a) impediments to domestic bank entry, (b) the degree of foreign bank ownership of the domestic banking industry, (c) an array of bank-specific characteristics, (c) banking sectorconcentration, and (d) various country traits. Using data on almost 1200 banks across 47 countries, the results suggest that restricting foreign bank entry boosts bank net interest margins. Also, restr...
Chinese firms and other emerging markets firms aim to select a favourable entry mode to start or expand their international business, thus this is significant issue for research. This study demonstrates that the Chinese firms’ choice of entry mode is determined by internal-factors, external-factors and transaction cost factors. This is illustrated through the investigation of a case study of the Haier Group’s choice of entry mode and internationalization. Therefore, four processes of choice o...
This paper argues that even temporary barriers to entry present at the very beginning of transition may lead to permanent extortion development. Entry restrictions, if binding, lead to excess profits, which create an incentive to extort. The emergence of extortionists reduces the expected profit from production, making producers expect extortion in the future. If, after this adaptation of expectations, the government removes the barriers to entry, only a few new firms will enter the market. H...
Hattori, Keisuke; Yoshikawa, Takeshi
We investigate the social desirability of free entry in the co-opetition model in which firms compete in a homogeneous product market while sharing common property resources that affect market size or consumers' willingness to pay for products. We show that free entry leads to socially excessive or insufficient entry into the market in the case of non-commitment co-opetition, depending on the magnitude of "business stealing" and "common property" effects of entry. On the other hand, in the ca...
Dyhr Ulrich, Anna Marie; Boyd, Britta; Hollensen, Svend
The aim of this article is to investigate the choice of entry modes for international markets in a control perspective. A survey from The Confederation of Danish Industry with 234 Danish small- and medium sized enterprises served as a data base. The entry modes are categorized into three groups...... depending on the control that the company has over its activities abroad. The paper examines selected factors that influence the ‘entry modes’ of Danish SMEs in different strategic settings. Results show that the most deciding factor for the choice of high control entry mode (subsidiary) was the factor...
Full Text Available This paper expands the discussion of the importance and function of adjusting entries for loan receivables. Discussion of the cyclical development of adjusting entries, their negative impact on the business cycle and potential solutions has intensified during the financial crisis. These discussions are still ongoing and continue to be relevant to members of the professional public, banking regulators and representatives of international accounting institutions. The objective of this paper is to evaluate a method of journaling dynamic adjusting entries under current accounting law. It also expresses the authors’ opinions on the potential for consistently implementing basic accounting principles in journaling adjusting entries for loan receivables under a dynamic model.
Conner, Sean D.; Schmid, Sandra L.
The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. `Endocytosis' encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.
Louw, Arnt Vestergaard
This article reports on methodological experiences obtained in an anthropologically inspired qualitative study among students of carpentry in Denmark. On the one hand the article deals with methodological issues of doing anthropological research among students of carpentry, while on the other...... it deals with the research findings that such a research design produced. As well as the methodological issues of researcher access, entry and participant position in the field, this article reports on the following questions: What kinds of implicit expectations of the students are embedded in the way...... the school introduces and initiates the programme? What kinds of effects does this have on the motivation of the students? How do the terms and professional language of the profession work on the individual students in including and excluding ways? These specific descriptions of classroom pedagogy, inspired...
Andersen, C.E.; Søgaard-Hansen, J.; Majborn, B.
A simple test structure for studies of radon entry into houses has been constructed at a field site at Riso National Laboratory. It consists of a 40 1, stainless-steel cylinder placed in a 0.52 m deep quadratic excavation with a side length of 2.4 m. The excavation is lined with an airtight...... membrane, and soil gas enters the cylinder through a changeable interface in the bottom. The depressurisation of the cylinder is controlled by a mass-flow controller, thereby limiting the influence of natural driving forces. Pressures, temperatures and radon concentrations are measured continuously...... in the cylinder and in selected locations in the soil. In this paper, the test structure is described, and initial results concerning the transport of soil gas and radon under steady-state conditions are reported. It is found that the soil in the vicinity of the structure is partially depleted with respect...
Two full-scale passive Earth Entry Vehicles (EEV) with realistic structure, surrogate sample container, and surrogate Thermal Protection System (TPS) were built at NASA Langley Research Center (LaRC) and tested at the Utah Test and Training Range (UTTR). The main test objective was to demonstrate structural integrity and investigate possible impact response deviations of the realistic vehicle as compared to rigid penetrometer responses. With the exception of the surrogate TPS and minor structural differences in the back shell construction, the two test vehicles were identical in geometry and both utilized the Integrated Composite Stiffener Structure (ICoSS) structural concept in the forward shell. The ICoSS concept is a lightweight and highly adaptable composite concept developed at NASA LaRC specifically for entry vehicle TPS carrier structures. The instrumented test vehicles were released from a helicopter approximately 400 m above ground. The drop height was selected such that at least 98% of the vehicles terminal velocity would be achieved. While drop tests of spherical penetrometers and a low fidelity aerodynamic EEV model were conducted at UTTR in 1998 and 2000, this was the first time a passive EEV with flight-like structure, surrogate TPS, and sample container was tested at UTTR for the purpose of complete structural system validation. Test results showed that at a landing vertical speed of approximately 30 m/s, the test vehicle maintained structural integrity and enough rigidity to penetrate the sandy clay surface thus attenuating the landing load, as measured at the vehicle CG, to less than 600 g. This measured deceleration was found to be in family with rigid penetrometer test data from the 1998 and 2000 test campaigns. Design implications of vehicle structure/soil interaction with respect to sample container and sample survivability are briefly discussed.
Mochida, Satoru; Rata, Scott; Hino, Hirotsugu; Nagai, Takeharu; Novák, Béla
The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex. Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15 [1, 2]. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible [2-4]. Here, we surprisingly find that Cdk1 auto-activation is dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). We show that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, we found a sharp Cdk1 threshold for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. Our results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry. Copyright © 2016 Elsevier Ltd. All rights reserved.
Robinson, Maxine F; Mihalopoulos, Cathrine; Merlin, Tracy; Roughead, Elizabeth
Australia relies on managed entry agreements (MEAs) for many medicines added to the national Pharmaceutical Benefits Scheme (PBS). Previous studies of Australian MEAs examined public domain documents and were not able to provide a comprehensive assessment of the types and operation of MEAs. This study used government documents approved for release to examine the implementation and administration of MEAs implemented January 2012 to May 2016. We accessed documents for medicines with MEAs on the PBS between January 2012 and May 2016. Data were extracted on Anatomical Therapeutic Classification (ATC), type of MEA (financial, financial with outcomes, outcomes, and subcategories within each group), implementation and administration methods, source of MEA recommendation, and type of economic analysis. Of all medication indication pairs (MIPs) recommended for listing, one-third had MEAs implemented. Our study of eighty-seven MIPs had 170 MEAs in place. The Government's expert health technology assessment (HTA) committee recommended MEAs for 90 percent of the eighty-seven MIPs. A total of 81 percent of MEAs were simple financial agreements: the majority either discounts (32 percent) or reimbursement caps (43 percent). Outcome-based MEAs were least common (5 percent). Ninety-two percent of MEAs were implemented and operated through legal agreements. Approximately half of the MIPs were listed on the basis of accepted claims of cost-minimization. Forty-nine percent of medicines were in ATC L group. Advice from HTA evaluations strongly influences the implementation of ways to manage uncertainties while providing access to medicines. The government relied primarily on simple financial agreements for the managed entry of medicines for which there were perceived risks.
Marvin O. Bates
Full Text Available Purpose – The purpose of this article is to describe the marketing-oriented market entry approaches that businesses are currently using across the three levels of the world economic pyramid (i.e., WEP. These levels are the Top-tier, the Middle-tier, and the Base of the Pyramid-tier (i.e., BoP-tier. Methodology – The literature of the BoP was reviewed, and market entry approaches were itemized across the three WEP levels. Secondly, BoP strategic theorists including Prahalad identified the need for a BoP marketing focus replacing the traditional 4Ps marketing approach (i.e., Product, Price, Place and Promotion with the BoP-specific 4As marketing approach (i.e., Awareness, Affordability, Access and Availability. This 4As marketing approach is discussed. Findings – New marketing-oriented market-entry approaches are proposed for each of the three WEP levels. These approaches are based on where in the WEP the firm currently exists, and where in the WEP the firm desires to refocus market-entry activities; identified approaches include: inter-country expansion, intra-country entry, adjacent market entry, and extended market entry. Secondly, the absence of a clearly articulated marketing strategy for middle-tier markets was observed. Practical implications – This article has two specific applications. First, it summarizes the evolving market entry perspectives to provide a context for future market research in both emerging markets and the pre-emerging BoP markets. Second, the future requirement for an articulated marketing strategy for middle-tier markets is suggested. Originality – This article examined existing market entry approaches across all three levels of the WEP, inclusive of the BoP economic level. The language used to clarify market entry movements was extended, providing a specificity of description not previously found in either the existing market entry or BoP literature.
Firms, whichhave to compete with global firms in domestic market, try to internationalizefor gaining competitive advantage. The aim of this study is to recommend a conceptual framework to explain thedynamics of internationalization process of leader and follower firms thatinternationalize from an emerging country. In this study, author tries tofigure out the determinants of entrymode decisions of follower firms. In addition, bandwagon effect of leaders'entry mode strategies on followers&...
Ju Guizhi; Yan Fengqin; Fu Shibo; Shen Bo; Sun Shilong; Yang Ying; Li Pengwu
Objective: To investigate the effect of RNAi p21 gene on uncoupling of EL-4 cells induced by X-irradiation. Methods: Construction of RNAi p21 plasmid of pSileneer3.1-H1 neo-p21 was performed. Lipofectamine transfection assay was used to transfer the p21siBNA into EL-4 cells. Fluorescent staining and flow cytometry (FCM) analysis were employed for measurement of protein expression. Fluorescent staining of propidium iodide (PI) and FCM were used for measurement of potyploid cells. Results: In dose-effect experiment it was found that the expression of P21 protein of EL-4 cells increased significantly 24 h after X- irradiation with different doses compared with sham-inadiated control. In time course experiment it was found that the expression of P21 protein of EL-4 cells increased significantly at 8 h to 72 h after 4.0 Gy X-irradiation compared with sham-irradiated control. The results showed that the number of polyploid cells in EL-4 cells was not changed markedly after X-irradiation with doses of 0.5-6.0 Gy. After RNA interference with p21 gene, the expression of P21 protein of EL-4 cells decreased significantly 24 h and 48 h after 4.0 Gy X-irradiation in transfection of plasmid of pSilencer3.1-H1 neo-p21 compared with transfection of plasmid of pSilencer3.1-H1 nco control. And at the same time, the number of polyploid cells in EL-4 cells was increased significantly in transfection of plasmid of pSilencer3.1-H1 neo-p21 compared with transfection of plasmid of pSilencer3.1-H1 nco control. Conclusions: Uncoupling could be induced by X-irradiation in EL-4 cells following BNAi p21 gene, suggesting that P21 protein may play an important role in uncoupling induced by X-rays. (authors)
Harms, Gary A. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
The US Department of Energy (DOE) Nuclear Energy Research Initiative funded the design and construction of the Seven Percent Critical Experiment (7uPCX) at Sandia National Laboratories. The start-up of the experiment facility and the execution of the experiments described here were funded by the DOE Nuclear Criticality Safety Program. The 7uPCX is designed to investigate critical systems with fuel for light water reactors in the enrichment range above 5% 235U. The 7uPCX assembly is a water-moderated and -reflected array of aluminum-clad square-pitched U(6.90%)O2 fuel rods.
Full Text Available Vaccinia virus (VACV, a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis.
Stephanie J DeWitte-Orr
Full Text Available Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (dsRNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNbeta. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II(-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as 'carriers', facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.
... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Proposed accounting... § 33.5 Proposed accounting entries. If the applicant is required to maintain its books of account in... present proposed accounting entries showing the effect of the transaction with sufficient detail to...
... 36 Parks, Forests, and Public Property 2 2010-07-01 2010-07-01 false Single entry sales or permits... MINERALS Disposal of Mineral Materials General Provisions § 228.54 Single entry sales or permits. The... plan which describes operating procedures and reclamation measures, unless the requirement is waived by...
... CERTAIN ANIMALS, BIRDS, FISH, AND POULTRY, AND CERTAIN ANIMAL, BIRD, AND POULTRY PRODUCTS; REQUIREMENTS FOR MEANS OF CONVEYANCE AND SHIPPING CONTAINERS Poultry Mexico 8 § 93.220 Inspection at port of entry. (a) All poultry offered for entry from Mexico, including such poultry intended for movement through...
... CERTAIN ANIMALS, BIRDS, FISH, AND POULTRY, AND CERTAIN ANIMAL, BIRD, AND POULTRY PRODUCTS; REQUIREMENTS FOR MEANS OF CONVEYANCE AND SHIPPING CONTAINERS Ruminants Mexico 10 § 93.426 Inspection at port of entry. (a) All ruminants offered for entry from Mexico, including such ruminants intended for movement...
Degryse, H.A.; Havrylchyk, O.; Jurzyk, E.; Kozak, S.
We employ a unique data set containing bank-specific information to explore how foreign bank entry determines credit allocation in emerging markets. We investigate the impact of the mode of foreign entry (greenfield or takeover) on banks’ portfolio allocation to borrowers with different degrees of
... 19 Customs Duties 1 2010-04-01 2010-04-01 false Procedures for permitted entry. 12.99 Section 12.99 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY SPECIAL CLASSES OF MERCHANDISE Switchblade Knives § 12.99 Procedures for permitted entry...
Ferreira, F. A.; Ferreira, Fl.
We study the effects of entry of a foreign firm on domestic welfare in the presence of licensing, when the entrant is technologically inferior to the incumbent. We show that foreign entry increases domestic welfare for intermediate (respectively, sufficiently large) technological differences between the firms under licensing with fixed fee (respectively, output royalty).
... 27 Alcohol, Tobacco Products and Firearms 3 2010-04-01 2010-04-01 false Right of entry and... Administrative and Miscellaneous Provisions § 478.23 Right of entry and examination. (a) Except as provided in... of a criminal investigation of a person or persons other than the licensee, (2) For insuring...
... 19 Customs Duties 2 2010-04-01 2010-04-01 false Entry for consumption. 146.63 Section 146.63... TREASURY (CONTINUED) FOREIGN TRADE ZONES Transfer of Merchandise From a Zone § 146.63 Entry for consumption... status may be entered for consumption from a zone. (b) Zone-restricted merchandise. Merchandise in a zone...
Kraaij, W.; Westerveld, T.H.W.; Hiemstra, Djoerd
An important class of searches on the world-wide-web has the goal to find an entry page (homepage) of an organisation. Entry page search is quite different from Ad Hoc search. Indeed a plain Ad Hoc system performs disappointingly. We explored three non-content features of web pages: page length,
... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY GENERAL AGENT'S RESPONSIBILITY IN CONNECTION WITH FOREIGN REPAIR CUSTOM'S ENTRIES Sec. 2 Submission of repair entries. At the... with the District Director of Customs as defined in 19 CFR 1.1(d) an affidavit on Custom's Form 3417...
Francis M. F. Nunes
Full Text Available RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP-derived double-stranded RNA (dsRNA-GFP is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.
Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P
RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.
Peng, Jamy C.; Karpen, Gary H.
In order to identify regulators of nuclear organization, Drosophila mutants in the Su(var)3-9 histone H3K9 methyltransferase, RNAi pathway components, and other regulators of heterochromatin-mediated gene silencing were examined for altered nucleoli and positioning of repeated DNAs. Animals lacking components of the H3K9 methylation and RNAi pathways contained disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared to wild type. We also observed a substantial increase in extrachromosomal repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 (Lig4), and ecc DNA formation is not induced by removal of cohesin. We conclude that H3K9 methylation of rDNA and satellites, maintained by Su(var)3-9, HP1, and the RNAi pathway, is necessary for the structural stability of repeated DNAs, which is mediated through suppression of non-homologous end joining (NHEJ). These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.
Liu, Guiqing; Wu, Qiang; Li, Jianwei; Zhang, Guifen; Wan, Fanghao
The transformer (tra) gene appears to act as the genetic switch that promotes female development by interaction with the transformer2 (tra-2) gene in several dipteran species including the Medfly, housefly and Drosophila melanogaster. In this study, we describe the isolation, expression and function of tra and tra-2 in the economically important agricultural pest, the oriental fruit fly, Bactrocera dorsalis (Hendel). Bdtra and Bdtra-2 are similar to their homologs from other tephritid species. Bdtra demonstrated sex-specific transcripts: one transcript in females and two transcripts in males. In contrast, Bdtra-2 only had one transcript that was common to males and females, which was transcribed continuously in different adult tissues and developmental stages. Bdtra-2 and the female form of Bdtra were maternally inherited in eggs, whereas the male form of Bdtra was not detectable until embryos of 1 and 2 h after egg laying. Function analyses of Bdtra and Bdtra-2 indicated that both were indispensable for female development, as nearly 100% males were obtained with embryonic RNAi against either Bdtra or Bdtra-2. The fertility of these RNAi-generated males was subsequently tested. More than 80% of RNAi-generated males could mate and the mated females could lay eggs, but only 40-48.6% males gave rise to progeny. In XX-reversed males and intersex individuals, no clear female gonadal morphology was observed after dissection. These results shed light on the development of a genetic sexing system with male-only release for this agricultural pest.
Full Text Available The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD, which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi has been used successfully to silence endogenous insect (including honey bee genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania. To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.
Adams, M.E.; Trau, M.; Gilbert, R.C.; Napper, D.R.
Mechanistic understanding of the processes governing the kinetics of emulsion polymerization has both scientific and technical interest. One component of this process that is poorly understood at present is that of free radical entry into latex particles. Measurements were made of the entry rate coefficient as a function of temperature for free radicals entering polystyrene latex particles in seeded emulsion polymerizations initiated by γ-rays. The activation energy for entry was found to be less than 24 ± 3 kJ mol -1 , consistent with entry being controlled by a physical (e.g. diffusional) rather than a chemical process. Measurement of the entry rate coefficient as a function of the γ-ray dose rate suggested that the factors that determine the entry rate when the primary free radicals are uncharged are similar to those that determine the entry rate for charged free radicals derived from chemical initiation by peroxydisulfate. This result was consistent with measurements of the entry rate coefficient of charged free radicals derived from peroxydisulfate; these data were found to be virtually independent of both the extent of the latex surface coverage by the anionic surfactant sodium dodecyl sulfate and the ionic strength of the continuous phase. The data refute several proposals given in the literature for the rate-determining step for entry, being inconsistent with control by collision of free radicals with the latex particles, surfactant desorption, and an electrostatic barrier arising from the colloidal nature of the entering free radical. The origin of the activation energy for entry remains obscure
Gotesman, M; Soliman, H; Besch, R; El-Matbouli, M
Spring viraemia of carp virus (SVCV) is an aetiological agent of a serious disease affecting carp farms in Europe and is a member of the Rhabdoviridae family of viruses. The genome of SVCV codes for five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). RNA-mediated interference (RNAi) by small interfering RNAs (siRNAs) is a powerful tool to inhibit gene transcription and is used to study genes important for viral replication. In previous studies regarding another member of Rhabdoviridae, siRNA inhibition of the rabies virus nucleoprotein gene provided in vitro and in vivo protection against rabies. In this study, synthetic siRNA molecules were designed to target SVCV-N and SVCV-P transcripts to inhibit SVCV replication and were tested in an epithelioma papulosum cyprini (EPC) cell line. Inhibition of gene transcription was measured by real-time quantitative reverse-transcription PCR (RT-qPCR). The efficacy of using siRNA for inhibition of viral replication was analysed by RT-qPCR measurement of a reporter gene (glycoprotein) expression and by virus endpoint titration. Inhibition of nucleoprotein and phosphoprotein gene expression by siRNA reduced SVCV replication. However, use of tandem siRNAs that target phosphoprotein and nucleoprotein worked best at reducing SVCV replication. © 2014 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.
Gotesman, M; Soliman, H; Besch, R; El-Matbouli, M
Spring viraemia of carp virus (SVCV) is an aetiological agent of a serious disease affecting carp farms in Europe and is a member of the Rhabdoviridae family of viruses. The genome of SVCV codes for five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). RNA-mediated interference (RNAi) by small interfering RNAs (siRNAs) is a powerful tool to inhibit gene transcription and is used to study genes important for viral replication. In previous studies regarding another member of Rhabdoviridae, siRNA inhibition of the rabies virus nucleoprotein gene provided in vitro and in vivo protection against rabies. In this study, synthetic siRNA molecules were designed to target SVCV-N and SVCV-P transcripts to inhibit SVCV replication and were tested in an epithelioma papulosum cyprini (EPC) cell line. Inhibition of gene transcription was measured by real-time quantitative reverse-transcription PCR (RT-qPCR). The efficacy of using siRNA for inhibition of viral replication was analysed by RT-qPCR measurement of a reporter gene (glycoprotein) expression and by virus endpoint titration. Inhibition of nucleoprotein and phosphoprotein gene expression by siRNA reduced SVCV replication. However, use of tandem siRNAs that target phosphoprotein and nucleoprotein worked best at reducing SVCV replication. PMID:24460815
Ding, L.; Wu, J.P.; Xu, G.; Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W.
Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis
Gellatly, Kyle J; Yoon, Kyong Sup; Doherty, Jeffery J; Sun, Weilin; Pittendrigh, Barry R; Clark, J Marshall
4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance. Copyright © 2015 Elsevier Inc. All rights reserved.
Miguel A Saldaña
Full Text Available Zika virus (ZIKV, a flavivirus transmitted primarily by Aedes aegypti, has recently spread globally in an unprecedented fashion, yet we have a poor understanding of host-microbe interactions in this system. To gain insights into the interplay between ZIKV and the mosquito, we sequenced the small RNA profiles in ZIKV-infected and non-infected Ae. aegypti mosquitoes at 2, 7 and 14 days post-infection. ZIKA induced an RNAi response in the mosquito with virus-derived short interfering RNAs and PIWI-interacting RNAs dramatically increased in abundance post-infection. Further, we found 17 host microRNAs (miRNAs that were modulated by ZIKV infection at all time points. Strikingly, many of these regulated miRNAs have been reported to have their expression altered by dengue and West Nile viruses, while the response was divergent from that induced by the alphavirus Chikungunya virus in mosquitoes. This suggests that conserved miRNA responses occur within mosquitoes in response to flavivirus infection. This study expands our understanding of ZIKV-vector interactions and provides potential avenues to be further investigated to target ZIKV in the mosquito host.
Chen, ChengShi; Liu, Rong; Wang, JianHua; Yan, ZhiPing; Qian, Sheng; Zhang, Wei
The obstruction of hepatic arterial blood flow results in tumor tissue hypoxia and elevated expression of hypoxia-inducible factor-1alpha (HIF-1α). Our study evaluated whether lentivirus-mediated short interference RNA against HIF-1α inhibits proliferation, invasion, and migration of hepatocellular carcinoma (HCC) cells under hypoxia. RNA interference knockdown of HIF-1α was achieved by HIF-1α-directed lentiviral shRNA, in a rat HCC cell line cultured under hypoxia condition for varying length of times. The expression levels of HIF-1α and vascular endothelial growth factor were examined using reverse transcription polymerase chain reaction and western blot analyses. Cell proliferation, migration, and invasion were measured by cell viability, transwell migration, and invasion assays, respectively. Inhibition of HIF-1α expression by shRNA suppressed vascular endothelial growth factor mRNA and protein levels under both normoxia and hypoxia. It also suppressed cell migration and invasion, which were enhanced under hypoxic conditions. RNAi knockdown of HIF-1α further suppressed hypoxia-mediated inhibition of the cell proliferation. These data suggest that shRNA of HIF-1α could antagonize the hypoxia-mediated increase in hepatic cancer cell migration and invasion, and synergize with hypoxia to inhibit the cell proliferation in HCC cells.
Full Text Available Most of our knowledge about the regulation of aging comes from mutants originally isolated for other phenotypes. To ask whether our current view of aging has been affected by selection bias, and to deepen our understanding of known longevity pathways, we screened a genomic Caenorhabditis elegans RNAi library for clones that extend lifespan. We identified 23 new longevity genes affecting signal transduction, the stress response, gene expression, and metabolism and assigned these genes to specific longevity pathways. Our most important findings are (i that dietary restriction extends C. elegans' lifespan by down-regulating expression of key genes, including a gene required for methylation of many macromolecules, (ii that integrin signaling is likely to play a general, evolutionarily conserved role in lifespan regulation, and (iii that specific lipophilic hormones may influence lifespan in a DAF-16/FOXO-dependent fashion. Surprisingly, of the new genes that have conserved sequence domains, only one could not be associated with a known longevity pathway. Thus, our current view of the genetics of aging has probably not been distorted substantially by selection bias.
Li, Hang; Jiang, Weihua; Zhang, Zan; Xing, Yanru; Li, Fei
The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1(st) to 5(th) instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4(th) instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20-94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (Ppest control.
Jeremy L Marshall
Full Text Available Postmating, prezygotic phenotypes, especially those that underlie reproductive isolation between closely related species, have been a central focus of evolutionary biologists over the past two decades. Such phenotypes are thought to evolve rapidly and be nearly ubiquitous among sexually reproducing eukaryotes where females mate with multiple partners. Because these phenotypes represent interplay between the male ejaculate and female reproductive tract, they are fertile ground for reproductive senescence--as ejaculate composition and female physiology typically change over an individual's life span. Although these phenotypes and their resulting dynamics are important, we have little understanding of the proteins that mediate these phenotypes, particularly for species groups where postmating, prezygotic traits are the primary mechanism of reproductive isolation. Here, we utilize proteomics, RNAi, mating experiments, and the Allonemobius socius complex of crickets, whose members are primarily isolated from one another by postmating, prezygotic phenotypes (including the ability of a male to induce a female to lay eggs, to demonstrate that one of the most abundant ejaculate proteins (a male accessory gland-biased protein similar to a trypsin-like serine protease decreases in abundance over a male's reproductive lifetime and mediates the induction of egg-laying in females. These findings represent one of the first studies to identify a protein that plays a role in mediating both a postmating, prezygotic isolation pathway and reproductive senescence.
Kanda, Atsuhiro; Ishizuka, Erdal Tan; Shibata, Atsushi; Matsumoto, Takahiro; Toyofuku, Hidekazu; Noda, Kousuke; Namba, Kenichi; Ishida, Susumu
The receptor-associated prorenin system (RAPS) refers to the pathogenic mechanism whereby prorenin binding to the (pro)renin receptor [(P)RR] dually activates the tissue renin-angiotensin system (RAS) and RAS-independent intracellular signaling. Here we revealed significant upregulation of prorenin and soluble (P)RR levels in the vitreous fluid of patients with uveitis compared to non-inflammatory controls, together with a positive correlation between these RAPS components and monocyte chemotactic protein-1 among several upregulated cytokines. Moreover, we developed a novel single-strand RNAi agent, proline-modified short hairpin RNA directed against human and mouse (P)RR [(P)RR-PshRNA], and we determined its safety and efficacy in vitro and in vivo. Application of (P)RR-PshRNA in mice caused significant amelioration of acute (uveitic) and chronic (diabetic) models of ocular inflammation with no apparent adverse effects. Our findings demonstrate the significant implication of RAPS in the pathogenesis of human uveitis and the potential usefulness of (P)RR-PshRNA as a therapeutic agent to reduce ocular inflammation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Ding, L.; Wu, J.P. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China); Xu, G. [Fudan University, Jinshan Hospital, Center Laboratory, Shanghai, China, Center Laboratory, Jinshan Hospital, Fudan University, Shanghai (China); Zhu, B.; Zeng, Q.M.; Li, D.F.; Lu, W. [Fudan University, Jinshan Hospital, Department of Orthopaedics, Shanghai, China, Department of Orthopaedics, Jinshan Hospital, Fudan University, Shanghai (China)
Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.
Full Text Available Most of our knowledge about the regulation of aging comes from mutants originally isolated for other phenotypes. To ask whether our current view of aging has been affected by selection bias, and to deepen our understanding of known longevity pathways, we screened a genomic Caenorhabditis elegans RNAi library for clones that extend lifespan. We identified 23 new longevity genes affecting signal transduction, the stress response, gene expression, and metabolism and assigned these genes to specific longevity pathways. Our most important findings are (i that dietary restriction extends C. elegans' lifespan by down-regulating expression of key genes, including a gene required for methylation of many macromolecules, (ii that integrin signaling is likely to play a general, evolutionarily conserved role in lifespan regulation, and (iii that specific lipophilic hormones may influence lifespan in a DAF-16/FOXO-dependent fashion. Surprisingly, of the new genes that have conserved sequence domains, only one could not be associated with a known longevity pathway. Thus, our current view of the genetics of aging has probably not been distorted substantially by selection bias.
Full Text Available Current studies find that degenerated cartilage endplates (CEP of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.
tenKate, LP; Verheij, JBGM; Wildhagen, MF; Hilderink, HBM; Kooij, L; Verzijl, JG; Habbema, JDF
Both single-entry two-step (SETS) couple screening and double-entry two-step (DETS) couple screening have been recommended as methods to screen for cystic fibrosis gene carriers. In this paper we compare the expected results from both types of screening. In general, DETS results in a higher
Peter J. Hume
Full Text Available Salmonella causes disease in humans and animals ranging from mild self-limiting gastroenteritis to potentially life-threatening typhoid fever. Salmonellosis remains a considerable cause of morbidity and mortality globally, and hence imposes a huge socio-economic burden worldwide. A key property of all pathogenic Salmonella strains is the ability to invade non-phagocytic host cells. The major determinant of this invasiveness is a Type 3 Secretion System (T3SS, a molecular syringe that injects virulence effector proteins directly into target host cells. These effectors cooperatively manipulate multiple host cell signaling pathways to drive pathogen internalization. Salmonella does not only rely on these injected effectors, but also uses several other T3SS-independent mechanisms to gain entry into host cells. This review summarizes our current understanding of the methods used by Salmonella for cell invasion, with a focus on the host signaling networks that must be coordinately exploited for the pathogen to achieve its goal.
Full Text Available To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1 circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl-3-trimethylammonium chitosan chloride (HTCC, and its hydrophobically-modified derivative (HM-HTCC as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses.
Vihinen-Ranta, Maija; Kalela, Anne; Mäkinen, Päivi; Kakkola, Laura; Marjomäki, Varpu; Vuento, Matti
The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18°C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membrane fusion steps in the entry process. Microinjection experiments showed that CPV particles which were injected directly into the cytoplasm, thus avoiding the endocytic pathway, were unable to initiate progeny virus production. CPV treated at pH 5.0 prior to microinjection was unable to initiate virus production, showing that factors of the endocytic route other than low pH are necessary for the initiation of infection by CPV. PMID:9420290
MSc. Florida Veljanoska
Full Text Available This paper investigates the effects of foreign bank entry in emerging markets. We developed a picture of a multinational bank in an emerging markets by combining statistics from several sources, in order to explore broad range of effects that brings foreign bank entry in the developing countries. Some impacts of foreign bank entry have been thoroughly studied, while others are hardly mention. Entry of foreign bank brings large benefits to host country’s financial system and economies at large. This paper is studying those benefits very carefully, by analyzing the impact of foreign bank entry on economy, government, monetary policy, large enterprises, small and medium size enterprises, domestic bank etc. But, we also consider the fact that at the same time, foreign investment in the financial sector, rises some concerns, and therefore we analyze the negative effects as well. At the end we must admit that although there are some negative consequences from foreign bank entry in emerging markets, the benefits that arise from foreign banks penetration are much more, and this trend of foreign bank entry has brought new positive economic impulse in developing world.
Viswanathan, Gopinath; Jafurulla, Md; Kumar, G Aditya; Raghunand, Tirumalai R; Chattopadhyay, Amitabha
Mycobacteria are intracellular pathogens that can invade and survive within host macrophages. Mycobacterial infections remain a major cause of mortality and morbidity worldwide, with serious concerns of emergence of multi and extensively drug-resistant tuberculosis. While significant advances have been made in identifying mycobacterial virulence determinants, the detailed molecular mechanism of internalization of mycobacteria into host cells remains poorly understood. Although several studies have highlighted the crucial role of sphingolipids in mycobacterial growth, persistence and establishment of infection, the role of sphingolipids in the entry of mycobacteria into host cells is not known. In this work, we explored the role of host membrane sphingolipids in the entry of Mycobacterium smegmatis into J774A.1 macrophages. Our results show that metabolic depletion of sphingolipids in host macrophages results in a significant reduction in the entry of M. smegmatis. Importantly, the entry of Escherichia coli into host macrophages under similar conditions remained invariant, implying the specificity of the requirement of sphingolipids in mycobacterial entry. To the best of our knowledge, our results constitute the first report demonstrating the role of host macrophage sphingolipids in the entry of mycobacteria. Our results could help in the development of novel therapeutic strategies targeting sphingolipid-mediated entry of mycobacteria into host cells. Copyright © 2018 Elsevier B.V. All rights reserved.
Tooley, Jeffrey; Desai, Prasun N.; Lynos, Daniel T.; Hirst, Edward A.; Wahl, Tom E.; Wawrzyniak, Georffery G.
Stardust is a comet sample return mission that successfully returned to Earth on January 15, 2006. Stardust's targeted landing area was the Utah Test and Training Range in the Northwest corner of Utah. Requirements for the risks associated with landing were levied on Stardust by the Utah Test and Training Range and NASA. This paper describes the analysis to verify that these requirements were met and and includes calculation of debris survivability, generation of landing site selection plots, and identification of keep-out zones, as well as appropriate selection of the landing site. Operationally the risk requirements were all met for both of the GOMO-GO polls, so entry was authorized.
Pichkhadze, K.; Vorontsov, V.; Polyakov, A.; Ivankov, A.; Taalas, P.; Pellinen, R.; Harri, A.-M.; Linkin, V.
Martian meteorological lander (MML) is intended for landing on the Martian surface in order to monitor the atmosphere at landing point for one Martian year. MMLs shall become the basic elements of a global network of meteorological mini-landers, observing the dynamics of changes of the atmospheric parameters on the Red Planet. The MML main scientific tasks are as follows: (1) Study of vertical structure of the Martian atmosphere throughout the MML descent; (2) On-surface meteorological observations for one Martian year. One of the essential factors influencing the lander's design is its entry, descent, and landing (EDL) sequence. During Phase A of the MML development, five different options for the lander's design were carefully analyzed. All of these options ensure the accomplishment of the above-mentioned scientific tasks with high effectiveness. CONCEPT A (conventional approach): Two lander options (with a parachute system + airbag and an inflatable airbrake + airbag) were analyzed. They are similar in terms of fulfilling braking phases and completely analogous in landing by means of airbags. CONCEPT B (innovative approach): Three lander options were analyzed. The distinguishing feature is the presence of inflatable braking units (IBU) in their configurations. SELECTED OPTION (innovative approach): Incorporating a unique design approach and modern technologies, the selected option of the lander represents a combination of the options analyzed in the framework of Concept B study. Currently, the selected lander option undergoes systems testing (Phase D1). Several MMLs can be delivered to Mars in frameworks of various missions as primary or piggybacking payload: (1) USA-led "Mars Scout" (2007); (2) France-led "NetLander" (2007/2009); (3) Russia-led "Mars-Deimos-Phobos sample return" (2007); (4) Independent mission (currently under preliminary study); etc.
Full Text Available In this study, we examine how initial public offerings (IPO entry rates are affected when stock markets are boundedly rational and IPO firms infer information from their counterparts in the market. We hypothesize a curvilinear relationship between the number of comparable stocks and initial public offerings (IPO entry rates into the NASDAQ Stock Exchange. Furthermore, we argue that trading volume and changes in stock returns partially mediates the relationship between the number of comparable stocks and IPO entry rates. The statistical evidence provides strong support for the hypotheses.
Full Text Available Japanese Foreign Direct Investment (FDI in the European Union and its performance were analysed in this work. Three different FDI or entry modes used by Japanese companies to enter the European market were compared, and the presence of a relationship between the selected entry mode and the performance of the subsidiary was investigated. We found that more than half of the Japanese investments in Europe took the form of new ventures, approximately 40% were joint ventures and less than 6% were acquisitions. We found that no specific entry mode performed better than another.
We discussed a second entry port for the Accelerator Test Facility (ATF) injection system at Brookhaven National Laboratory, which consists of a photocathode rf gun and a straight - ahead beamline directly into the 50 MeV linac. The proposed second entry port should improve the beam quality and lower the emittance needed for FEL (Free Electron Laser), and laser - acceleration experiments. A discussion on the laser driven high brightness photoelectrons through the primary entry port (a low energy 180 degrees achromatic double bend transport line) now in operation, and a beam analysis for the proposed secondary port is also given
Entry, Descent and Landing (EDL) Modeling and Simulation (MS) is an enabling capability for complex NASA entry missions such as MSL and Orion. MS is used in every mission phase to define mission concepts, select appropriate architectures, design EDL systems, quantify margin and risk, ensure correct system operation, and analyze data returned from the entry. In an environment where it is impossible to fully test EDL concepts on the ground prior to use, accurate MS capability is required to extrapolate ground test results to expected flight performance.
A new research effort at NASA Ames Research Center has been initiated in Planetary Defense, which integrates the disciplines of planetary science, atmospheric entry physics, and physics-based risk assessment. This paper describes work within the new program and is focused on meteor entry and breakup.Over the last six decades significant effort was expended in the US and in Europe to understand meteor entry including ablation, fragmentation and airburst (if any) for various types of meteors ranging from stony to iron spectral types. These efforts have produced primarily empirical mathematical models based on observations. Weaknesses of these models, apart from their empiricism, are reliance on idealized shapes (spheres, cylinders, etc.) and simplified models for thermal response of meteoritic materials to aerodynamic and radiative heating. Furthermore, the fragmentation and energy release of meteors (airburst) is poorly understood.On the other hand, flight of human-made atmospheric entry capsules is well understood. The capsules and their requisite heatshields are designed and margined to survive entry. However, the highest speed Earth entry for capsules is 13 kms (Stardust). Furthermore, Earth entry capsules have never exceeded diameters of 5 m, nor have their peak aerothermal environments exceeded 0.3 atm and 1 kW/sq cm. The aims of the current work are: (i) to define the aerothermal environments for objects with entry velocities from 13 to 20 kms; (ii) to explore various hypotheses of fragmentation and airburst of stony meteors in the near term; (iii) to explore the possibility of performing relevant ground-based tests to verify candidate hypotheses; and (iv) to quantify the energy released in airbursts. The results of the new simulations will be used to anchor said risk assessment analyses. With these aims in mind, state-of-the-art entry capsule design tools are being extended for meteor entries. We describe: (i) applications of current simulation tools to
Claudinéia Pereira Costa
Full Text Available Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk, thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs β bursicon genes prevented the complete formation and tanning (melanization/sclerotization of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs β and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs β and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.
Mehta, Adi; Shervington, Amal; Howl, John; Jones, Sarah; Shervington, Leroy
Heat shock protein 90 promotes tumor progression and survival and has emerged as a vital therapeutic target. Previously we reported that the combinatorial treatment of 17AAG/sihsp90α significantly downregulated Hsp90α mRNA and protein levels in Glioblastoma Multiforme (GBM). Here we investigated the ability of cell penetrating peptide (Tat48-60 CPP)-mediated siRNA-induced hsp90α knockdown as a single agent and in combination with 17-allylamino-17-demethoxygeldanamycin (17-AAG) to induce tumor growth inhibition in GBM and whether it possessed therapeutic implications. GBM and non-tumorigenic cells exposed to siRNA and/or 17-AAG were subsequently assessed by qRT-PCR, immunofluorescence, FACS analysis, quantitative Akt, LDH leakage and cell viability assays. PAGE was performed for serum stability assessment. A combination of siRNA/17-AAG treatment significantly induced Hsp90α gene and protein knockdown by 95% and 98%, respectively, concomitant to 84% Akt kinase activity attenuation, induced cell cycle arrest and tumor-specific cytotoxicity by 88%. Efficient complex formation between CPP and siRNA exhibited improved serum stability of the siRNA with minimal intrinsic toxicity in vitro. The preliminary in vivo results showed that combination therapy induced hsp90α knockdown and attenuated Akt kinase activity in intracranial glioblastoma mouse models. The results imply that RNAi-mediated hsp90α knockdown increases 17-AAG treatment efficacy in GBM. In addition, the cytotoxic response observed was the consequence of downregulation of hsp90α gene expression, reduced Akt kinase activity and S-G2/M cell cycle arrest. These results are novel and highlight the ability of Tat to efficiently deliver siRNA in GBM and suggest that the dual inhibition of Hsp90 has therapeutic potentials.
Sugahara, Ryohei; Tanaka, Seiji; Shiotsuki, Takahiro
The Halloween gene SPOOK (SPO) is involved in the production of the active metabolite of ecdysteroid, 20-hydroxyecdysone (20E), in insects. A previous study showed that RNAi-mediated knockdown of SPO in Schistocerca gregaria last instar nymphs markedly reduced the hemolymph 20E titer, but did not affect metamorphosis. In the present study, the effects of SPO interference on development were re-examined in this locust. Injections of SPO double-stranded RNA (dsSPO) into nymphs at mid and late instars significantly delayed nymphal development and interfered with molting. The 20E levels of dsSPO-treated nymphs were generally low, with a delayed, small peak, suggesting that disturbance of the 20E levels caused the above developmental abnormalities. A small proportion of the dsSPO-injected nymphs metamorphosed precociously, producing adults and adultoids. Precocious adults were characterized by small body size, short wings with abbreviated venation, and normal reproductive activity. Fourth instar nymphs that precociously metamorphosed at the following instar exhibited temporal expression patterns of ecdysone-induced protein 93F and the juvenile hormone (JH) early-inducible gene Krüppel homolog 1 similar to those observed at the last instar in normal nymphs. Adultoids displayed mating behavior and adultoid females developed eggs, but never laid eggs. JH injection around the expected time of the 20E peak in the dsSPO-injected nymphs completely inhibited the appearance of adultoids, suggesting that appearance of adultoids might be due to a reduced titer of JH rather than of 20E. These results suggest that SPO plays an important role in controlling morphogenesis, metamorphosis, and reproduction in S. gregaria. Copyright © 2017 Elsevier Inc. All rights reserved.
National Aeronautics and Space Administration — The innovative adpative deployable entry and placement technology (ADEPT), also known as transformable entry system technology (TEST) concept, akin to an umbrella,...
Dillman, Robert; Corliss, James
NASA's Mars Sample Return (MSR) project will bring Mars surface and atmosphere samples back to Earth for detailed examination. Langley Research Center's MSR Earth Entry Vehicle (EEV) is a core part of the mission, protecting the sample container during atmospheric entry, descent, and landing. Planetary protection requirements demand a higher reliability from the EEV than for any previous planetary entry vehicle. An overview of the EEV design and preliminary analysis is presented, with a follow-on discussion of recommended future design trade studies to be performed over the next several years in support of an MSR launch in 2018 or 2020. Planned topics include vehicle size for impact protection of a range of sample container sizes, outer mold line changes to achieve surface sterilization during re-entry, micrometeoroid protection, aerodynamic stability, thermal protection, and structural materials selection.
... AIR COMMERCE REGULATIONS Private Aircraft § 122.26 Entry and clearance. Private aircraft, as defined... information as set forth in § 122.22(c), and grants electronic clearance via electronic mail or telephone...
Gold, David L.
Reviews issues to be considered in determining the order of meanings for a lexeme in a dictionary entry and compares techniques for deciding order. Types of ordering include importance, frequency, logical ordering, dominant meaning, syntactic, and historical. (MSE)
Sklarin, Nancy T.; Granovsky, Svetlana; O'Reilly, Eileen M.; Zelenetz, Andrew D.
Implementation of computerized provider order entry for complex chemotherapy regimens supported Memorial Sloan-Kettering Cancer Center's strategic plan to successfully establish a distributive, networked health care delivery system.
... conditions as he may prescribe to assure that allowing the entry of such articles will be consistent with the... inventory controls are maintained at all times. Inedible egg products may be processed in official plants...
Gerard de Valence
Full Text Available This research looks at the significance of barriers that firms considering entry into the construction industry might face. Drawing on the microeconomic characteristics of imperfectly competitive and oligopolistic markets the analysis finds that there are a dozen barriers to entry that affect the industry, but their significance depends on the product type. The discussion covers the question of product homogeneity in construction and evidence for the existence of barriers to entry in concentration levels. Barriers to entry specific to construction are then identified, which leads to an analysis of how they operate and their significance (high, medium or low in different market types, thus increasing our understanding of construction industry dynamics.
The increased demand for freight movements through international ports of entry and the signing of the North American Free Trade Agreement (NAFTA) have increased freight traffic at border ports of entry. The State-of-the-Art Port of Entry Workshop initiated a dialogue among technologists and stakeholders to explore the potential uses of technology at border crossings and to set development priorities. International ports of entry are both information and labor intensive, and there are many promising technologies that could be used to provide timely information and optimize inspection resources. Participants universally held that integration of technologies and operations is critical to improving port services. A series of Next Steps was developed to address stakeholder issues and national priorities, such as the National Transportation Policy and National Drug Policy. This report documents the views of the various stakeholders and technologists present at the workshop and outlines future directions of study.
Apr 4, 2018 ... ... made in superior and inferior entry points at supine position at 0° .... Trendelenburg degree, taking all risks into consideration. As a result of our .... patient's position influences the incidence of dysrhythmias during pulmonary.
... of consignee—(1) Authorized agent with knowledge of the facts. When entry is made in a consignee's name by an agent who has knowledge of the facts and who is authorized under a proper power of attorney...
Borrow, P; Oldstone, M B
The path that the arenavirus lymphocytic choriomeningitis virus (LCMV) uses to enter rodent fibroblastic cell lines was dissected by infectivity and inhibition studies and immunoelectron microscopy. Lysosomotropic weak bases (chloroquine and ammonium chloride) and carboxylic ionophores (monensin and nigericin) inhibited virus entry, assessed as virus nucleoprotein expression at early times post-infection, indicating that the entry process involved a pH-dependent fusion step in intracellular vesicles. That entry occurred in vesicles rather than by direct fusion of virions with the plasma membrane was confirmed by immunoelectron microscopy. The vesicles involved were large (150-300 nm diameter), smooth-walled, and not associated with clathrin. Unlike classical phagocytosis, virus uptake in these vesicles was a microfilament-independent process, as it was not blocked by cytochalasins. LCMV entry into rodent fibroblast cell lines thus involves viropexis in large smooth-walled vesicles, followed by a pH-dependent fusion event inside the cell.
Heilimo, J.; Harri, A.-M.; Aleksashkin, S.; Koryanov, V.; Guerrero, H.; Schmidt, W.; Haukka, H.; Finchenko, V.; Martynov, M.; Ostresko, B.; Ponomarenko, A.; Kazakovtsev, V.; Arruego, I.; Martin, S.; Siili, T.
In 2001 - 2011 an inflatable Entry, Descent and Landing System (EDLS) for Martian atmosphere was developed by FMI and the MetNet team. This MetNet Mars Lander EDLS is used in both the initial deceleration during atmospheric entry and in the final deceleration before the semi-hard impact of the penetrator to Martian surface. The EDLS design is ingenious and its applicability to Earth's atmosphere is studied in the on-going project. In particular, the behavior of the system in the critical transonic aerodynamic (from hypersonic to subsonic) regime will be investigated. This project targets to analyze and test the transonic behavior of this compact and light weight payload entry system to Earth's atmosphere . Scaling and adaptation for terrestrial atmospheric conditions, instead of a completely new design, is a favorable approach for providing a new re-entry vehicle for terrestrial space applications.
A selected list of references in the fields of astronautics and re-entry mechanics is classified and discussed, and a comprehensive subject and author index is included for ease in locating the references. (Author)
.... Airline deregulation has led to lower fares and better service for most air travelers largely because of increased competition spurred by the entry of new airlines into the industry and established...
...) of the Intelligence Reform and Terrorism Prevention Act of 2004 (IRTPA), 118 Stat. 3638, as amended... major airport authority printed its own signs for Global Entry and strategically placed them throughout...
Bastida Virgili, B.; Krag, H.; Lips, T.; De Pasquale, E.
The first ATV was launched on 9th March 2008 and, after a successful mission, the last phase was a controlled destructive re-entry on 29th September 2008, shortly after 13:30 UTC, in which the remains of the ATV and its load fell into the South Pacific Ocean. In order to better understand the re-entry processes, an insitu optical observation campaign was launched to record and analyze the ATV controlled re-entry with several instruments on board of two airplanes and also from the ISS. This observation campaign was successful and triggered several different still-ongoing studies on the extraction and analysis of data to draw conclusions on the adequacy of the re-entry break-up and explosion models used for the safety analysis of the ATV re-entry. This paper addresses the validation process for ESA’s model for re-entry survivability and on-ground risk assessment for explosive re-entry events using the observation data. The underlying rationale is to improve the models for the benefit of planning and execution of future controlled re-entries and in risk calculation in case of uncontrolled ones. The re-entry trajectory of the ATV, the explosive event and the trajectories of the fragments are simulated with the existing ESA tools and the EVOLVE explosion model. Additional software has been developed to simulate airborne sensor field of view(FOV) crossings based on the aircraft trajectories, attitude profile, sensor mounts and FOVs. Sensor performance and object radiation are modeled in order to generate synthetic images for the different sensors in the ISS and the two airplanes. These synthetic images and synthetic videos are compared with the available reentry observations of the ATV. This paper will present the software and techniques to generate synthetic imagery. It will give results of the comparison between the simulated and the real trajectories and fragmentation and explain the subsequent validation process of the ESA re-entry tools and the potential
The subject of presented bachelor thesis is Franchising as a method of entry to the market. The first part of this thesis deals with characteristics of franchising such as the definition of franchising and its advantages and disadvantages for both parties involved. Then there is also provided the current situation of franchising in the Czech Republic. The second part deals with entry into the market by franchising. In this section franchise agreement is mainly mentioned, how to start a franch...
Singh, Mamta K.
Although the educational evaluation process is useful and valuable and is supported by the Higher Education Act, a strong research base for program evaluation of college entry-level science courses is still lacking. Studies in science disciplines such as, biology, chemistry, and physics have addressed various affective and demographic factors and their relationships to student achievement. However, the literature contains little information that specifically addresses student biology content knowledge skills (basics and higher order thinking skills) and identifies factors that affect students' success in entry-level college science courses. These gate-keeping courses require detailed evaluation if the goal of an institution is to increase students' performance and success in these courses. These factors are, in fact, a stepping stone for increasing the number of graduates in Science, Technology, Engineering, and Mathematics (STEM) majors. The present study measured students' biology content knowledge and investigated students' performance and success in college biology, chemistry, and physics entry-level courses. Seven variables---gender, ethnicity, high school Grade Point Average (GPA), high school science, college major, school financial aid support, and work hours were used as independent variables and course final performance as a dichotomous dependent variable. The sample comprised voluntary student participants in entry-level science courses. The study attempted to explore eight research questions. Content knowledge assessments, demographic information analysis, multiple regression analysis, and binary logistic regression analysis were used to address research questions. The results suggested that high school GPA was a consistently good predictor of students' performance and success in entry-level science courses. Additionally, high school chemistry was a significant predictor variable for student success in entry-level biology and chemistry courses
Ferri, F.; Lewis, S. R.; Withers, P.; Aboudan, A.; Bettanini, C.; Colombatti, G.; Debei, S.; Golombek, M.; Harri, A. M.; Komatsu, G.; Leese, M. R.; Mäkinen, T.; Müller-Wodarg, I.; Ori, G. G.; Patel, M. R.
The entry, descent and landing of ExoMars offer a rare (once-per-mission) opportunity to perform in situ investigation of the martian environment over a wide altitude range. Entry, Descent and Landing System (EDLS) measurements can provide essential data for atmospheric scientific investigations.\\ud \\ud We intend to perform atmospheric science measurements by exploiting data from EDLS engineering sensors and exploiting their readings beyond the expected engineering information.
Örten, Remzi; Kurt, Ganite; Torun, Salih
It is known that accounting is applied since the existence of human being. When historical development of accounting is examined, recording methods may be summarized as single sided and double sided. The first method used in accounting is the single entry bookkeeping. According to this entry, not all of information related to financial events but important part of them are recorded in a single way. We can specify follow-up of receivable, payable, incomes and expenses as single sided recording...
The purpose of this thesis was to carry out a market entry planning by franchising Kungfu catering in Finland. Kungfu catering was a typical Chinese fast food restaurant, of which there was a gap in Finnish fast food market. McDonald’s and Hesburger were succesful examples of franchise business, experiences of those 2 restaurants were used for reference to create a suitable franchise strategy for Kungfu Finland. Finnish market entry planning of Kungfu Finland has been explained from several a...
Multinationals’ mode of foreign expansion may depend on whether or not they expect technological externalities or spillovers to generate new competition. Existing models where ex-post spillovers affect the ex-ante entry choice usually study the choice between exporting and FDI with horizontal spillovers. I consider a monopoly firm with a vertical production structure that has four possible modes of entry, one of which includes outsourcing of intermediate input production to a h...
Ajay Kalra; Surendra Rajiv; Kannan Srinivasan
Empirical studies examining responses to new product entries come to the puzzling conclusion that, in general, an incumbent reacts to a new entrant after a significant delay. Even easy-to-implement price cuts are observed after significant lag following entry. These findings seem to contradict the existing literature that either implicitly assumes or strongly advocates immediate defensive responses to limit competitive encroachment. When a competing firm enters the market, consumers may be un...
Features of an entry control system designed to automatically control access to nuclear facilities is described. Control independent of variable human factors is stressed, but security force action is required for assessment and response as a result of an alarm. A design based on a distributed processing capability is utilized. Flexibility and generality are emphasized in an effort to maximize applicability to the entry-control problem faced by nuclear facilities upgrading security as a result of the Safeguards Program
This paper investigates the gender wage gap in entry wages and in the early career for German skilled workers in the period 1975-1990. We use a new administrative longitudinal data source that allows to observe complete work and skill accumulation histories from the beginning for up to 13 years in the labour market. Descriptives show an entry wage differential of 22 percent between male and female full-time workers. Furthermore, the differential stays almost constant throughout the first 8 ei...
Jessica Camara Siqueira
Full Text Available Language dictionaries present the meaning of an entry in a way not restricted to linguistic information, since we also have interdisciplinary and contextual aspects that dialogue with socio-cultural issues in which the entry is inserted. Considering the potential character of the study of contextual meanings, the “death” entry was chosen to carry out a comparative analysis among language dictionaries. The choice was motivated by the recurrence of this entry in ancient and contemporary dictionaries, and the fact of its having a concept that allows various historical, social and ideological discussions. The goal is to reveal the diﬀerent nuances of the “death” entry in language dictionaries, observing four main aspects: etymology, diachrony, synonymic construction and meaning of “death” in school dictionaries. Based on lexicographical studies of classical authors, we did a comparative analysis of meanings of “death” in diﬀerent types of language dictionaries. We found that in each group dictionary we may ﬁnd traces of ideological choices in the construction of the contextual meaning of the “death” entry.
Full Text Available This paper analyzes the effects of investment in advertising in the three-stage entry game model with one incumbent and one potential entrant firm. It is shown that if a game theory is applied, under particular conditions, advertising can be used as a strategic weapon in the market entry game. Depending on the level of the advertising interaction factor, conditions for over-investment in advertising for strategic purposes are given. Furthermore, three specific cases are analyzed: strictly predatory advertising, informative advertising and the case when one firm’s advertising cannot directly influence the other firm's profit. For each of them, depending on the costs of advertising and marginal costs, equilibrium is determined, and conditions under which it is possible to deter the entry are given. It is shown that if the value of the advertising interaction factor increases, power of using advertising as a weapon to deter entry into the market decreases. Thus, in the case of informative advertising, advertising cannot be used as a tool for deterring entry into the market, while in the case of predatory advertising, it can. Also, we have proved that in the case of strictly informative advertising an over-investment never occurs, while in the two other cases, there is always over-investment either to deter or to accommodate the entry.
Full Text Available The entry of a multinational into a cluster, a geographic agglomeration in a given place or region of predominantly small and medium enterprises specialized in a given industry or related industries, impacts the incumbent in the cluster. Aiming to identify the main effects of a multinational entry on the firms’ strategy in a cluster, it was analyzed the entry of IKEA, a Swedish multinational, into the cluster of furniture production in Paços de Ferreira and Paredes, in Portugal. In this study, the data collection technique to access primary data was a survey. The sample has small enterprises, which is similar to the structure of firms in the studied cluster. Results show that more than half the sample thinks that the entry of the multinational had not affected them. However, the firms that acknowledge a significant impact, assess that impact as negative. The competitiveness factors that have improved more significantly after IKEA’s entry were new product development and exporting strategies. The main responses of incumbent firms to the multinational entry were internationalization and the development of generic strategies of differentiation and focus based on differentiation.
Full Text Available Herpes Simplex virus (HSV is associated with a variety of diseases such as genital herpes and numerous ocular diseases. At the global level, high prevalence of individuals who are seropositive for HSV, combined with its inconspicuous infection, remains a cause for major concern. At the molecular level, HSV entry into a host cell involves multiple steps, primarily the interaction of viral glycoproteins with various cell surface receptors, many of which have alternate substitutes. The molecular complexity of the virus to enter a cell is also enhanced by the existence of different modes of viral entry. The availability of many entry receptors, along with a variety of entry mechanisms, has resulted in a virus that is capable of infecting virtually all cell types. While HSV uses a wide repertoire of viral and host factors in establishing infection, current therapeutics aimed against the virus are not as diversified. In this particular review, we will focus on the initial entry of the virus into the cell, while highlighting potential novel therapeutics that can control this process. Virus entry is a decisive step and effective therapeutics can translate to less virus replication, reduced cell death, and detrimental symptoms.
The NASA Langley Research Center has been performing studies to address the feasibility of various single-stage to orbit concepts for use by NASA and the commercial launch industry to provide a lower cost access to space. Some work on the conceptual design of a typical lifting body concept vehicle, designated VentureStar(sup TM) has been conducted in cooperation with the Lockheed Martin Skunk Works. This paper will address the results of a preliminary flight controls assessment of this vehicle concept during the atmospheric entry phase of flight. The work includes control analysis from hypersonic flight at the atmospheric entry through supersonic speeds to final approach and landing at subsonic conditions. The requirements of the flight control effectors are determined over the full range of entry vehicle Mach number conditions. The analysis was performed for a typical maximum crossrange entry trajectory utilizing angle of attack to limit entry heating and providing for energy management, and bank angle to modulation of the lift vector to provide downrange and crossrange capability to fly the vehicle to a specified landing site. Sensitivity of the vehicle open and closed loop characteristics to CG location, control surface mixing strategy and wind gusts are included in the results. An alternative control surface mixing strategy utilizing a reverse aileron technique demonstrated a significant reduction in RCS torque and fuel required to perform bank maneuvers during entry. The results of the control analysis revealed challenges for an early vehicle configuration in the areas of hypersonic pitch trim and subsonic longitudinal controllability.
Full Text Available Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.
Full Text Available Systematic, genome-wide RNA interference (RNAi analysis is a powerful approach to identify gene functions that support or modulate selected biological processes. An emerging challenge shared with some other genome-wide approaches is that independent RNAi studies often show limited agreement in their lists of implicated genes. To better understand this, we analyzed four genome-wide RNAi studies that identified host genes involved in influenza virus replication. These studies collectively identified and validated the roles of 614 cell genes, but pair-wise overlap among the four gene lists was only 3% to 15% (average 6.7%. However, a number of functional categories were overrepresented in multiple studies. The pair-wise overlap of these enriched-category lists was high, ∼19%, implying more agreement among studies than apparent at the gene level. Probing this further, we found that the gene lists implicated by independent studies were highly connected in interacting networks by independent functional measures such as protein-protein interactions, at rates significantly higher than predicted by chance. We also developed a general, model-based approach to gauge the effects of false-positive and false-negative factors and to estimate, from a limited number of studies, the total number of genes involved in a process. For influenza virus replication, this novel statistical approach estimates the total number of cell genes involved to be ∼2,800. This and multiple other aspects of our experimental and computational results imply that, when following good quality control practices, the low overlap between studies is primarily due to false negatives rather than false-positive gene identifications. These results and methods have implications for and applications to multiple forms of genome-wide analysis.
Ogita, Shinjiro; Uefuji, Hirotaka; Morimoto, Masayuki; Sano, Hiroshi
The caffeine biosynthetic pathway in coffee plants has been proposed to involve three distinct N -methyltransferases, xanthosine methyltransferase (XMT), 7- N -methylxanthine methyltransferase (MXMT; theobromine synthase), and 3,7-dimethylxanthine methyltransferase (DXMT; caffeine synthase). We previously isolated all corresponding cDNAs designated as CaXMT1 , CaMXMT1 , CaMXMT2 and CaDXMT1 , respectively, and showed that caffeine was indeed synthesized in vitro by the combination of their gene products. In order to regulate caffeine biosynthesis in planta , we suppressed expression of CaMXMT1 by the double stranded RNA interference (RNAi) method. For this purpose, we first established a protocol for efficient somatic embryogenesis of Coffea arabica and C. canephora , and then Agrobacterium -mediated transformation techniques. The RNAi transgenic lines of embryogenic tissues derived from C. arabica and transgenic plantlets of C. canephora demonstrated a clear reduction in transcripts for CaMXMT1 in comparison with the control plants. Transcripts for CaXMT1 and CaDXMT1 were also reduced in the most cases. Both embryonic tissues and plantlets exhibited a concomitant reduction of theobromine and caffeine contents to a range between 30% and 50% of that of the control. These results suggest that the CaMXMT1 -RNAi sequence affected expression of not only CaMXMT1 itself, but also CaXMT1 and CaDXMT1 , and that, since the reduction in theobromine content was proportional to that for caffeine, it is involved in the major synthetic pathway in coffee plants. The results also indicate that the method can be practically applied to produce decaffeinated coffee plants.
Full Text Available Enhancement of micronutrient bioavailability is crucial to address the malnutrition in the developing countries. Various approaches employed to address the micronutrient bioavailability are showing promising signs, especially in cereal crops. Phytic acid (PA is considered as a major antinutrient due to its ability to chelate important micronutrients and thereby restricting their bioavailability. Therefore, manipulating PA biosynthesis pathway has largely been explored to overcome the pleiotropic effect in different crop species. Recently, we reported that functional wheat inositol pentakisphosphate kinase (TaIPK1 is involved in PA biosynthesis, however, the functional roles of the IPK1 gene in wheat remains elusive. In this study, RNAi-mediated gene silencing was performed for IPK1 transcripts in hexaploid wheat. Four non-segregating RNAi lines of wheat were selected for detailed study (S3-D-6-1; S6-K-3-3; S6-K-6-10 and S16-D-9-5. Homozygous transgenic RNAi lines at T4 seeds with a decreased transcript of TaIPK1 showed 28–56% reduction of the PA. Silencing of IPK1 also resulted in increased free phosphate in mature grains. Although, no phenotypic changes in the spike was observed but, lowering of grain PA resulted in the reduced number of seeds per spikelet. The lowering of grain PA was also accompanied by a significant increase in iron (Fe and zinc (Zn content, thereby enhancing their molar ratios (Zn:PA and Fe:PA. Overall, this work suggests that IPK1 is a promising candidate for employing genome editing tools to address the mineral accumulation in wheat grains.
Enright Anton J
Full Text Available Abstract Background RNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs, causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s. Results We identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death. Conclusions We have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences.
Paulsen, Aksel; Overgaard, Søren; Lauritsen, Jens Martin
The clinical and scientific usage of patient-reported outcome measures is increasing in the health services. Often paper forms are used. Manual double entry of data is defined as the definitive gold standard for transferring data to an electronic format, but the process is laborious. Automated...
Elbireer, Ali; Le Chasseur, Julie; Jackson, Brooks
The Uganda Makerere University provides clinical laboratory support to over 70 clients in Uganda. With increased volume, manual data entry errors have steadily increased, prompting laboratory managers to employ the Six Sigma method to evaluate and reduce their problems. The purpose of this paper is to describe how laboratory data entry quality was improved by using Six Sigma. The Six Sigma Quality Improvement (QI) project team followed a sequence of steps, starting with defining project goals, measuring data entry errors to assess current performance, analyzing data and determining data-entry error root causes. Finally the team implemented changes and control measures to address the root causes and to maintain improvements. Establishing the Six Sigma project required considerable resources and maintaining the gains requires additional personnel time and dedicated resources. After initiating the Six Sigma project, there was a 60.5 percent reduction in data entry errors from 423 errors a month (i.e. 4.34 Six Sigma) in the first month, down to an average 166 errors/month (i.e. 4.65 Six Sigma) over 12 months. The team estimated the average cost of identifying and fixing a data entry error to be $16.25 per error. Thus, reducing errors by an average of 257 errors per month over one year has saved the laboratory an estimated $50,115 a year. The Six Sigma QI project provides a replicable framework for Ugandan laboratory staff and other resource-limited organizations to promote quality environment. Laboratory staff can deliver excellent care at a lower cost, by applying QI principles. This innovative QI method of reducing data entry errors in medical laboratories may improve the clinical workflow processes and make cost savings across the health care continuum.
Full Text Available Brian Sims,1,2,* Linlin Gu,3,* Alexandre Krendelchtchikov,3 Qiana L Matthews3,4 1Division of Neonatology, Department of Pediatrics, 2Department of Cell, Developmental, and Integrative Biology, 3Division of Infectious Diseases, Department of Medicine, 4Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL, USA *These authors contributed equally to this work Background: Viruses enter host cells through interactions of viral ligands with cellular receptors. Viruses can also enter cells in a receptor-independent fashion. Mechanisms regarding the receptor-independent viral entry into cells have not been fully elucidated. Exosomal trafficking between cells may offer a mechanism by which viruses can enter cells.Methods: To investigate the role of exosomes on cellular viral entry, we employed neural stem cell-derived exosomes and adenovirus type 5 (Ad5 for the proof-of-principle study. Results: Exosomes significantly enhanced Ad5 entry in Coxsackie virus and adenovirus receptor (CAR-deficient cells, in which Ad5 only had very limited entry. The exosomes were shown to contain T-cell immunoglobulin mucin protein 4 (TIM-4, which binds phosphatidylserine. Treatment with anti-TIM-4 antibody significantly blocked the exosome-mediated Ad5 entry.Conclusion: Neural stem cell-derived exosomes mediated significant cellular entry of Ad5 in a receptor-independent fashion. This mediation may be hampered by an antibody specifically targeting TIM-4 on exosomes. This set of results will benefit further elucidation of virus/exosome pathways, which would contribute to reducing natural viral infection by developing therapeutic agents or vaccines. Keywords: neural stem cell-derived exosomes, adenovirus type 5, TIM-4, viral entry, phospholipids
Moller-Tank, Sven; Albritton, Lorraine M.; Rennert, Paul D.
ABSTRACT T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. IMPORTANCE T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral
Leavitt, James Aaron
The design and performance evaluation of an entry guidance algorithm for future space transportation vehicles is presented. The algorithm performs two functions: on-board trajectory planning and trajectory tracking. The planned longitudinal path is followed by tracking drag acceleration, as is done by the Space Shuttle entry guidance. Unlike the Shuttle entry guidance, lateral path curvature is also planned and followed. A new trajectory planning function for the guidance algorithm is developed that is suitable for suborbital entry and that significantly enhances the overall performance of the algorithm for both orbital and suborbital entry. In comparison with the previous trajectory planner, the new planner produces trajectories that are easier to track, especially near the upper and lower drag boundaries and for suborbital entry. The new planner accomplishes this by matching the vehicle's initial flight path angle and bank angle, and by enforcing the full three-degree-of-freedom equations of motion with control derivative limits. Insights gained from trajectory optimization results contribute to the design of the new planner, giving it near-optimal downrange and crossrange capabilities. Planned trajectories and guidance simulation results are presented that demonstrate the improved performance. Based on the new planner, a method is developed for approximating the landing footprint for entry vehicles in near real-time, as would be needed for an on-board flight management system. The boundary of the footprint is constructed from the endpoints of extreme downrange and crossrange trajectories generated by the new trajectory planner. The footprint algorithm inherently possesses many of the qualities of the new planner, including quick execution, the ability to accurately approximate the vehicle's glide capabilities, and applicability to a wide range of entry conditions. Footprints can be generated for orbital and suborbital entry conditions using a pre
Richard K Plemper
Full Text Available Measles virus (MeV, a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H and fusion (F proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family.
Lafleur, Jarret M.
One of the most important metrics characterizing an atmospheric entry trajectory in preliminary design is the size of its predicted landing ellipse. Often, requirements for this ellipse are set early in design and significantly influence both the expected scientific return from a particular mission and the cost of development. Requirements typically specify a certain probability level (6-level) for the prescribed ellipse, and frequently this latter requirement is taken at 36. However, searches for the justification of 36 as a robustness requirement suggest it is an empirical rule of thumb borrowed from non-aerospace fields. This paper presents an investigation into the sensitivity of trajectory performance to varying robustness (6-level) requirements. The treatment of robustness as a distinct objective is discussed, and an analysis framework is presented involving the manipulation of design variables to effect trades between performance and robustness objectives. The scenario for which this method is illustrated is the ballistic entry of an MSL-class Mars entry vehicle. Here, the design variable is entry flight path angle, and objectives are parachute deploy altitude performance and error ellipse robustness. Resulting plots show the sensitivities between these objectives and trends in the entry flight path angles required to design to these objectives. Relevance to the trajectory designer is discussed, as are potential steps for further development and use of this type of analysis.
Full Text Available Interferon-induced BST2/Tetherin prevents budding of vpu-deficient HIV-1 by tethering mature viral particles to the plasma membrane. BST2 also inhibits release of other enveloped viruses including Ebola virus and Kaposi's sarcoma associated herpesvirus (KSHV, indicating that BST2 is a broadly acting antiviral host protein. Unexpectedly however, recovery of human cytomegalovirus (HCMV from supernatants of BST2-expressing human fibroblasts was increased rather than decreased. Furthermore, BST2 seemed to enhance viral entry into cells since more virion proteins were released into BST2-expressing cells and subsequent viral gene expression was elevated. A significant increase in viral entry was also observed upon induction of endogenous BST2 during differentiation of the pro-monocytic cell line THP-1. Moreover, treatment of primary human monocytes with siRNA to BST2 reduced HCMV infection, suggesting that BST2 facilitates entry of HCMV into cells expressing high levels of BST2 either constitutively or in response to exogenous stimuli. Since BST2 is present in HCMV particles we propose that HCMV entry is enhanced via a reverse-tethering mechanism with BST2 in the viral envelope interacting with BST2 in the target cell membrane. Our data suggest that HCMV not only counteracts the well-established function of BST2 as inhibitor of viral egress but also employs this anti-viral protein to gain entry into BST2-expressing hematopoietic cells, a process that might play a role in hematogenous dissemination of HCMV.
Peter J Gaskill
Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.
Dudleenamjil, Enkhmart; Lin, Chin-Yo; Dredge, Devin; Murray, Byron K; Robison, Richard A; Johnson, F Brent
Entry events of bovine parvovirus (BPV) were studied. Transmission electron micrographs of infected cells showed virus particles in cytoplasmic vesicles. Chemical inhibitors that block certain aspects of the cellular machinery were employed to assess viral dependency upon those cellular processes. Chlorpromazine, ammonium chloride, chloroquine and bafilamicin A1 were used to inhibit acidification of endosomes and clathrin-associated endocytosis. Nystatin was used as an inhibitor of the caveolae pathway. Cytochalasin D and ML-7 were used to inhibit actin and myosin functions, respectively. Nocodazole and colchicine were employed to inhibit microtubule activity. Virus entry was assessed by measuring viral transcription using real-time PCR, synthesis of capsid protein and assembly of infectious progeny virus in the presence of inhibitor blockage. The results indicated that BPV entry into embryonic bovine trachael cells utilizes endocytosis in clathrin-coated vesicles, is dependent upon acidification, and appears to be associated with actin and microtubule dependency. Evidence for viral entry through caveolae was not obtained. These findings provide a fuller understanding of the early cell-entry events of the replication cycle for members of the genus Bocavirus.