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Sample records for monitor rna accumulation

  1. Telomerase RNA accumulates in Cajal bodies in human cancer cells.

    Science.gov (United States)

    Zhu, Yusheng; Tomlinson, Rebecca L; Lukowiak, Andrew A; Terns, Rebecca M; Terns, Michael P

    2004-01-01

    Telomerase synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. The RNA component of the enzyme (hTR) provides the template for telomere synthesis, which is catalyzed by telomerase reverse transcriptase (hTERT). Little is known regarding the subcellular localization of hTR and hTERT and the pathway by which telomerase is assembled. Here we report the first glimpse of the detailed subcellular localization of endogenous hTR in human cells, which we obtained by fluorescence in situ hybridization (FISH). Our studies have revealed a distinctive hTR localization pattern in cancer cells. We have found that hTR accumulates within intranuclear foci called Cajal bodies in all typical tumor-derived cell lines examined (in which telomerase is active), but not in primary or ALT cells (where little or no hTERT is present). Accumulation of hTR in the Cajal bodies of primary cells is induced when hTERT is ectopically expressed. Moreover, we report that hTERT is also found in Cajal bodies. Our data suggest that Cajal bodies are involved in the assembly and/or function of human telomerase.

  2. Chloroplasts continuously monitor photoreceptor signals during accumulation movement.

    Science.gov (United States)

    Tsuboi, Hidenori; Wada, Masamitsu

    2013-07-01

    Under low light conditions, chloroplasts gather at a cell surface to maximize light absorption for efficient photosynthesis, which is called the accumulation response. Phototropin1 (phot1) and phototropin2 (phot2) were identified as blue light photoreceptors in the accumulation response that occurs in Arabidopsis thaliana and Adiantum capillus-veneris with neochrome1 (neo1) as a red light photoreceptor in A. capillus-veneris. However, the signal molecule that is emitted from the photoreceptors and transmitted to the chloroplasts is not known. To investigate this topic, the accumulation response was induced by partial cell irradiation with a microbeam of red, blue and far-red light in A. capillus-veneris gametophyte cells. Chloroplasts moved towards the irradiated region and were able to sense the signal as long as its signal flowed. The signal from neo1 had a longer life than the signal that came from phototropins. When two microbeams with the same wavelength and the same fluence rate were placed 20 μm apart from each other and were applied to a dark-adapted cell, chloroplasts at an equidistant position always moved towards the center (midpoint) of the two microbeams, but not towards either one. This result indicates that chloroplasts are detecting the concentration of the signal but not the direction of signal flow. Chloroplasts repeatedly move and stop at roughly 10 s intervals during the accumulation response, suggesting that they monitor the intermittent signal waves from photoreceptors.

  3. Accuracy of helium accumulation fluence monitor for fast reactor dosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Chikara; Aoyama, Takafumi [Power Reactor and Nuclear Fuel Development Corp., Oarai, Ibaraki (Japan). Oarai Engineering Center

    1998-03-01

    A helium (He) accumulation fluence monitor (HAFM) has been developed for fast reactor dosimetry. In order to evaluate the measurement accuracy of neutron fluence by the HAFM method, the HAFMs of enriched boron (B) and beryllium (Be) were irradiated in the Fast Neutron Source Reactor `YAYOI`. The number of He atoms produced in the HAFMs were measured and compared with the calculated values. As a result of this study, it was confirmed that the neutron fluence could be measured within 5 % by the HAFM method, and that met the required accuracy for fast reactor dosimetry. (author)

  4. Anemia induces accumulation of erythropoietin mRNA in the kidney and liver.

    OpenAIRE

    Bondurant, M C; Koury, M J

    1986-01-01

    Regulation of the production of erythropoietin occurs in the kidney and liver largely through control of accumulation of erythropoietin mRNA. Erythropoietin mRNA was first detected in kidneys at 1.5 h postanemia and reached a plateau value at least 200-fold above the control value by 4 to 8 h. A 20-base sequence immediately upstream from the reported erythropoietin mRNA initiation site is complementary to a hypervariable sequence in 18S rRNA.

  5. Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

    Science.gov (United States)

    Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W

    2011-09-01

    Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.

  6. Accumulation of catechins in tea in relation to accumulation of mRNA from genes involved in catechin biosynthesis.

    Science.gov (United States)

    Eungwanichayapant, P D; Popluechai, S

    2009-02-01

    Catechins are a group of polyphenols found in tea (Camellia sinensis var. sinensis) at high levels. They are beneficial for health. From the study on accumulation of catechins in shoots and mature leaves of a tea cultivar, Oolong No. 17, using high-performance liquid chromatography (HPLC), it was found that the amounts of most catechins in the shoots were higher than those in the mature leaves, with an exception of catechins gallate (CG) that was found in trace amounts in both the shoots and mature leaves. mRNA accumulation of genes involved in catechin synthesis was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the mRNA accumulation of the genes were higher in the shoots than in the mature leaves. These genes included genes of phenylalanine ammonia-lyase 1 (PAL1; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), leucoanthocyanidin reductase (LCR; EC 1.17.1.3), and flavanone 3-hydroxylase (F3H; EC 1.14.11.9).

  7. RNA-dependent RNA polymerase 6 delays accumulation and precludes meristem invasion of a viroid that replicates in the nucleus.

    Science.gov (United States)

    Di Serio, Francesco; Martínez de Alba, Angel-Emilio; Navarro, Beatriz; Gisel, Andreas; Flores, Ricardo

    2010-03-01

    The detection of viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs (siRNAs, 21 to 24 nucleotides [nt]) in plants infected by nuclear-replicating members of the family Pospiviroidae (type species, Potato spindle tuber viroid [PSTVd]) indicates that they are inducers and targets of the RNA-silencing machinery of their hosts. RNA-dependent RNA polymerase 6 (RDR6) catalyzes an amplification circuit producing the double-stranded precursors of secondary siRNAs. Recently, the role of RDR6 in restricting systemic spread of certain RNA viruses and precluding their invasion of the apical growing tip has been documented using RDR6-silenced Nicotiana benthamiana (NbRDR6i) plants. Here we show that RDR6 is also engaged in regulating PSTVd levels: accumulation of PSTVd genomic RNA was increased in NbRDR6i plants with respect to the wild-type controls (Nbwt) early in infection, whereas this difference decreased or disappeared in later infection stages. Moreover, in situ hybridization revealed that RDR6 is involved in restricting PSTVd access in floral and vegetative meristems, thus providing firm genetic evidence for an antiviroid RNA silencing mechanism. RNA gel blot hybridization and deep sequencing showed in wt and RDR6i backgrounds that PSTVd sRNAs (i) accumulate to levels paralleling their genomic RNA, (ii) display similar patterns with prevailing 22- or 21-nt plus-strand species, and (iii) adopt strand-specific hot spot profiles along the genomic RNA. Therefore, the surveillance mechanism restraining entry of some RNA viruses into meristems likely also controls PSTVd access in N. benthamiana. Unexpectedly, deep sequencing also disclosed in NbRDR6i plants a profile of RDR6-derived siRNA dominated by 21-nt plus-strand species mapping within a narrow window of the hairpin RNA stem expressed transgenically for silencing RDR6, indicating that minus-strand siRNAs silencing the NbRDR6 mRNA represent a minor fraction of the total siRNA population.

  8. New insights into pri-miRNA processing and accumulation in plants.

    Science.gov (United States)

    Zhang, Shuxin; Liu, Yuhui; Yu, Bin

    2015-01-01

    MicroRNAs (miRNAs) regulate many biological processes such as development, metabolism, and others. They are processed from their primary transcripts called primary miRNA transcripts (pri-miRNAs) by the processor complex containing the RNAse III enzyme, DICER-LIKE1 (DCL1), in plants. Consequently, miRNA biogenesis is controlled through altering pri-miRNA accumulation and processing, which is crucial for plant development and adaptation to environmental changes. Plant pri-miRNAs are transcribed by DNA-dependent RNA polymerase II (Pol II) and their levels are determined through transcription and degradation, whereas pri-miRNA processing is affected by its structure, splicing, alternative splicing, loading to the processor and the processor activity, which involve in many accessory proteins. Here, we summarize recent progresses related to pri-miRNA transcription, stability, and processing in plants.

  9. mRNA accumulation in the Cajal bodies of the diplotene larch microsporocyte.

    Science.gov (United States)

    Smoliński, Dariusz Jan; Kołowerzo, Agnieszka

    2012-02-01

    In microsporocytes of the European larch, we demonstrated the presence of several mRNAs in spherical nuclear bodies. In the nuclei of microsporocytes, we observed up to 12 bodies, ranging from 0.5 to 6 μm in diameter, during the prophase of the first meiotic division. Our previous studies revealed the presence of polyadenylated RNA (poly(A) RNA) in these bodies, but did not confirm the presence of nascent transcripts or splicing factors of the SR family. The lack of these molecules precludes the bodies from being the sites of synthesis and early maturation of primary transcripts (Kołowerzo et al., Protoplasma 236:13-19, 2009). However, the bodies serve as sites for the accumulation of splicing machinery, including the Sm proteins and small nuclear RNAs. Characteristic ultrastructures and the molecular composition of the nuclear bodies, which contain poly(A) RNA, are indicative of Cajal bodies (CBs). Here, we demonstrated the presence of several housekeeping gene transcripts--α-tubulin, pectin methylesterase, peroxidase and catalase, ATPase, and inositol-3-phosphate synthase--in CBs. Additionally, we observed transcripts of the RNA polymerase II subunits RPB2 and RPB10 RNA pol II and the core spliceosome proteins mRNA SmD1, SmD2, and SmE. The co-localization of nascent transcripts and mRNAs indicates that mRNA accumulation/storage, particularly in CBs, occurs in the nucleus of microsporocytes.

  10. Extragenic accumulation of RNA polymerase II enhances transcription by RNA polymerase III.

    Directory of Open Access Journals (Sweden)

    Imke Listerman

    2007-11-01

    Full Text Available Recent genomic data indicate that RNA polymerase II (Pol II function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III. Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated approximately 300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was alpha-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon alpha-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.

  11. Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs.

    Science.gov (United States)

    Shu, Yi; Haque, Farzin; Shu, Dan; Li, Wei; Zhu, Zhenqi; Kotb, Malak; Lyubchenko, Yuri; Guo, Peixuan

    2013-06-01

    Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed "toolkits" utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.

  12. Heat shock increases lifetime of a small RNA and induces its accumulation in cells.

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    Tatosyan, Karina A; Kramerov, Dmitri A

    2016-08-01

    4.5SH and 4.5SI RNA are two abundant small non-coding RNAs specific for several related rodent families including Muridae. These RNAs have a number of common characteristics such as the short length (about 100nt), transcription by RNA polymerase III, and origin from Short Interspersed Elements (SINEs). However, their stabilities in cells substantially differ: the half-life of 4.5SH RNA is about 20min, while that of 4.5SI RNA is 22h. Here we studied the influence of cell stress such as heat shock or viral infection on these two RNAs. We found that the level of 4.5SI RNA did not change in stressed cells; whereas heat shock increased the abundance of 4.5SH RNA 3.2-10.5 times in different cell lines; and viral infection, 5 times. Due to the significant difference in the turnover rates of these two RNAs, a similar activation of their transcription by heat shock increases the level of the short-lived 4.5SH RNA and has minor effect on the level of the long-lived 4.5SI RNA. In addition, the accumulation of 4.5SH RNA results not only from the induction of its transcription but also from a substantial retardation of its decay. To our knowledge, it is the first example of a short-lived non-coding RNA whose elongated lifetime contributes significantly to its accumulation in stressed cells.

  13. Sense and Antisense DMPK RNA Foci Accumulate in DM1 Tissues during Development.

    Directory of Open Access Journals (Sweden)

    Lise Michel

    Full Text Available Myotonic dystrophy type 1 (DM1 is caused by an unstable expanded CTG repeat located within the DMPK gene 3'UTR. The nature, severity and age at onset of DM1 symptoms are very variable in patients. Different forms of the disease are described, among which the congenital form (CDM is the most severe. Molecular mechanisms of DM1 are well characterized for the adult form and involve accumulation of mutant DMPK RNA forming foci in the nucleus. These RNA foci sequester proteins from the MBNL family and deregulate CELF proteins. These proteins are involved in many cellular mechanisms such as alternative splicing, transcriptional, translational and post-translational regulation miRNA regulation as well as mRNA polyadenylation and localization. All these mechanisms can be impaired in DM1 because of the deregulation of CELF and MBNL functions. The mechanisms involved in CDM are not clearly described. In order to get insight into the mechanisms underlying CDM, we investigated if expanded RNA nuclear foci, one of the molecular hallmarks of DM1, could be detected in human DM1 fetal tissues, as well as in embryonic and neonatal tissues from transgenic mice carrying the human DMPK gene with an expanded CTG repeat. We observed very abundant RNA foci formed by sense DMPK RNA and, to a lesser extent, antisense DMPK RNA foci. Sense DMPK RNA foci clearly co-localized with MBNL1 and MBNL2 proteins. In addition, we studied DMPK sense and antisense expression during development in the transgenic mice. We found that DMPK sense and antisense transcripts are expressed from embryonic and fetal stages in heart, muscle and brain and are regulated during development. These results suggest that mechanisms underlying DM1 and CDM involved common players including toxic expanded RNA forming numerous nuclear foci at early stages during development.

  14. The ribosomal RNA transcription unit of Entamoeba invadens: accumulation of unprocessed pre-rRNA and a long non coding RNA during encystation.

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    Ojha, Sandeep; Singh, Nishant; Bhattacharya, Alok; Bhattacharya, Sudha

    2013-01-01

    The ribosomal RNA genes in Entamoeba spp. are located on extrachromosomal circular molecules. Unlike model organisms where rRNA transcription stops during growth stress, Entamoeba histolytica continues transcription; but unprocessed pre-rRNA accumulates during stress, along with a novel class of circular transcripts from the 5'-external transcribed spacer (ETS). To determine the fate of rRNA transcription during stage conversion between trophozoite to cyst we analyzed Entamoeba invadens, a model system for differentiation studies in Entamoeba. We characterized the complete rDNA transcription unit by mapping the ends of pre-rRNA and mature rRNAs. The 3' end of mature 28S rRNA was located 321 nt downstream of the end predicted by sequence homology with E. histolytica. The major processing sites were mapped in external and internal transcribed spacers. The promoter located within 146 nt upstream of 5' ETS was used to transcribe the pre-rRNA. On the other hand, a second promoter located at the 3' end of 28S rDNA was used to transcribe almost the entire intergenic spacer into a long non coding (nc) RNA (>10 kb). Interestingly we found that the levels of pre-rRNA and long ncRNA, measured by northern hybridization, decreased initially in cells shifted to encystation medium, after which they began to increase and reached high levels by 72 h when mature cysts were formed. Unlike E. histolytica, no circular transcripts were found in E. invadens. E. histolytica and E. invadens express fundamentally different ncRNAs from the rDNA locus, which may reflect their adaptation to different hosts (human and reptiles, respectively). This is the first description of rDNA organization and transcription in E. invadens, and provides the framework for further studies on regulation of rRNA synthesis during cyst formation.

  15. Satellite RNA-mediated Reduction of Cucumber Mosaic Virus Genomic RNAs Accumulation in Nicotiana tabacum

    Institute of Scientific and Technical Information of China (English)

    Qiansheng LIAO; Liping ZHU; Zhiyou DU; Rong ZENG; Junli FENG; Jishuang CHEN

    2007-01-01

    Satellite RNAs (satRNAs) are molecular parasites that interfere with the pathogenesis of the helper viruses.In this study,the relative accumulation of cucumber mosaic virus (CMV)-Fny genomic RNAs with or without satRNAs were quantitatively analyzed by real-time RT-PCR.The results showed that satRs apparently attenuated the symptoms of CMV-Fny on Nicotiana tabacum by depressing the accumulation of CMV-Fny genomic RNAs,tested as open reading frames.The accumulation of CMV-Fny la,2a,2b,3a,and CP genes was much higher than that of CMV-Fny with satRs added(CMV-Fsat),at different inoculation times.CMV-Fny△2b,in which the complete 2b gene and 41 amino acids at the C-terminal of the 2a gene were deleted,caused only a slight mosaic effect on N.tabacum seedlings,similar to that of CMVFsat,but the addition of satRs to CMV-Fny△2b showed further decrease in the accumulation of CMVFny△2b genomic RNAs.Our results indicated that the attenuation of CMV,by adding satRs or deleting the 2b gene,was due to the low accumulation of CMV genomic RNAs,and that satRNA-mediated reduction of CMV genomic RNAs accumulation in N.tabacum was possibly related to the 2b gene.

  16. Monitoring nitrogen accumulation in mosses in central European forests

    Energy Technology Data Exchange (ETDEWEB)

    Pesch, Roland [Chair of Landscape Ecology, University of Vechta, PO 1553, D-49356 Vechta (Germany)], E-mail: rpesch@iuw.uni-vechta.de; Schroeder, Winfried [Chair of Landscape Ecology, University of Vechta, PO 1553, D-49356 Vechta (Germany)], E-mail: wschroeder@iuw.uni-vechta.de; Schmidt, Gunther [Chair of Landscape Ecology, University of Vechta, PO 1553, D-49356 Vechta (Germany)], E-mail: gschmidt@iuw.uni-vechta.de; Genssler, Lutz [North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection (Germany)], E-mail: lutz.genssler@lanuv.nrw.de

    2008-10-15

    In order to assess whether nitrogen (N) loads in mosses reflect different land uses, 143 sites in North Rhine-Westphalia, the Weser-Ems Region and the Euro Region Nissa were sampled between 2000 and 2005. The data were analysed statistically with available surface information on land use and forest conditions. N bioaccumulation in mosses in the Weser-Ems Region with high densities of agricultural land use and livestock exceeded the concentrations in the more industrialised Euro Region Nissa. In all three study areas agricultural and livestock spatial densities were found to be positively correlated with N bioaccumulation in mosses. In North Rhine-Westphalia, the N concentrations in mosses was also moderately correlated with N concentrations in leaves and needles of forest trees. The moss method proved useful to assess the spatial patterns of N bioaccumulation due to land use. - Nitrogen accumulation in mosses from forests in central Europe is spatially correlated with the density of agricultural land use.

  17. Monitoring Damage Accumulation in Ceramic Matrix Composites Using Electrical Resistivity

    Science.gov (United States)

    Smith, Craig E.; Morscher, Gregory N.; Xia, Zhenhai H.

    2008-01-01

    The electric resistance of woven SiC fiber reinforced SiC matrix composites were measured under tensile loading conditions. The results show that the electrical resistance is closely related to damage and that real-time information about the damage state can be obtained through monitoring of the resistance. Such self-sensing capability provides the possibility of on-board/in-situ damage detection and accurate life prediction for high-temperature ceramic matrix composites. Woven silicon carbide fiber-reinforced silicon carbide (SiC/SiC) ceramic matrix composites (CMC) possess unique properties such as high thermal conductivity, excellent creep resistance, improved toughness, and good environmental stability (oxidation resistance), making them particularly suitable for hot structure applications. In specific, CMCs could be applied to hot section components of gas turbines [1], aerojet engines [2], thermal protection systems [3], and hot control surfaces [4]. The benefits of implementing these materials include reduced cooling air requirements, lower weight, simpler component design, longer service life, and higher thrust [5]. It has been identified in NASA High Speed Research (HSR) program that the SiC/SiC CMC has the most promise for high temperature, high oxidation applications [6]. One of the critical issues in the successful application of CMCs is on-board or insitu assessment of the damage state and an accurate prediction of the remaining service life of a particular component. This is of great concern, since most CMC components envisioned for aerospace applications will be exposed to harsh environments and play a key role in the vehicle s safety. On-line health monitoring can enable prediction of remaining life; thus resulting in improved safety and reliability of structural components. Monitoring can also allow for appropriate corrections to be made in real time, therefore leading to the prevention of catastrophic failures. Most conventional nondestructive

  18. piRNA-mediated nuclear accumulation of retrotransposon transcripts in the Drosophila female germline.

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    Chambeyron, Séverine; Popkova, Anna; Payen-Groschêne, Geneviève; Brun, Christine; Laouini, Dorsaf; Pelisson, Alain; Bucheton, Alain

    2008-09-30

    Germline silencing of transposable elements is essential for the maintenance of genome integrity. Recent results indicate that this repression is largely achieved through a RNA silencing pathway that involves Piwi-interacting RNAs (piRNAs). However the repressive mechanisms are not well understood. To address this question, we used the possibility to disrupt the repression of the Drosophila I element retrotransposon by hybrid dysgenesis. We show here that the repression of the functional I elements that are located in euchromatin requires proteins of the piRNA pathway, and that the amount of ovarian I element piRNAs correlates with the strength of the repression in the female germline. Antisense RNAs, which are likely used to produce antisense piRNAs, are transcribed by heterochromatic defective I elements, but efficient production of these antisense small RNAs requires the presence in the genome of euchromatic functional I elements. Finally, we demonstrate that the piRNA-induced silencing of the functional I elements is at least partially posttranscriptional. In a repressive background, these elements are still transcribed, but some of their sense transcripts are kept in nurse cell nuclear foci together with those of the Doc retrotransposon. In the absence of I element piRNAs, either in dysgenic females or in mutants of the piRNA silencing pathway, sense I element transcripts are transported toward the oocyte where retrotransposition occurs. Our results indicate that piRNAs are involved in a posttranscriptional gene-silencing mechanism resulting in RNA nuclear accumulation.

  19. Nitrogen accumulation in forests. Exposure monitoring by mosses.

    Science.gov (United States)

    Pesch, Roland; Schröder, Winfried; Schmidt, Gunther

    2007-03-21

    At present, there is still little information on nitrogen (N) accumulation in forests contrasting with the crucial importance of N in forest ecosystems. This work analyzes the N bioaccumulation in mosses from forested areas from Lower Saxony and North Rhine-Westphalia (two of 16 federal states of Germany), the Weser Ems Region (part of Lower Saxony), and the Euro Region Nissa (covering the Czech Republic, Germany, Poland). The studies involved samples collected from 190 sites between 1998 and 2005. Different spatial scales and regional differences in land use were chosen to assess the factors affecting N bioaccumulation in forested areas. A continuous reduction of N bioaccumulation was found from Lower Saxony (a region where agriculture is most predominant) to North Rhine-Westphalia (mostly urban). The Weser Ems Region (an agricultural region) showed a higher N concentration in mosses than the Euroregion Nissa (a former industrial region). Statistical analyses performed at the different spatial scales revealed that the areas showing greater agricultural and livestock spatial densities favor N bioaccumulation in mosses. N concentration in mosses was moderately correlated with the N concentration in the leaves and needles of the surrounding trees. No significant relationships were found regarding the crown density of forest trees or N deposition estimations from a combination of atmospheric models and deposition measurements.

  20. Nitrogen Accumulation in Forests. Exposure Monitoring by Mosses

    Directory of Open Access Journals (Sweden)

    Roland Pesch

    2007-01-01

    Full Text Available At present, there is still little information on nitrogen (N accumulation in forests contrasting with the crucial importance of N in forest ecosystems. This work analyzes the N bioaccumulation in mosses from forested areas from Lower Saxony and North Rhine-Westphalia (two of 16 federal states of Germany, the Weser Ems Region (part of Lower Saxony, and the Euro Region Nissa (covering the Czech Republic, Germany, Poland. The studies involved samples collected from 190 sites between 1998 and 2005. Different spatial scales and regional differences in land use were chosen to assess the factors affecting N bioaccumulation in forested areas. A continuous reduction of N bioaccumulation was found from Lower Saxony (a region where agriculture is most predominant to North Rhine-Westphalia (mostly urban. The Weser Ems Region (an agricultural region showed a higher N concentration in mosses than the Euroregion Nissa (a former industrial region. Statistical analyses performed at the different spatial scales revealed that the areas showing greater agricultural and livestock spatial densities favor N bioaccumulation in mosses. N concentration in mosses was moderately correlated with the N concentration in the leaves and needles of the surrounding trees. No significant relationships were found regarding the crown density of forest trees or N deposition estimations from a combination of atmospheric models and deposition measurements.

  1. RNA Interference-mediated Silencing of Phytochelatin Synthase Gene Reduce Cadmium Accumulation in Rice Seeds

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Phytochelatins (PCs) play an important role in heavy metal resistance and accumulation. To reduce the accumulation of cadmium (Cd) in rice seeds, the expression of phytochelatin synthase (PCS) gene OsPCS1 was suppressed by RNA interference (RNAi). A hairpin construct of a PCS fragment was designed in the pRNAi-OsPCS1 under the control of ZMM1, a seed-specific promoter from maize. The construct was introduced into rice (japonica) through Agrobacterium tumefaciens. The RNAi rice plantlets were selected and cultivated in pots exposured to 10 mg/kg Cd. The transcriptional level of OsPCS1 declined in seeds of some RNAi rice compared to the wild type. As a result Cd accumulation was reduced by about half in the seeds of RNAi rice. As expected, no apparent difference of growth appeared between RNAi and wild-type plants. The results suggest that this new approach can be used to control heavy metal accumulation in crops.

  2. MicroRNA-124 promotes hepatic triglyceride accumulation through targeting tribbles homolog 3

    OpenAIRE

    Xing Liu; Jiejie Zhao; Qi Liu; Xuelian Xiong; Zhijian Zhang; Yang Jiao; Xiaoying Li; Bin Liu; Yao Li; Yan Lu

    2016-01-01

    An increase in hepatic triglyceride (TG) contents usually results in non-alcoholic fatty liver disease (NAFLD) and related metabolic diseases. However, the mechanisms underlying perturbations of hepatic TG homeostasis remain largely unknown. Here, we showed that MicroRNA-124 was up-regulated in the livers of C57BL/6 mice fed a short-term high-fat-diet (HFD). Adenoviral overexpression of miR-124 in C57BL/6 mice led to accumulation of excessive triglycerides and up-regulation of lipogenic genes...

  3. Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

    Energy Technology Data Exchange (ETDEWEB)

    Hannas, Bethany R.; Wang, Ying H.; Thomson, Susanne; Kwon, Gwijun; Li Hong [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States); LeBlanc, Gerald A., E-mail: Gerald_LeBlanc@ncsu.edu [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States)

    2011-01-25

    The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

  4. Immune Monitoring Using mRNA-Transfected Dendritic Cells

    DEFF Research Database (Denmark)

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by m......RNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate...... and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA....

  5. Monitoring of the aquatic environment by species accumulator of pollutants: a review

    Directory of Open Access Journals (Sweden)

    Oscar RAVERA

    2001-09-01

    Full Text Available This paper is a short review on the biomonitoring of aquatic environments by animal and plant species accumulators of toxic pollutants ("scavengers". This monitoring is based on the relationship between the pollutant concentration in the organism and that in its environment, and not on alterations produced by pollution on the biota. The latter is the basis of other types of biomonitoring, such as those based on the biotic and diversity indices and saprobic scale. The various aspects of monitoring by pollutant accumulators are illustrated; for example, the uptake and loss of pollutants, the "critical organs" and "tissues", the detoxification mechanisms and the most common factors (C.F., BAF, BSAF for establishing a connection between the pollutant concentration in the organism and that in its environment. Several examples of this monitoring on heavy metals, radioisotopes and organic micropollutants are reported. The advantages of this monitoring, the characteristics of the species to be used as bioaccumulators and some practical suggestions are listed. A close collaboration between the scientific teams working on the biomonitoring based on accumulator organisms and on the chemical monitoring is recommended from the scientific and economic point of view.

  6. High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

    Institute of Scientific and Technical Information of China (English)

    SHEN Wan-xia; Neil A Smith; ZHOU Chang-yong; WANG Ming-bo

    2014-01-01

    RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the speciifc yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not signiifcantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no signiifcant impact on normal plant development.

  7. Midkine accumulated in nucleolus of HepG2 cells involved in rRNA transcription

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Jian-Zhong Shao; Li-Shan Min; Yong-Tao Xiao; Li-Xin Xiang; Zhi-Hong Ma

    2008-01-01

    AIM: To invesgate the ultrastructural location of midkine (MK) in nucleolus and function corresponding to its location. METHODS: To investigate the ultrastructural location of MK in nucleolus with immunoelectronic microscopy. To study the role that MK plays in ribosomal biogenesis by real-time PCR. The effect of MK on anti-apoptotic activity of HepG2 cells was studied with FITC-conjugated annexin V and propidium iodide PI double staining through FACS assay. RESULTS: MK mainly localized in the granular component (GC), dense fibrillar component (DFC) and the border between the DF-C and fibrillar center (FC). The production of 45S precursor rRNA level was decreased significantly in the presence of IK antisense oligonucleotide in the HepG2 cells. Furthermore, it was found that exogenous MK could protect HepG2 from apoptosis significantly. CONCLUSION: NK was constitutively translocated to the nucleolus of HepG2 cells, where it accumulated and mostly distributed at DFC, GC components and at the region between FC and DFC, MK played an important role in rRNA transcription, ribosome biogenesis, and cell proliferation in HepG2 cells. MK might serve as a molecular target for therapeutic intervention of human carcinomas.

  8. Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

    Science.gov (United States)

    Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

    2012-07-01

    Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  9. Control charts for monitoring accumulating adverse event count frequencies from single and multiple blinded trials.

    Science.gov (United States)

    Gould, A Lawrence

    2016-12-30

    Conventional practice monitors accumulating information about drug safety in terms of the numbers of adverse events reported from trials in a drug development program. Estimates of between-treatment adverse event risk differences can be obtained readily from unblinded trials with adjustment for differences among trials using conventional statistical methods. Recent regulatory guidelines require monitoring the cumulative frequency of adverse event reports to identify possible between-treatment adverse event risk differences without unblinding ongoing trials. Conventional statistical methods for assessing between-treatment adverse event risks cannot be applied when the trials are blinded. However, CUSUM charts can be used to monitor the accumulation of adverse event occurrences. CUSUM charts for monitoring adverse event occurrence in a Bayesian paradigm are based on assumptions about the process generating the adverse event counts in a trial as expressed by informative prior distributions. This article describes the construction of control charts for monitoring adverse event occurrence based on statistical models for the processes, characterizes their statistical properties, and describes how to construct useful prior distributions. Application of the approach to two adverse events of interest in a real trial gave nearly identical results for binomial and Poisson observed event count likelihoods. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Stress-induced Nuclear Bodies Are Sites of Accumulation of Pre-mRNA Processing Factors

    Science.gov (United States)

    Denegri, Marco; Chiodi, Ilaria; Corioni, Margherita; Cobianchi, Fabio; Riva, Silvano; Biamonti, Giuseppe

    2001-01-01

    Heterogeneous nuclear ribonucleoprotein (hnRNP) HAP (hnRNP A1 interacting protein) is a multifunctional protein with roles in RNA metabolism, transcription, and nuclear structure. After stress treatments, HAP is recruited to a small number of nuclear bodies, usually adjacent to the nucleoli, which consist of clusters of perichromatin granules and are depots of transcripts synthesized before stress. In this article we show that HAP bodies are sites of accumulation for a subset of RNA processing factors and are related to Sam68 nuclear bodies (SNBs) detectable in unstressed cells. Indeed, HAP and Sam68 are both present in SNBs and in HAP bodies, that we rename “stress-induced SNBs.” The determinants required for the redistribution of HAP lie between residue 580 and 788. Different portions of this region direct the recruitment of the green fluorescent protein to stress-induced SNBs, suggesting an interaction of HAP with different components of the bodies. With the use of the 580–725 region as bait in a two-hybrid screening, we have selected SRp30c and 9G8, two members of the SR family of splicing factors. Splicing factors are differentially affected by heat shock: SRp30c and SF2/ASF are efficiently recruited to stress-induced SNBs, whereas the distribution of SC35 is not perturbed. We propose that the differential sequestration of splicing factors could affect processing of specific transcripts. Accordingly, the formation of stress-induced SNBs is accompanied by a change in the splicing pattern of the adenovirus E1A transcripts. PMID:11694584

  11. Calibration of a He accumulation fluence monitor for fast reactor dosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Chikara [Power Reactor and Nuclear Fuel Development Corp., Oarai, Ibaraki (Japan). Oarai Engineering Center

    1997-03-01

    The helium accumulation fluence monitor (HAFM) has been developed for a fast reactor dosimetry. The HAFM measurement system was calibrated using He gas and He implanted samples and the measurement accuracy was confirmed to be less than 5%. Based on the preliminary irradiation test in JOYO, the measured He in the {sup 10}B type HAFM agreed well with the calculated values using the JENDL-3.2 library. (author)

  12. Long noncoding RNA TUG1 alleviates extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ in diabetic nephropathy.

    Science.gov (United States)

    Duan, Li-Jun; Ding, Min; Hou, Li-Jun; Cui, Yuan-Tao; Li, Chun-Jun; Yu, De-Min

    2017-03-11

    Long noncoding RNA taurine-upregulated gene 1 (lncRNA TUG1) has been reported to play a key role in the progression of diabetic nephropathy (DN). However, the role of lncRNA TUG1 in the regulation of diabetic nephropathy remains largely unknown. The aim of the present study is to identify the regulation of lncRNA TUG1 on extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ, and investigate the underlying mechanisms in progression of DN. Microarray was performed to screen differentially expressed miRNAs in db/db DN mice. Afterwards, computational prediction programs (TargetScan, miRanda, PicTar and miRGen) was applied to predict the target gene of miRNAs. The complementary binding of miRNA and lncRNA was assessed by luciferase assays. Protein and mRNA expression were detected by western blot and real time quantitate PCR. MiRNA-377 was screened by miRNA microarray and differentially up-regulated in db/db DN mice. PPARγ was predicted to be the target of miR-377 and the prediction was verified by luciferase assays. Expression of miR-377 was up-regulated in mesangial cell treated with high glucose (25 mM), and overexpression of miR-377 inhibited PPARγ expression and promoted PAI-1 and TGF-β1 expression. The expression of TUG1 antagonized the effect of miR-377 on the downregulation of its target PPARγ and inhibited extracellular matrix accumulation, including PAI-1, TGF-β1, fibronectin (FN) and collagen IV (Col IV), induced by high glucose. LncRNA TUG1 acts as an endogenous sponge of miR-377 and downregulates miR-377 expression levels, and thereby relieving the inhibition of its target gene PPARγ and alleviates extracellular matrix accumulation of mesangial cells, which provides a novel insight of diabetic nephropathy pathogenesis.

  13. Genome-wide RNA polymerase II profiles and RNA accumulation reveal kinetics of transcription and associated epigenetic changes during diurnal cycles.

    Directory of Open Access Journals (Sweden)

    Gwendal Le Martelot

    Full Text Available Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver.

  14. NMR monitoring of the SELEX process to confirm enrichment of structured RNA.

    Science.gov (United States)

    Amano, Ryo; Aoki, Kazuteru; Miyakawa, Shin; Nakamura, Yoshikazu; Kozu, Tomoko; Kawai, Gota; Sakamoto, Taiichi

    2017-03-21

    RNA aptamers are RNA molecules that bind to a target molecule with high affinity and specificity using uniquely-folded tertiary structures. RNA aptamers are selected from an RNA pool typically comprising up to 10(15) different sequences generated by iterative steps of selection and amplification known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Over several rounds of SELEX, the diversity of the RNA pool decreases and the aptamers are enriched. Hence, monitoring of the enrichment of these RNA pools is critical for the successful selection of aptamers, and several methods for monitoring them have been developed. In this study, we measured one-dimensional imino proton NMR spectra of RNA pools during SELEX. The spectrum of the initial RNA pool indicates that the RNAs adopt tertiary structures. The structural diversity of the RNA pools was shown to depend highly on the design of the primer-binding sequence. Furthermore, we demonstrate that enrichment of RNA aptamers can be monitored using NMR. The RNA pools can be recovered from the NMR tube after measurement of NMR spectra. We also can monitor target binding in the NMR tubes. Thus, we propose using NMR to monitor the enrichment of structured aptamers during the SELEX process.

  15. Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria.

    Science.gov (United States)

    Kaper, Thijs; Lager, Ida; Looger, Loren L; Chermak, Diane; Frommer, Wolf B

    2008-06-03

    Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production. An Escherichia coli expression vector compatible with phage lambda recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates. The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating

  16. Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria

    Directory of Open Access Journals (Sweden)

    Looger Loren L

    2008-06-01

    Full Text Available Abstract Background Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production. Results An Escherichia coli expression vector compatible with phage λ recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates. Conclusion The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo

  17. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling.

    Science.gov (United States)

    Hartshorn, Cristina; Rice, John E; Wangh, Lawrence J

    2003-01-01

    Xist gene expression begins at the late 2-cell stage in female mouse embryos and by the third division results in the accumulation of an average 100 copies of Xist RNA per cell, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). In the blastocyst, the trophectoderm maintains the paternally imprinted pattern of Xist expression present during early development, while either the maternal or the paternal X chromosome can express Xist among cells of the inner mass. Fluorescent in situ hybridization (FISH) has previously established that Xist transcripts are localized on the silenced X chromosome, forming aggregates of variable dimensions in blastomeres of 8-cell embryos. This observation and the fact that Xist RNA accumulation per cell sharply decreases after morula stage raise the possibility that cells of cleaving embryos contain different levels of Xist RNA, perhaps linked to their subsequent developmental fates. We show here that Xist RNA is efficiently recovered from single blastomeres isolated from 8-cell embryos following laser zona drilling. Sexing of the samples and simultaneous quantification of Xist RNA in individual cells is achieved with a multiplex Xist/Sry real-time RT-PCR assay sensitive to the single-copy level. This analysis reveals that Xist RNA is indeed accumulated to substantially different levels in individual blastomeres of the same 8-cell embryo and that two blastomeres contain most of the molecules per embryo. These results support the conclusion that cells of the early mammalian embryo are not all functionally equivalent. Differential Xist gene expression could arise from differences in DNA methylation, or the order in which cells divide. Copyright 2003 Wiley-Liss, Inc.

  18. Damage accumulation in cyclically-loaded glass-ceramic matrix composites monitored by acoustic emission.

    Science.gov (United States)

    Aggelis, D G; Dassios, K G; Kordatos, E Z; Matikas, T E

    2013-01-01

    Barium osumilite (BMAS) ceramic matrix composites reinforced with SiC-Tyranno fibers are tested in a cyclic loading protocol. Broadband acoustic emission (AE) sensors are used for monitoring the occurrence of different possible damage mechanisms. Improved use of AE indices is proposed by excluding low-severity signals based on waveform parameters, rather than only threshold criteria. The application of such improvements enhances the accuracy of the indices as accumulated damage descriptors. RA-value, duration, and signal energy follow the extension cycles indicating moments of maximum or minimum strain, while the frequency content of the AE signals proves very sensitive to the pull-out mechanism.

  19. Damage Accumulation in Cyclically-Loaded Glass-Ceramic Matrix Composites Monitored by Acoustic Emission

    Directory of Open Access Journals (Sweden)

    D. G. Aggelis

    2013-01-01

    Full Text Available Barium osumilite (BMAS ceramic matrix composites reinforced with SiC-Tyranno fibers are tested in a cyclic loading protocol. Broadband acoustic emission (AE sensors are used for monitoring the occurrence of different possible damage mechanisms. Improved use of AE indices is proposed by excluding low-severity signals based on waveform parameters, rather than only threshold criteria. The application of such improvements enhances the accuracy of the indices as accumulated damage descriptors. RA-value, duration, and signal energy follow the extension cycles indicating moments of maximum or minimum strain, while the frequency content of the AE signals proves very sensitive to the pull-out mechanism.

  20. Use of Acoustic Emission to Monitor Progressive Damage Accumulation in Kevlar (R) 49 Composites

    Science.gov (United States)

    Waller, Jess M.; Saulsberry, Regor L.; Andrade, Eduardo

    2009-01-01

    Acoustic emission (AE) data acquired during intermittent load hold tensile testing of epoxy impregnated Kevlar(Registeres TradeMark) 49 (K/Ep) composite strands were analyzed to monitor progressive damage during the approach to tensile failure. Insight into the progressive damage of K/Ep strands was gained by monitoring AE event rate and energy. Source location based on energy attenuation and arrival time data was used to discern between significant AE attributable to microstructural damage and spurious AE attributable to noise. One of the significant findings was the observation of increasing violation of the Kaiser effect (Felicity ratio < 1.0) with damage accumulation. The efficacy of three different intermittent load hold stress schedules that allowed the Felicity ratio to be determined analytically is discussed.

  1. Putrescine accumulation does not affect RNA metabolism in the lichen Evernia prunastri.

    OpenAIRE

    Escribano, M. Isabel; Legaz, María Estrella

    1985-01-01

    Evernia prunastri thaiIi are able to accumulate exogenous putrescine from the media. This accumulation is optimal at pH 9.15 although the highest uptake rate is achieved at pH 5.0 in parallel to the highest production of endogenous diamine. The lowest values of RNase activity seems to be related to alkaline pH values rather than accumulation of free putrescine.

  2. Monitoring intracellular polyphosphate accumulation in enhanced biological phosphorus removal systems by quantitative image analysis.

    Science.gov (United States)

    Mesquita, Daniela P; Amaral, A Luís; Leal, Cristiano; Carvalheira, Mónica; Cunha, Jorge R; Oehmen, Adrian; Reis, Maria A M; Ferreira, Eugénio C

    2014-01-01

    A rapid methodology for intracellular storage polyphosphate (poly-P) identification and monitoring in enhanced biological phosphorus removal (EBPR) systems is proposed based on quantitative image analysis (QIA). In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluorescence in situ hybridization to evaluate the microbial community. The proposed monitoring technique is based on a QIA procedure specifically developed for determining poly-P inclusions within a biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature regarding DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation occurred. Digital images were then acquired and processed by means of image processing and analysis. A correlation was found between average poly-P intensity values and the analytical determination. The proposed methodology can be seen as a promising alternative procedure for quantifying intracellular poly-P accumulation in a faster and less labour-intensive way.

  3. Brome mosaic virus Infection of Rice Results in Decreased Accumulation of RNA1.

    Science.gov (United States)

    Kitayama, Masahiko; Hoover, Haley; Middleton, Stefani; Kao, C Cheng

    2015-05-01

    Brome mosaic virus (BMV) (the Russian strain) infects monocot plants and has been studied extensively in barley and wheat. Here, we report BMV can systemically infect rice (Oryza sativa var. japonica), including cultivars in which the genomes have been determined. The BMV capsid protein can be found throughout the inoculated plants. However, infection in rice exhibits delayed symptom expression or no symptoms when compared with wheat (Triticum aestivum). The sequences of BMV RNAs isolated from rice did not reveal any nucleotide changes in RNA1 or RNA2, while RNA3 had only one synonymous nucleotide change from the inoculum sequence. Preparations of purified BMV virions contained RNA1 at a significantly reduced level relative to the other two RNAs. Analysis of BMV RNA replication in rice revealed that minus-strand RNA1 was replicated at a reduced rate when compared with RNA2. Thus, rice appears to either inhibit RNA1 replication or lacks a sufficient amount of a factor needed to support efficient RNA1 replication.

  4. siRNA delivery targeting to the lung via agglutination-induced accumulation and clearance of cationic tetraamino fullerene

    Science.gov (United States)

    Minami, Kosuke; Okamoto, Koji; Doi, Kent; Harano, Koji; Noiri, Eisei; Nakamura, Eiichi

    2014-05-01

    The efficient treatment of lung diseases requires lung-selective delivery of agents to the lung. However, lung-selective delivery is difficult because the accumulation of micrometer-sized carriers in the lung often induces inflammation and embolization-related toxicity. Here we demonstrate a lung-selective delivery system of small interfering RNA (siRNA) by controlling the size of carrier vehicle in blood vessels. The carrier is made of tetra(piperazino)fullerene epoxide (TPFE), a water-soluble cationic tetraamino fullerene. TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles. The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery. We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

  5. Cadmium-induced accumulation of metallothionein messenger RNA in rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Ohi, S.; Cardenosa, G.; Pine, R.; Huang, P.C.

    1981-03-10

    Multiple injections of nontoxic levels of cadmium to a rat result in much higher level of metallothionein (MT) production in the liver than does the single injection. In order to understand the underlying mechanisms we have quantitated and compared the metallothionein-specific messenger RNA contents in the livers following the two induction regimens. Cell-free translation assays coupled with specific immunoprecipitation of MT revealed that MT-mRNA activity in livers of animals multiply injected with Cd is 7- to 10-fold higher than that in livers 4 h after a single Cd-induction. By oligo(dT)-cellulose chromatography, sucrose density gradient centrifugation, and methylmercuric hydroxide-agarose gel electrophoresis this mRNA has been enriched approximately 100-fold from the total RNA. The size of the mRNA is about 400 nucleotides. Hybridization assays with a complementary DNA probe synthesized against the enriched MT-mRNA showed a 4-fold difference in the level of MT-mRNA between the two induction regimens in agreement with the results obtained by the cell-free translation assays. The possible mechanisms for these observations in consideration of the short lived nature of MT-mRNA are discussed.

  6. Monitoring RNA release from human rhinovirus by dynamic force microscopy.

    Science.gov (United States)

    Kienberger, Ferry; Zhu, Rong; Moser, Rosita; Blaas, Dieter; Hinterdorfer, Peter

    2004-04-01

    Human rhinoviruses were imaged under physiological conditions by dynamic force microscopy. Topographical images revealed various polygonal areas on the surfaces of the 30-nm viral particles. RNA release was initiated by exposure to a low-pH buffer. The lengths of the RNAs that were released but still connected to the virus capsid varied between 40 and 330 nm, whereas RNA molecules that were completely released from the virus were observed with lengths up to 1 micro m. Fork-like structure elements with 30-nm extensions were sometimes resolved at one end of the RNA molecules. They possibly correspond to the characteristic multi-stem-loop conformation, the internal ribosomal entry site, located at the 5' region of the genome. This study demonstrates that dynamic force microscopy can be used to study viral RNA release in situ under physiological conditions.

  7. Control of. cap alpha. -amylase mRNA accumulation by gibberellic acid and calcium in barley aleurone layers

    Energy Technology Data Exchange (ETDEWEB)

    Deikman, J.; Jones, R.L.

    1985-01-01

    Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA/sub 3/) with or without 5 millimolar CaCl/sub 2/ shows that ..cap alpha..-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca/sup 2 +/. No difference was observed in ..cap alpha..-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA/sub 3/ with 5 millimolar CaCl/sub 2/ and layers incubated in GA/sub 3/ alone. RNA isolated from layers incubated for 12 hours in GA/sub 3/ with and without CA/sup 2 +/. A cDNA clone for ..cap alpha..-amylase was isolated and used to measure ..cap alpha..-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca/sup 2 +/ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca/sup 2 +/ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for ..cap alpha..-amylase synthesized in Ca/sup 2 +/-deprived aleurone layers was translatable. Ca/sup 2 +/ is required for the synthesis of ..cap alpha..-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.

  8. Nuclear accumulation of CDH1 mRNA in hepatocellular carcinoma cells

    Science.gov (United States)

    Ghafoory, S; Mehrabi, A; Hafezi, M; Cheng, X; Breitkopf-Heinlein, K; Hick, M; Huichalaf, M; Herbel, V; Saffari, A; Wölfl, S

    2015-01-01

    Expression of E-cadherin has a central role in maintaining epithelial morphology. In solid tumors, reduction of E-cadherin results in disruption of intercellular contacts. Consequently, cells lose adhesive properties and gain more invasive mesenchymal properties. Nevertheless, the mechanism of E-cadherin regulation is not completely elucidated. Here we analyzed the distribution of E-cadherin expression at the cell level in human hepatocellular carcinoma, in which human liver paraffin blocks from 25 hepatocellular carcinoma patients were prepared from cancerous (CA) and noncancerous areas (NCA). In situ hybridization (ISH) was performed to detect E-cadherin and hypoxia-induced factor-1α (HIF1α) mRNAs and immunohistochemistry to stain E-cadherin protein. In parallel, RNA was extracted from CA and NCA, and E-cadherin and HIF1α were quantified by quantitative reverse transcription PCR. ISH revealed abundant E-cadherin mRNA in nuclei of hepatocellular carcinoma cells (HCCs), whereas immunohistochemistry showed depletion of E-cadherin protein from these areas. In sections of NCA, E-cadherin mRNA was also found in the cytosol, and E-cadherin protein was detected on the membrane of cells. Experiments in cell lines confirmed E-cadherin mRNA in nuclei of cells negative for E-cadherin protein. HIF1α expression is elevated in CAs, which is associated with a clear cytosolic staining for this mRNA. Our results demonstrate that E-caderhin mRNA is selectively retained in nuclei of HCCs, whereas other mRNAs are still exported, suggesting that translocation of E-cadherin mRNA from nuclei to cytoplasm has a role in regulating E-cadherin protein levels during epithelial mesenchymal transition. PMID:26029826

  9. Standard Test Method for Application and Analysis of Helium Accumulation Fluence Monitors for Reactor Vessel Surveillance, E706 (IIIC)

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2007-01-01

    1.1 This test method describes the concept and use of helium accumulation for neutron fluence dosimetry for reactor vessel surveillance. Although this test method is directed toward applications in vessel surveillance, the concepts and techniques are equally applicable to the general field of neutron dosimetry. The various applications of this test method for reactor vessel surveillance are as follows: 1.1.1 Helium accumulation fluence monitor (HAFM) capsules, 1.1.2 Unencapsulated, or cadmium or gadolinium covered, radiometric monitors (RM) and HAFM wires for helium analysis, 1.1.3 Charpy test block samples for helium accumulation, and 1.1.4 Reactor vessel (RV) wall samples for helium accumulation. This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

  10. miRNA-133a attenuates lipid accumulation via TR4-CD36 pathway in macrophages.

    Science.gov (United States)

    Peng, Xiao-Ping; Huang, Lei; Liu, Zhi-Hong

    2016-08-01

    lipid metabolism is the major causes of atherosclerosis. There is increasing evidence that miR-133a plays an important role in atherosclerosis. However, the regulatory mechanism of miR-133a in macrophages is still unclear. Several lines of evidence indicate that loss of TR4 leads to reduce lipid accumulation in liver and adipose tissues, etc, and lesional macrophages-derived TR4 can greatly increase the foam cell formation through increasing the CD36-mediated the uptake of ox-LDL. Interestingly, computational analysis suggests that TR4 may be a target gene of miR-133a. Here, we examined whether miR-133a regulates TR4 expression in ox-LDL-induced mouse RAW 264.7 macrophages, thereby affecting lipid accumulation. Using ox-LDL-treatment RAW 264.7 macrophages transfected with miR-133a mimics or inhibitors, we have showed that miR-133a can directly regulate the expression of TR4 in RAW 264.7 cells, thereby attenuates CD36-medide lipid accumulation. Furthermore, our studies suggest an additional explanation for the regulatory mechanism of miR-133a regulation to its functional target, TR4 in RAW 264.7 macrophages. Thus, our findings suggest that miR-133a may regulate lipid accumulation in ox-LDL-stimulated RAW 264.7 macrophages via TR4-CD36 pathway.

  11. RNA interference reduces aflatoxin accumulation by Aspergillus flavus in peanut seeds

    Science.gov (United States)

    Aflatoxins are among the most powerful carcinogens in nature. They are produced by the fungal pathogen Aspergillus flavus Link and other Aspergillus species. Aflatoxins accumulate in many crops, including rice, wheat, oats, pecans, pistachios, soybean, cassava, almonds, peanuts, beans, corn and cot...

  12. From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

    Directory of Open Access Journals (Sweden)

    Mireia Ferrer

    2016-08-01

    Full Text Available In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV and complex human immunodeficiency virus type 1 (HIV-1 in mind, the techniques were described in order to benefit to a larger community.

  13. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation

    OpenAIRE

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-01-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21–22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, di...

  14. The Living Filter: Monitoring Nitrate Accumulation after 50 Years of Wastewater Irrigation

    Science.gov (United States)

    Hagedorn, J.

    2015-12-01

    As global freshwater sources decline due to environmental contamination and a growing population, more sustainable wastewater renovation techniques will need to be applied to ensure freshwater for future generations. One such example of a sustainable solution is called the Living Filter, located on the campus of Pennsylvania State University. For fifty years, Pennsylvania State University has sprayed treated wastewater onto agricultural fields and forest ecosystems, leaving natural processes to further filter the wastewater. This cyclical process is deemed sustainable because the freshwater is recycled, providing drinking water to an increasing university population and nutrients to agricultural crops, without causing major environmental catastrophes such as fish kills, eutrophication or groundwater contamination. At first glance this project seems sustainable and effective, but for how long can this setup continue without nutrient overloading and environmental contamination? To be truly declared sustainable, the hopeful answer to this question is indefinitely. Using a combination of soil core and monitoring tools, ecosystem indicators such as soil nutrient capacities, moisture levels, and soil characteristics were measured. Comparing data from the initial system installation to present data collected from soil cores showed how ecosystems changed over time. Results revealed that nitrate concentrations were elevated through the profile in all land use types, but the concentrations were below EPA threshold. Soil characteristic analysis including particle size distribution, soil elemental composition, and texture yielded inconclusive results regarding which factors control the nitrate accumulation most significantly. The nitrate depth profile findings suggest that spray irrigation at the Living Filter under the current rates of application has not caused the ultimate stage of nitrogen saturation in the spray irrigation site. Variations in land use present interesting

  15. Monitoring RNA-ligand interactions using isothermal titration calorimetry.

    Science.gov (United States)

    Gilbert, Sunny D; Batey, Robert T

    2009-01-01

    Isothermal titration calorimetry (ITC) is a biophysical technique that measures the heat evolved or absorbed during a reaction to report the enthalpy, entropy, stoichiometry of binding, and equilibrium association constant. A significant advantage of ITC over other methods is that it can be readily applied to almost any RNA-ligand complex without having to label either molecule and can be performed under a broad range of pH, temperature, and ionic concentrations. During our application of ITC to investigate the thermodynamic details of the interaction of a variety of compounds with the purine riboswitch, we have explored and optimized experimental parameters that yield the most useful and reproducible results for RNAs. In this chapter, we detail this method using the titration of an adenine-binding RNA with 2,6-diaminopurine (DAP) as a practical example. Our insights should be generally applicable to observing the interactions of a broad range of molecules with structured RNAs.

  16. RNA Futile Cycling in Model Persisters Derived from MazF Accumulation (Open Access)

    Science.gov (United States)

    2015-11-17

    MazF and its antitoxin MazE, bacterial populations that were almost entirely tolerant to fluoroquinolone and -lactam antibiotics were obtained upon...of transcription. Thus, in MazF model persisters, futile cycles of RNA synthesis and degradation result in both significant metabolic demands and...they remained highly metabolically active. We further uncovered a futile cycle driven by continued transcription and MazF-mediated transcript degradation

  17. Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Krochko, J E; Pramanik, S K; Bewley, J D

    1992-05-01

    During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media

  18. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    Science.gov (United States)

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  19. Circulating miRNA-155 is realted to blood pressure monitoring parameters

    Institute of Scientific and Technical Information of China (English)

    WU Ying; HUANG Yu-qing; HUANG Cheng; LI Jie; CAI An-ping; YU Xue-ju; ZHOU Dan

    2016-01-01

    Background Studies have shown that miRNA-155 played an important role in the process of development of hypertension.However,there is no date about miRNA-155 and blood pressure monitoring parameters.Therefore,we examined whether in hypertensive patients the expression level of plasma miRNA-155 related to 24-h ambulatory blood pressure monitoring (ABPM) parameters.Methods A cohort of adult patients scheduled to receive physical examination,office and ambulatory blood pressure monitoring.Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of selected miRNA-155.The miRNA-155 expression level correlation between blood pressure parameters was assessed using the Spearman correlation coefficient.Results Fifty four essential hypertension patients (25 men;mean age,53.28 ± 9.52 years) and thirty healthy volunteers (15 men;mean age,53.03 ± 5.87 years) were included.We observed higher expression level of miRNA-155 (32.31 ± 2.85 vs 27.21 ± 1.59,P < 0.001) in hypertensive patients compared to healthy control individuals.MiRNA-155 expression level showed significant positive correlation with 24 h Daytime SBP (r =0.681,P < 0.001),24 h Daytime DBP (r =0.473,P < 0.001),24 h Daytime PP (r =0.565,P < 0.001) and dipping (r =0.257,P =0.018),respectively.Conclusions Our study showed that miRNA-155 expression level was associated positively with daytime blood pressure monitoring parameters,as well as blood pressure variability,indicating a possible implication of miRNA-155 in the pathogenesis of hypertension.

  20. Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum.

    Science.gov (United States)

    Yu, Huilin; Cong, Ling; Zhu, Zhenxing; Wang, Chunyu; Zou, Jianqiu; Tao, Chengguang; Shi, Zhensheng; Lu, Xiaochun

    2015-10-25

    MicroRNAs (miRNAs) have been shown to play important roles in plant development, growth and stress response. Sweet sorghum [Sorghum bicolor (L.) Moench] is an important source of bioenergy due to the high sugar content in its stems. However, it is not clear how the miRNA is involved in sugar accumulation in sorghum stems. In order to identify the miRNAs in the stems and the leaves of sweet sorghum, we extracted RNAs of the stems and leaves of sweet sorghum (Rio) and grain sorghum (BTx623) at the heading and dough stages for high-throughput sequencing. A total of 179279048 reads were obtained from Illumina-based sequencing. Further analysis identified nine known miRNAs and twelve novel miRNAs that showed significantly and specifically differentially expressed in the stems of sweet sorghum. The target genes of the differentially expressed novel miRNAs include the transcription factor, glucosyltransferase, protein kinase, cytochrome P450, transporters etc. GO enrichment analysis showed that the predicted targets of these differentially expressed miRNAs participated in diverse physiological and metabolic processes. We performed RT-qRCR analysis on these miRNAs across eight different libraries to validate the miRNAs. Finally, we screened stem-specifically expressed novel miRNA and a leaf-specifically expressed novel miRNA in sweet sorghum comparing with grain sorghum. Our results provide a basis for further investigation of the potential role of these individual miRNAs in sugar accumulation.

  1. Regulation of polyhydroxybutyrate accumulation in Sinorhizobium meliloti by the trans-encoded small RNA MmgR.

    Science.gov (United States)

    Lagares, Antonio; Ceizel Borella, Germán; Linne, Uwe; Becker, Anke; Valverde, Claudio

    2017-02-06

    Riboregulation has a major role in the fine-tuning of multiple bacterial processes. Among the RNA players, trans-encoded untranslated small RNAs (sRNAs) regulate complex metabolic networks by tuning expression from multiple target genes in response to numerous signals. In Sinorhizobium meliloti, over 400 sRNAs are expressed under different stimuli. The sRNA MmgR-standing for Makes more granules Regulator-has been of particular interest to us since its sequence and structure are highly conserved among the α-proteobacteria, and its expression is regulated by the amount and quality of the bacterium's available nitrogen source. In this work, we explored the biological role of MmgR in S. meliloti 2011 by characterizing the effect of a deletion of the internal conserved core of mmgR (mmgR(Δ33-51)). This mutation resulted in higher amounts of polyhydroxybutyrate (PHB) distributed into more intracellular granules than are found in the wild-type strain. This phenotype was expressed upon cessation of balanced growth owing to a nitrogen depletion in the presence of surplus carbon (i. e., at a carbon:nitrogen molar ratio greater than 10). The normal PHB accumulation was complemented with a wild-type mmgR copy, but not with unrelated sRNA genes. Furthermore, the expression of mmgR limited PHB accumulation in the wild-type, regardless of the magnitude of the C surplus. Quantitative proteomic profiling and qRT-PCR revealed that the absence of MmgR results in a posttranscriptional overexpression of both PHB-phasin proteins (PhaP1, PhaP2). All together, our results indicate that the widely conserved α-proteobacterial MmgR sRNA fine-tunes the regulation of PHB storage in S. meliloti IMPORTANCE: High-throughput RNA sequencing has recently uncovered an overwhelming number of trans-encoded small RNAs (sRNAs) in diverse prokaryotes. In the nitrogen-fixing α-proteobacterial symbiont of alfalfa root nodules Sinorhizobium meliloti, only four out of hundreds of identified sRNA genes

  2. Rapid monitoring of RNA degradation activity in vivo for mammalian cells.

    Science.gov (United States)

    Tani, Hidenori; Sato, Hiroaki; Torimura, Masaki

    2017-04-01

    We have developed a rapid fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA degradation activity in mammalian cells. In this technique, double-stranded RNA (dsRNA) fluorescent probes are used. The dsRNA fluorescent probes consist of a 5' fluorophore-labeled strand hybridized to a 3' quencher-labeled strand, and the fluorescent dye is quenched by a quencher dye. When the dsRNA is degraded by nascent RNases in cells, the fluorescence emission of the fluorophore is induced following the degradation of the double strands. The degradation rates of the dsRNA are decelerated in response to chemical or environmental toxicity; therefore, in the case of cellular toxicity, the dsRNA is not degraded and remains intact, thus quenching the fluorescence. Unlike in conventional cell-counting assays, this new assay eliminates time-consuming steps, and can be used to simply evaluate the cellular toxicity via a single reaction. Our results demonstrate that this assay can rapidly quantify the RNA degradation rates in vivo within 4 h for three model chemicals. We propose that this assay will be useful for monitoring cellular toxicity in high-throughput applications.

  3. Low cytotoxicity fluorescent PAMAM dendrimer as gene carriers for monitoring the delivery of siRNA

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Lingmei [Sichuan University, State Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, College of Life Sciences (China); Huang, Saipeng [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China); Chen, Zhao [Xi’an Jiaotong University, School of Science (China); Li, Yanchao [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China); Liu, Ke [Sichuan University, State Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, College of Life Sciences (China); Liu, Yang, E-mail: yliu@iccas.ac.cn; Du, Libo, E-mail: dulibo@iccas.ac.cn [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China)

    2015-09-15

    Visual detection of gene vectors has attracted a great deal of attention due to the application of these vectors in monitoring and evaluating the effect of gene carriers in living cells. A non-viral vector, the fluorescent PAMAM dendrimer (F-PAMAM), was synthesized through conjugation of PAMAM dendrimers and fluorescein. In vitro and ex vivo experiments show that F-PAMAM exhibits superphotostability, low cytotoxicity and facilitates endocytosis by A549 cells. The vector has a high siRNA binding affinity and it increases the efficiency of cy5-siRNA delivery in A549 cells, in comparison with a cy5-siRNA monomer. Our results provide a new method for simultaneously monitoring the delivery of siRNA and its non-viral carriers in living cells.

  4. Monitoring Protein and Starch Accumulation in Wheat Grains with Leaf SPAD and Canopy Spectral Reflectance

    Institute of Scientific and Technical Information of China (English)

    TIAN Yong-chao; ZHU yan; CAO Wei-xing; FAN Xue-mei; LIU Xiao-jun

    2003-01-01

    The research was conducted to determine the relationships of protein and starch accumulation dynamics in grains of wheat to post-heading leaf SPAD values and canopy spectral reflectance. The results showed that leaf nitrogen accumulation was exponentially related to leaf SPAD values and linearly related to canopy spectral reflectance, and that there was negative linear relationship between leaf nitrogen accumulation and grain protein accumulation, but positive linear relationship between post-heading leaf nitrogen translocation and grain protein accumulation at maturity. In addition, leaf SPAD values were parabolically related with and ratio indices R(1 500,610) and R(1 220,560) were exponentially related with protein and starch accumulation in grains. These results indicate that leaf SPAD values and canopy spectral reflectance should be good indicators of quality formation dynamics in wheat grains.

  5. The HEX1 gene of Fusarium graminearum is required for fungal asexual reproduction and pathogenesis and for efficient viral RNA accumulation of Fusarium graminearum virus 1.

    Science.gov (United States)

    Son, Moonil; Lee, Kyung-Mi; Yu, Jisuk; Kang, Minji; Park, Jin Man; Kwon, Sun-Jung; Kim, Kook-Hyung

    2013-09-01

    The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus.

  6. Differential mRNA Accumulation upon Early Arabidopsis thaliana Infection with ORMV and TMV-Cg Is Associated with Distinct Endogenous Small RNAs Level.

    Science.gov (United States)

    Zavallo, Diego; Debat, Humberto Julio; Conti, Gabriela; Manacorda, Carlos Augusto; Rodriguez, Maria Cecilia; Asurmendi, Sebastian

    2015-01-01

    Small RNAs (sRNAs) play important roles in plant development and host-pathogen interactions. Several studies have highlighted the relationship between viral infections, endogenous sRNA accumulation and transcriptional changes associated with symptoms. However, few studies have described a global analysis of endogenous sRNAs by comparing related viruses at early stages of infection, especially before viral accumulation reaches systemic tissues. An sRNA high-throughput sequencing of Arabidopsis thaliana leaf samples infected either with Oilseed rape mosaic virus (ORMV) or crucifer-infecting Tobacco mosaic virus (TMV-Cg) with slightly different symptomatology at two early stages of infection (2 and 4 dpi) was performed. At early stages, both viral infections strongly alter the patterns of several types of endogenous sRNA species in distal tissues with no virus accumulation suggesting a systemic signaling process foregoing to virus spread. A correlation between sRNAs derived from protein coding genes and the associated mRNA transcripts was also detected, indicating that an unknown recursive mechanism is involved in a regulatory circuit encompassing this sRNA/mRNA equilibrium. This work represents the initial step in uncovering how differential accumulation of endogenous sRNAs contributes to explain the massive alteration of the transcriptome associated with plant-virus interactions.

  7. Differential mRNA Accumulation upon Early Arabidopsis thaliana Infection with ORMV and TMV-Cg Is Associated with Distinct Endogenous Small RNAs Level.

    Directory of Open Access Journals (Sweden)

    Diego Zavallo

    Full Text Available Small RNAs (sRNAs play important roles in plant development and host-pathogen interactions. Several studies have highlighted the relationship between viral infections, endogenous sRNA accumulation and transcriptional changes associated with symptoms. However, few studies have described a global analysis of endogenous sRNAs by comparing related viruses at early stages of infection, especially before viral accumulation reaches systemic tissues. An sRNA high-throughput sequencing of Arabidopsis thaliana leaf samples infected either with Oilseed rape mosaic virus (ORMV or crucifer-infecting Tobacco mosaic virus (TMV-Cg with slightly different symptomatology at two early stages of infection (2 and 4 dpi was performed. At early stages, both viral infections strongly alter the patterns of several types of endogenous sRNA species in distal tissues with no virus accumulation suggesting a systemic signaling process foregoing to virus spread. A correlation between sRNAs derived from protein coding genes and the associated mRNA transcripts was also detected, indicating that an unknown recursive mechanism is involved in a regulatory circuit encompassing this sRNA/mRNA equilibrium. This work represents the initial step in uncovering how differential accumulation of endogenous sRNAs contributes to explain the massive alteration of the transcriptome associated with plant-virus interactions.

  8. Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

    Science.gov (United States)

    Vian, A.; Henry-Vian, C.; Davies, E.

    1999-01-01

    It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

  9. A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms

    Science.gov (United States)

    Hedenstierna, K. O.; Lee, Y. H.; Yang, Y.; Fox, G. E.

    1993-01-01

    A prototype stable RNA identification cassette for monitoring genetically engineered plasmids carried by strains of Escherichia coli has been developed. The cassette consists of a Vibrio proteolyticus 5S ribosomal RNA (rRNA) gene surrounded by promoters and terminators from the rrnB operon of Escherischia coli. The identifier RNA is expressed and successfully processed so that approximately 30% of the 5S rRNA isolated from either whole cells or 70S ribosomes is of the V. proteolyticus type. Cells carrying the identifier are readily detectable by hybridization. Accurate measurements show that the identification cassette has little effect on fitness compared to a strain containing an analogous plasmid carrying wild type E. coli 5S rRNA, and the V. proteolyticus 5S rRNA gene is not inactivated after prolonged growth. These results demonstrate the feasibility of developing small standardized identification cassettes that can utilize already existing highly sensitive rRNA detection methods. Cassettes of this type could in principle be incorporated into either the engineered regions of recombinant plasmids or their hosts.

  10. Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples.

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    Full Text Available There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA tubes and PAXgene Blood RNA Tubes (PAXgene tubes. These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.

  11. 16S rRNA beacons for bacterial monitoring during human space missions.

    Science.gov (United States)

    Larios-Sanz, Maia; Kourentzi, Katerina D; Warmflash, David; Jones, Jeffrey; Pierson, Duane L; Willson, Richard C; Fox, George E

    2007-04-01

    Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.

  12. Phylogenetic distribution and evolutionary pattern of an α-proteobacterial small RNA gene that controls polyhydroxybutyrate accumulation in Sinorhizobium meliloti.

    Science.gov (United States)

    Lagares, Antonio; Roux, Indra; Valverde, Claudio

    2016-06-01

    It has become clear that sRNAs play relevant regulatory functions in bacteria. However, a comprehensive understanding of their biological roles considering evolutionary aspects has not been achieved for most of them. Thus, we have characterized the evolutionary and phylogenetic aspects of the Sinorhizobium meliloti mmgR gene encoding the small RNA MmgR, which has been recently reported to be involved in the regulation of polyhydroxybutyrate accumulation in this bacterium. We constructed a covariance model from a multiple sequence and structure alignment of mmgR close homologs that allowed us to extend the search and to detect further remote homologs of the sRNA gene. From our results, mmgR seemed to evolve from a common ancestor of the α-proteobacteria that diverged from the order of Rickettsiales. We have found mmgR homologs in most current species of α-proteobacteria, with a few exceptions in which genomic reduction events or gene rearrangements seem to explain its absence. Furthermore, a strong microsyntenic relationship was found between a large set of mmgR homologs and homologs of a gene encoding a putative N-formyl glutamate amidohydrolase (NFGAH) that allowed us to trace back the evolutionary path of this group of mmgR orthologs. Among them, structure and sequence traits have been completely conserved throughout evolution, namely a Rho-independent terminator and a 10-mer (5'-UUUCCUCCCU-3') that is predicted to remain in a single-stranded region of the sRNA. We thus propose the definition of the new family of α-proteobacterial sRNAs αr8, as well as the subfamily αr8s1 which encompass S. meliloti mmgR orthologs physically linked with the downstream open reading frame encoding a putative NFGAH. So far, mmgR is the trans-encoded small RNA with the widest phylogenetic distribution of well recognized orthologs among α-proteobacteria. Expression of the expected MmgR transcript in rhizobiales other than S. meliloti (Sinorhizobium fredii, Rhizobium

  13. Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP.

    Science.gov (United States)

    Tsutsui, Motomu; Hasegawa, Hitoki; Adachi, Koichi; Miyata, Maiko; Huang, Peng; Ishiguro, Naoki; Hamaguchi, Michinari; Iwamoto, Takashi

    2008-08-08

    Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.

  14. The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins.

    Science.gov (United States)

    López-Carrasco, Amparo; Flores, Ricardo

    2017-07-01

    Avocado sunblotch viroid (ASBVd), the type member of the family Avsunviroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246-250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (-) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2'-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (-) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber viroid, the type member of nuclear viroids (family Pospiviroidae), indicating that two different viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected avocado leaves.

  15. Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation.

    Science.gov (United States)

    Bermejo, B; Remacha, M; Ortiz-Reyes, B; Santos, C; Ballesta, J P

    1994-02-11

    Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included. The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted. Deletions in the 5' region of the two genes have been found to oppositely affect their expression. Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell. A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell. The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation. Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect. The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers. In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide. YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44').

  16. Monitoring of an RNA Multistep Folding Pathway by Isothermal Titration Calorimetry

    Science.gov (United States)

    Reymond, Cédric; Bisaillon, Martin; Perreault, Jean-Pierre

    2009-01-01

    Abstract Isothermal titration calorimetry was used to monitor the energetic landscape of a catalytic RNA, specifically that of the hepatitis delta virus ribozyme. Using mutants that isolated various tertiary interactions, the thermodynamic parameters of several ribozyme-substrate intermediates were determined. The results shed light on the impact of several tertiary interactions on the global structure of the ribozyme. In addition, the data indicate that the formation of the P1.1 pseudoknot is the limiting step of the molecular mechanism. Last, as illustrated here, isothermal titration calorimetry appears to be a method of choice for the elucidation of an RNA's folding pathway. PMID:19134473

  17. Gold-nanobeacons for real-time monitoring of RNA synthesis.

    Science.gov (United States)

    Rosa, João; Conde, João; de la Fuente, Jesus M; Lima, João C; Baptista, Pedro V

    2012-01-01

    Measuring RNA synthesis and, when required, the level of inhibition, is crucial towards the development of practical strategies to evaluate silencing efficiency of gene silencing approaches. We developed a direct method to follow RNA synthesis in real time based on gold nanoparticles (AuNPs) functionalized with a fluorophore labeled hairpin-DNA, i.e. gold-nanobeacon (Au-nanobeacon). Under hairpin configuration, proximity to gold nanoparticles leads to fluorescence quenching; hybridization to a complementary target restores fluorescence emission due to the Au-nanobeacons' conformational reorganization that causes the fluorophore and the AuNP to part from each other, yielding a quantitative response. With this reporter Au-nanobeacon we were able to measure the rate of in vitro RNA synthesis (~10.3 fmol of RNA per minute). Then, we designed a second Au-nanobeacon targeting the promoter sequence (inhibitor) so as to inhibit transcription whilst simultaneously monitor the number of promoters being silenced. Using the two Au-nanobeacons in the same reaction mixture, we are capable of quantitatively assess in real time the synthesis of RNA and the level of inhibition. The biosensor concept can easily be extended and adapted to situations when real-time quantitative assessment of RNA synthesis and determination of the level of inhibition are required. In fact, this biosensor may assist the in vitro evaluation of silencing potential of a given sequence to be later used for in vivo gene silencing.

  18. Fluorescence assays for monitoring RNA-ligand interactions and riboswitch-targeted drug discovery screening.

    Science.gov (United States)

    Liu, J; Zeng, C; Zhou, S; Means, J A; Hines, J V

    2015-01-01

    Riboswitches and other noncoding regulatory RNA are intriguing targets for the development of therapeutic agents. A significant challenge in the drug discovery process, however, is the identification of potent compounds that bind the target RNA specifically and disrupt its function. Essential to this process is an effectively designed cascade of screening assays. A screening cascade for identifying compounds that target the T box riboswitch antiterminator element is described. In the primary assays, moderate to higher throughput screening of compound libraries is achieved by combining the sensitivity of fluorescence techniques with functionally relevant assays. Active compounds are then validated and the binding to target RNA further characterized in secondary assays. The cascade of assays monitor ligand-induced changes in the steady-state fluorescence of an attached dye or internally incorporated 2-aminopurine; the fluorescence anisotropy of an RNA complex; and, the thermal denaturation fluorescence profile of a fluorophore-quencher labeled RNA. While the assays described have been developed for T box riboswitch-targeted drug discovery, the fluorescence methods and screening cascade design principles can be applied to drug discovery efforts targeted toward other medicinally relevant noncoding RNA.

  19. In vivo Monitoring of microRNA Biogenesis Using Reporter Gene Imaging

    OpenAIRE

    2013-01-01

    MicroRNAs are small noncoding RNAs regulating gene expression, through base paring with their target mRNAs, which have been actively investigated as key regulators in a wide range of biological processes. Conventional methods such as Northern blot are generally time-consuming, non-repeatable, and cannot be applied in vivo due to the requirement for cell fixation. Therefore, a noninvasive imaging system is required for the monitoring of microRNA biogenesis to understand the versatile functions...

  20. Monitoring the accumulated water soluble airborne compounds deposited on surfaces of showcases and walls in museums, archives and historical buildings

    DEFF Research Database (Denmark)

    Skytte, Lilian; Rasmussen, Kaare Lund; Svensmark, Bo

    2017-01-01

    to implement by curators and conservators, who can the send the flush water to specialized laboratories. Brief summary: A new methodology capable of monitoring the accumulated airborne deposits on surfaces in showcases and historic buildings is presented and tested. The method is cheap and is easy to implement...... themselves. This might make the compounds seem absent from analyses of indoor air samples. Context and purpose of the study: A new method of detecting water soluble pollutants without taking samples from the interior walls or from the CH objects themselves has been developed. The method involves sampling...... the pollutants accumulated on a surface near the CH object, e.g. a nearby wall or an interior glass surface of a showcase. The samples were obtained by gently flushing the surface with deionised water to collect the ions readily removed from the surface. The method was tested on a variety of surfaces. Results...

  1. Specific binding of Fusarium graminearum Hex1 protein to untranslated regions of the genomic RNA of Fusarium graminearum virus 1 correlates with increased accumulation of both strands of viral RNA.

    Science.gov (United States)

    Son, Moonil; Choi, Hoseong; Kim, Kook-Hyung

    2016-02-01

    The HEX1 gene of Fusarium graminearum was previously reported to be required for the efficient accumulation of Fusarium graminearum virus 1 (FgV1) RNA in its host. To investigate the molecular mechanism underlying the production of FgHEX1 and the replication of FgV1 viral RNA, we conducted electrophoretic mobility shift assays (EMSA) with recombinant FgHex1 protein and RNA sequences derived from various regions of FgV1 genomic RNA. These analyses demonstrated that FgHex1 and both the 5'- and 3'-untranslated regions of plus-strand FgV1 RNA formed complexes. To determine whether FgHex1 protein affects FgV1 replication, we quantified accumulation viral RNAs in protoplasts and showed that both (+)- and (-)-strands of FgV1 RNAs were increased in the over-expression mutant and decreased in the deletion mutant. These results indicate that the FgHex1 functions in the synthesis of both strands of FgV1 RNA and therefore in FgV1 replication probably by specifically binding to the FgV1 genomic RNA. Copyright © 2016. Published by Elsevier Inc.

  2. Osteocalcin and matrix GLA protein in developing teleost teeth: identification of sites of mRNA and protein accumulation at single cell resolution.

    Science.gov (United States)

    Ortiz-Delgado, J B; Simes, D C; Gavaia, P; Sarasquete, C; Cancela, M L

    2005-08-01

    In this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence of BGP and MGP mRNA in teeth was revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules, while mRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP and MGP gene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.

  3. Mungbean yellow mosaic virus (MYMV) AC4 suppresses post-transcriptional gene silencing and an AC4 hairpin RNA gene reduces MYMV DNA accumulation in transgenic tobacco.

    Science.gov (United States)

    Sunitha, Sukumaran; Shanmugapriya, Gnanasekaran; Balamani, Veluthambi; Veluthambi, Karuppannan

    2013-06-01

    Mungbean yellow mosaic virus (MYMV) is a legume-infecting geminivirus that causes yellow mosaic disease in blackgram, mungbean, soybean, Frenchbean and mothbean. AC4/C4, which is nested completely within the Rep gene, is less conserved among geminiviruses. Much less is known about its role in viral pathogenesis other than its known role in the suppression of host-mediated gene silencing. Transient expression of MYMV AC4 by agroinfiltration suppressed post-transcriptional gene silencing in Nicotiana benthamiana 16c expressing green fluorescence protein, at a level comparable to MYMV TrAP expression. AC4 full-length gene and an inverted repeat of AC4 (comprising the full-length AC4 sequence in sense and antisense orientations with an intervening intron) which makes a hairpin RNA (hpRNA) upon transcription were introduced into tobacco by Agrobacterium-mediated leaf disc transformation. Leaf discs of the transgenic plants were agroinoculated with partial dimers of MYMV and used to study the effect of the AC4-sense and AC4 hpRNA genes on MYMV DNA accumulation. Leaf discs of two transgenic plants that express the AC4-sense gene displayed an increase in MYMV DNA accumulation. Leaf discs of six transgenic plants containing the AC4 hpRNA gene accumulated small-interfering RNAs (siRNAs) specific to AC4, and upon agroinoculation with MYMV they exhibited a severe reduction in the accumulation of MYMV DNA. Thus, the MYMV AC4 hpRNA gene has emerged as a good candidate to engineer resistance against MYMV in susceptible plants.

  4. β-catenin accumulation in nuclei of hepatocellular carcinoma cells up-regulates glutathione-s-transferase M3 mRNA

    Institute of Scientific and Technical Information of China (English)

    Yu-Sang Li; Min Liu; Yoshihiro Nakata; He-Bin Tang

    2011-01-01

    AIM: To identify the differentially over-expressed genes associated with β-catenin accumulation in nuclei of hepatocellular carcinoma (HCC) cells.METHODS: Differentially expressed genes were identified in radiation-induced B6C3 F1 mouse HCC cells by mRNA differential display, Northern blot and RT-PCR,respectively. Total glutathione-s-transferase (GST) activity was measured by GST activity assay and β-catenin localization was detected with immunostaining in radiation-induced mouse HCC cells and in HepG2 cell lines.RESULTS: Two up-regulated genes, glutamine synthetase and glutathione-s-transferase M3 (GSTM3), were identified in radiation-induced mouse HCC cells. Influence of β-catenin accumulation in nuclei of HCC cells on upregulation of GSTM3 mRNA was investigated. The nearby upstream domain of GSTM3 contained the β-catenin/Tcf-Lef consensus binding site sequences [5'-(A/T)(A/T)CAAAG-3'], and the total GST activity ratio was considerably higher in B6C3F1 mouse HCC cells with β-cateninaccumulation in nuclei of HCC cells than in those withoutβ-catenin accumulation (0.353 ± 0.117 vs 0.071 ± 0.064,P < 0.001). The TWS119 (a distinct GSK-3β inhibitor)-induced total GST activity was significantly higher in HepG2 cells with β-catenin accumulation than in those withoutβ-catenin accumulation in nuclei of HCC cells. Additionally,the GSTM3 mRNA level was significantly higher at 24 h than at 12 h in TWS119-treated HepG2 cells.CONCLUSION: β-catenin accumulation increases GST activity in nuclei of HCC cells, and GSTM3 may be a novel target gene of the β-catenin/Tcf-Lef complex.

  5. Accumulation of mercury by aufwuchs in Wisconsin seepage lakes: Implications for monitoring

    Science.gov (United States)

    Cope, W. Gregory; Rada, Ronald G.

    1992-01-01

    We examined temporal variation in the total Hg content of aufwuchs (attached algae) collected from artificial substrates in 11 seepage lakes in north-central Wisconsin and its relation to the Hg content of resident yellow perch Perca flavescens from the lakes. Dry weight concentrations of Hg in aufwuchs varied temporally, as follows: summer/fall 1985 > summer 1985 > spring/summer 1986. Areal concentrations of Hg during the 1985 sampling periods were greater than concentrations during the spring/summer 1986 period, but did not differ from each other. The analyses of aufwuchs do not indicate potential accumulation of Hg in fish in these lakes.

  6. A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication

    Directory of Open Access Journals (Sweden)

    Stoltzfus C Martin

    2006-02-01

    Full Text Available Abstract Background Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1 primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss, which are positively regulated by exonic splicing enhancers (ESE and negatively regulated by exonic splicing silencers (ESS and intronic splicing silencers (ISS. We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. Results Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. Conclusion These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non

  7. Toxic cyanobacterial breakthrough and accumulation in a drinking water plant: a monitoring and treatment challenge.

    Science.gov (United States)

    Zamyadi, Arash; MacLeod, Sherri L; Fan, Yan; McQuaid, Natasha; Dorner, Sarah; Sauvé, Sébastien; Prévost, Michèle

    2012-04-01

    The detection of cyanobacteria and their associated toxins has intensified in recent years in both drinking water sources and the raw water of drinking water treatment plants (DWTPs). The objectives of this study were to: 1) estimate the breakthrough and accumulation of toxic cyanobacteria in water, scums and sludge inside a DWTP, and 2) to determine whether chlorination can be an efficient barrier to the prevention of cyanotoxin breakthrough in drinking water. In a full scale DWTP, the fate of cyanobacteria and their associated toxins was studied after the addition of coagulant and powdered activated carbon, post clarification, within the clarifier sludge bed, after filtration and final chlorination. Elevated cyanobacterial cell numbers (4.7 × 10(6)cells/mL) and total microcystins concentrations (up to 10 mg/L) accumulated in the clarifiers of the treatment plant. Breakthrough of cells and toxins in filtered water was observed. Also, a total microcystins concentration of 2.47 μg/L was measured in chlorinated drinking water. Cyanobacterial cells and toxins from environmental bloom samples were more resistant to chlorination than results obtained using laboratory cultured cells and dissolved standard toxins.

  8. Seasonal ERT monitoring of subsurface processes connected to freezing, thawing, snow accumulation and melt cycles

    Science.gov (United States)

    Krzeminska, Dominika; Starkloff, Torsten; Bloem, Esther; Stolte, Jannes

    2016-04-01

    For a better understanding of processes that influence snowmelt infiltration and runoff, and their consequences on soil erosion during spring periods, we established a long-term winter-spring ERT transect in the Gryteland catchment (Norway). The ERT transect is 71 m long, with 1 m spacing between the electrodes. It covers a depression with a north and south facing slope. The readings are collected once a week and, if needed, after a sudden change in weather conditions. Additionally, the soil transect is equipped with six TDR profiles, which register soil moisture and soil temperature every thirty minutes, at five depths (5, 10, 20, 30, 40 cm), for quantifying the ERT readings. The measurements performed during winter 2014/2015 gave promising results and showed the potential of ERT monitoring for understanding the soil thermal and hydraulic processes occurring during a winter and early spring. Moreover, there are visible differences in temporal trends and spatial variations in observed ERT patterns on the opposite facing slopes, which are of special interest. With the on-going experiment, we are aiming to understand the reoccurrence of the observed processes as well as to quantify soil moisture patterns. Herein, we would like to present the preliminary result of two ERT experiments (2014/2015 and 2015/2016) and discuss the advantages and limitations of our experiments. Moreover, we would like to stimulate the discussion about the potential of ERT for spatial and temporal monitoring of soil hydraulic and thermal processes and indirect measurements of soil water content.

  9. A diaCEST MRI approach for monitoring liposomal accumulation in tumors.

    Science.gov (United States)

    Chan, Kannie W Y; Yu, Tao; Qiao, Yuan; Liu, Qiang; Yang, Ming; Patel, Himatkumar; Liu, Guanshu; Kinzler, Kenneth W; Vogelstein, Bert; Bulte, Jeff W M; van Zijl, Peter C M; Hanes, Justin; Zhou, Shibin; McMahon, Michael T

    2014-04-28

    Nanocarrier-based chemotherapy allows preferential delivery of therapeutics to tumors and has been found to improve the efficacy of cancer treatment. However, difficulties in tracking nanocarriers and evaluating their pharmacological fates in patients have limited judicious selection of patients to those who might most benefit from nanotherapeutics. To enable the monitoring of nanocarriers in vivo, we developed MRI-traceable diamagnetic Chemical Exchange Saturation Transfer (diaCEST) liposomes. The diaCEST liposomes were based on the clinical formulation of liposomal doxorubicin (i.e. DOXIL®) and were loaded with barbituric acid (BA), a small, organic, biocompatible diaCEST contrast agent. The optimized diaCEST liposomal formulation with a BA-to-lipid ratio of 25% exhibited 30% contrast enhancement at B1=4.7μT in vitro. The contrast was stable, with ~80% of the initial CEST signal sustained over 8h in vitro. We used the diaCEST liposomes to monitor the response to tumor necrosis factor-alpha (TNF-α), an agent in clinical trials that increases vascular permeability and uptake of nanocarriers into tumors. After systemic administration of diaCEST liposomes to mice bearing CT26 tumors, we found an average diaCEST contrast at the BA frequency (5ppm) of 0.4% at B1=4.7μT while if TNF-α was co-administered the contrast increased to 1.5%. This novel approach provides a non-radioactive, non-metallic, biocompatible, semi-quantitative, and clinically translatable approach to evaluate the tumor targeting of stealth liposomes in vivo, which may enable personalized nanomedicine.

  10. Mutations that alter a repeated ACCA element located at the 5' end of the Potato virus X genome affect RNA accumulation.

    Science.gov (United States)

    Park, Mi-Ri; Kwon, Sun-Jung; Choi, Hong-Soo; Hemenway, Cynthia L; Kim, Kook-Hyung

    2008-08-15

    The repeated ACCA or AC-rich sequence and structural (SL1) elements in the 5' non-translated region (NTR) of the Potato virus X (PVX) RNA play vital roles in the PVX life cycle by controlling translation, RNA replication, movement, and assembly. It has already been shown that the repeated ACCA or AC-rich sequence affect both gRNA and sgRNA accumulation, while not affecting minus-strand RNA accumulation, and are also required for host protein binding. The functional significance of the repeated ACCA sequence elements in the 5' NTR region was investigated by analyzing the effects of deletion and site-directed mutations on PVX replication in Nicotiana benthamiana plants and NT1 protoplasts. Substitution (ACCA into AAAA or UUUU) mutations introduced in the first (nt 10-13) element in the 5' NTR of the PVX RNA significantly affected viral replication, while mutations introduced in the second (nt 17-20) and third (nt 20-23) elements did not. The fourth (nt 29-32) ACCA element weakly affected virus replication, whereas mutations in the fifth (nt 38-41) significantly reduced virus replication due to the structure disruption of SL1 by AAAA and/or UUUU substitutions. Further characterization of the first ACCA element indicated that duplication of ACCA at nt 10-13 (nt 10-17, ACCAACCA) caused severe symptom development as compared to that of wild type, while deletion of the single element (nt 10-13), DeltaACCA) or tripling of this element caused reduced symptom development. Single- and double-nucleotide substitutions introduced into the first ACCA element revealed the importance of CC located at nt positions 11 and 12. Altogether, these results indicate that the first ACCA element is important for PVX replication.

  11. Natural Radioactivity Accumulated in the Arctic from Long-range Atmospheric Transport - Observations in Canadian Monitoring Stations

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jing; Zhang, Weihua [Radiation Protection Bureau, Health Canada, 775 Brookfield Road, Ottawa K1A 1C1 (Canada)

    2014-07-01

    In the environment, the main sources of naturally occurring radionuclides come from radionuclides in the uranium decay series. Activity concentrations of uranium decay series radionuclides may vary considerably from place to place depending on the geological characteristics at the location. Their releases to the atmosphere are mainly through radon ({sup 222}Rn), a radioactive noble gas occurring naturally as an indirect decay product of uranium in soils and rocks. Due to the abundance of uranium, radon continuously emanates from continental land masses. With radon as the main source of naturally occurring radioactivity in the environment, one would think that the Arctic should be an area of low background radiation, because a considerable area of the Arctic is covered by glaciers and permafrost, and radon emanation rate has been reported to be negligible from those glacier and permafrost areas. However, available data have shown the opposite. The elevated level of naturally occurring radioactivity in the Arctic is due to natural sources outside of the Arctic, mainly through long-range atmospheric transport of radon and radon progeny. In some cases, natural radioactivity can accumulate to relatively high levels and become a health concern or a limiting factor of country food consumption. By definition, contaminants are undesirable substances which can cause harm to the environment, the biota, and humans. We can call these naturally accumulating radiological burdens to the Arctic 'natural contaminants' to distinguish them from the traditional meaning of contamination, the 'artificial contaminants' which are attributable to industrial or man-made sources. This paper reviews information available in the literature, analyses long-term atmospheric monitoring data in the Canadian high Arctic, sub-Arctic and mid-latitude sites, and provides discussion on research needed to address questions, such as how heavily the Arctic has been impacted by the

  12. RNA-Seq analysis uncovers non-coding small RNA system of Mycobacterium neoaurum in the metabolism of sterols to accumulate steroid intermediates.

    Science.gov (United States)

    Liu, Min; Zhu, Zhan-Tao; Tao, Xin-Yi; Wang, Feng-Qing; Wei, Dong-Zhi

    2016-04-25

    Understanding the metabolic mechanism of sterols to produce valuable steroid intermediates in mycobacterium by a noncoding small RNA (sRNA) view is still limited. In the work, RNA-seq was implemented to investigate the noncoding transcriptome of Mycobacterium neoaurum (Mn) in the transformation process of sterols to valuable steroid intermediates, including 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA). A total of 263 sRNA candidates were predicted from the intergenic regions in Mn. Differential expression of sRNA candidates was explored in the wide type Mn with vs without sterol addition, and the steroid intermediate producing Mn strains vs wide type Mn with sterol addition, respectively. Generally, sRNA candidates were differentially expressed in various strains, but there were still some shared candidates with outstandingly upregulated or downregulated expression in these steroid producing strains. Accordingly, four regulatory networks were constructed to reveal the direct and/or indirect interactions between sRNA candidates and their target genes in four groups, including wide type Mn with vs without sterol addition, 9OHAD, ADD, and BNA producing strains vs wide type Mn with sterol addition, respectively. Based on these constructed networks, several highly focused sRNA candidates were discovered to be prevalent in the networks, which showed comprehensive regulatory roles in various cellular processes, including lipid transport and metabolism, amino acid transport and metabolism, signal transduction, cell envelope biosynthesis and ATP synthesis. To explore the functional role of sRNA candidates in Mn cells, we manipulated the overexpression of candidates 131 and 138 in strain Mn-9OHAD, which led to enhanced production of 9OHAD from 1.5- to 2.3-fold during 6 d' fermentation and a slight effect on growth rate. This study revealed the complex and important regulatory

  13. NCPDP recommendations for dose accumulation monitoring in the inpatient setting: Acetaminophen case model, version 1.0.

    Science.gov (United States)

    2016-08-01

    Best practices and guidance are provided for improved electronic detection and alerting of inadvertent supratherapeutic cumulative doses of acetaminophen and other medications with narrow therapeutic ranges in inpatient settings. Despite the use of medication safety technologies, overdosage and associated sentinel events continue to be serious problems in many inpatient settings. The tools needed to monitor and employ dose alerts, accumulators, and warning systems are available to reduce inadvertent overdose. Required are staff training and the implementation of processes that provide guidance and documentation of the drug reconciliation process from admittance to discharge for safe patient passage through the various transitions of care. Recommendations to achieve optimal patient safety outcomes include the adoption and integration of available technologies with full functionality configured to meet the institution's policies and processes, initial training and retraining of all staff who use these systems, continuing education of the patient care staff on the dosing safety requirements, and assigning a prominent role to the clinical pharmacist in the entire drug-use and reconciliation process. The key factors contributing to inadvertent overdosage in inpatient settings include a lack of recognition of recommended maximum daily dosages; failure to optimally communicate medication information at transitions of care; failure to optimally implement medication safety technologies, particularly dose accumulator calculation features and associated alerts; and alert fatigue and override. Copyright © 2016 by the National Council for Prescription Drug Programs (NCPDP).

  14. Met-myoglobin formation, accumulation, degradation, and myoglobin oxygenation monitoring based on multiwavelength attenuance measurement in porcine meat

    Science.gov (United States)

    Nguyen, Thien; Phan, Kien Nguyen; Lee, Jee-Bum; Kim, Jae Gwan

    2016-05-01

    We propose a simple, rapid, and nondestructive method to investigate formation, accumulation, and degradation of met-myoglobin (met-Mb) and myoglobin oxygenation from the interior of porcine meat. For the experiment, color photos and attenuance spectra of porcine meat (well-bled muscle, fat, and mixed) were collected daily to perform colorimetric analysis and to obtain the differences of attenuance between 578 and 567 nm (A578-A567) and between 615 and 630 nm (A630-A615), respectively. Oxy-, deoxy-, and met-myoglobin concentration changes over storage time were also calculated using Beer-Lamberts' law with reflectance intensities at 557, 582, and 630 nm. The change of A578-A567 was well matched with the change of myoglobin oxygenation, and the change of A630-A615 corresponded well with the formation and degradation of met-Mb. In addition, attenuation differences, A578-A567 and A630-A615, were able to show the formation of met-Mb earlier than colorimetric analysis. Therefore, the attenuance differences between wavelengths can be indicators for estimating myoglobin oxygenation and met-Mb formation, accumulation, and degradation, which enable us to design a simple device to monitor myoglobin activities in porcine meat.

  15. Effect of temperature on the pathogenesis, accumulation of viral and satellite RNAs and on plant proteome in peanut stunt virus and satellite RNA-infected plants

    Directory of Open Access Journals (Sweden)

    Aleksandra eObrępalska-Stęplowska

    2015-10-01

    Full Text Available Temperature is an important environmental factor influencing plant development in natural and diseased conditions. The growth rate of plants grown at 27°C is more rapid than for plants grown at 21°C. Thus, temperature affects the rate of pathogenesis progression in individual plants. We have analyzed the effect of temperature conditions (either 21°C or 27°C during the day on the accumulation rate of the virus and satellite RNA (satRNA in Nicotiana benthamiana plants infected by peanut stunt virus (PSV with and without its satRNA, at four time points. In addition, we extracted proteins from PSV and PSV+satRNA-infected plants harvested at 21 dpi, when disease symptoms began to appear on plants grown at 21°C and were well developed on those grown at 27°C, to assess the proteome profile in infected plants compared to mock-inoculated plants grown at these two temperatures, using 2D-gel electrophoresis and mass spectrometry approaches. The accumulation rate of the viral RNAs and satRNA was more rapid at 27°C at the beginning of the infection and then rapidly decreased in PSV-infected plants. At 21 dpi, PSV and satRNA accumulation was higher at 21°C and had a tendency to increase further. In all studied plants grown at 27°C, we observed a significant drop in the identified proteins participating in photosynthesis and carbohydrate metabolism at the proteome level, in comparison to plants maintained at 21°C. On the other hand, the proteins involved in protein metabolic processes were all more abundant in plants grown at 27°C. This was especially evident when PSV-infected plants were analyzed, where increase in abundance of proteins involved in protein synthesis, degradation, and folding was revealed. In mock-inoculated and PSV-infected plants we found an increase in abundance of the majority of stress-related differently-regulated proteins and those associated with protein metabolism. In contrast, in PSV+satRNA-infected plants the shift in the

  16. Zinc-bearing zeolite clinoptilolite improves tissue zinc accumulation in laying hens by enhancing zinc transporter gene mRNA abundance.

    Science.gov (United States)

    Li, Linfeng; Li, Ping; Chen, Yueping; Wen, Chao; Zhuang, Su; Zhou, Yanmin

    2015-08-01

    A study was conducted to investigate effects of zinc-bearing zeolite clinoptilolite (ZnCP), as an alternative for zinc sulfate (ZnSO4), on laying performance, tissue Zn accumulation and Zn transporter genes expression in laying hens. Hy-Line Brown laying hens were allocated to three treatments, each of which had six replicates with 15 hens per replicate, receiving basal diet supplemented with ZnSO4 (control, 80 mg Zn/kg diet), 0.23% ZnCP (40.25 mg Zn/kg diet) and 0.46% ZnCP (80.50 mg Zn/kg diet) for 8 weeks, respectively. Compared with control, hens fed diet containing 0.23% ZnCP had similar Zn content in measured tissues (P > 0.05). A higher ZnCP inclusion (0.46%) enhanced Zn accumulation in liver (P < 0.05) and pancreas (P < 0.05). In addition, ZnCP inclusion increased blood iron (Fe) content (P < 0.05). ZnCP supplementation enhanced jejunal metallothionein-4 (MT-4) messenger RNA (mRNA) abundance (P < 0.05). ZnCP inclusion at a higher level (0.46%) increased mRNA expression of MT-4 in pancreas (P < 0.05) and zinc transporter-1 (ZnT-1) in jejunum (P < 0.05). The highest ZnT-2 mRNA abundance in jejunum was found in hens fed 0.23% ZnCP inclusion diet (P < 0.05). The results indicated that ZnCP reached a higher bioavailability as compared with ZnSO4 as evidenced by enhanced tissue Zn accumulation and Zn transporter genes expression.

  17. Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing

    National Research Council Canada - National Science Library

    Meyer, Rikke Louise; Saunders, Aaron Marc; Blackall, Linda Louise

    2006-01-01

    ...), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed...

  18. MicroRNA profiling analysis throughout tomato fruit development and ripening reveals potential regulatory role of RIN on microRNAs accumulation.

    Science.gov (United States)

    Gao, Chao; Ju, Zheng; Cao, Dongyan; Zhai, Baiqiang; Qin, Guozheng; Zhu, Hongliang; Fu, Daqi; Luo, Yunbo; Zhu, Benzhong

    2015-04-01

    The development and ripening of tomato fruit are complex processes involving many gene regulatory pathways at the transcriptional and post-transcriptional level. Ripening inhibitor (RIN) is a vital transcription factor, which targets numerous ripening-related genes at the transcriptional level during tomato fruit ripening. MicroRNAs (miRNAs) are a class of short noncoding RNAs that play important roles in post-transcriptional gene regulation. To elucidate the potential regulatory relationship between rin and miRNAs during fruit development and ripening, we identified known miRNAs and profiled their expression in wild-type tomato and rin mutant using a deep sequencing approach combined with quantitative RT-PCR. A total of 33 known miRNA families were identified, of which 14 miRNA families were differently accumulated. Subsequent promoter analysis showed that possible RIN-binding motifs (CArG-box) tended to occur frequently in the promoter regions of partial differently expressed miRNAs. In addition, ethylene may participate in the regulation of miRNAs accumulation during tomato fruit ripening. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay confirmed the direct binding of RIN to the promoter of MIR172a. Collectively, these results showed a close correlation between miRNA expression and RIN as well as ethylene, which further elucidated the regulatory roles of miRNAs during fruit development and ripening and enriched the regulatory network of RIN in tomato fruit.

  19. Transgenic accumulation of a defective cucumber mosaic virus (CMV) replicase derived double stranded RNA modulates plant defence against CMV strains O and Y in potato.

    Science.gov (United States)

    Ntui, Valentine Otang; Kynet, Kong; Azadi, Pejman; Khan, Raham Sher; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro

    2013-12-01

    Cucumber mosaic virus is an important plant pathogen with a broad host range encompassing many plant species. This study demonstrates the production of transgenic potato lines exhibiting complete resistance to cucumber mosaic virus strain O and Y by post transcriptional gene silencing. Two constructs were used, one, pEKH2IN2CMVai, contains inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O), while the other, TRV-based VIGS vector (pTRV2CMVai), contains the same fragment of the replicase gene, but without inverted repeat. These constructs were used to produce transgenic potato lines of cultivar 'Danshaku', a susceptible genotype to CMV. Transgenic lines derived from pEKH2IN2CMVai accumulated small interfering RNA (siRNA) before and after virus challenge, whereas those derived from pTRV2CMVai showed siRNA expression after virus challenge. When transgenic lines were challenged with CMV-O or CMV-Y, four lines exhibited complete (100%) resistance to both strains, whereas the other lines had high levels of resistance. Infectivity of CMV-O was lower than that of CMV-Y in the highly resistant plants. There were no significant differences with regard to resistance between plants derived from pEKH2IN2CMVai and those obtained from pTRV2CMVai. The presence of CMV-specific siRNA in the resistant phenotypes indicates that the resistance was acquired through RNA silencing.

  20. Automatic semi-continuous accumulation chamber for diffuse gas emissions monitoring in volcanic and non-volcanic areas

    Science.gov (United States)

    Lelli, Matteo; Raco, Brunella; Norelli, Francesco; Virgili, Giorgio; Continanza, Davide

    2016-04-01

    Since various decades the accumulation chamber method is intensively used in monitoring activities of diffuse gas emissions in volcanic areas. Although some improvements have been performed in terms of sensitivity and reproducibility of the detectors, the equipment used for measurement of gas emissions temporal variation usually requires expensive and bulky equipment. The unit described in this work is a low cost, easy to install-and-manage instrument that will make possible the creation of low-cost monitoring networks. The Non-Dispersive Infrared detector used has a concentration range of 0-5% CO2, but the substitution with other detector (range 0-5000 ppm) is possible and very easy. Power supply unit has a 12V, 7Ah battery, which is recharged by a 35W solar panel (equipped with charge regulator). The control unit contains a custom programmed CPU and the remote transmission is assured by a GPRS modem. The chamber is activated by DataLogger unit, using a linear actuator between the closed position (sampling) and closed position (idle). A probe for the measure of soil temperature, soil electrical conductivity, soil volumetric water content, air pressure and air temperature is assembled on the device, which is already arranged for the connection of others external sensors, including an automatic weather station. The automatic station has been tested on the field at Lipari island (Sicily, Italy) during a period of three months, performing CO2 flux measurement (and also weather parameters), each 1 hour. The possibility to measure in semi-continuous mode, and at the same time, the gas fluxes from soil and many external parameters, helps the time series analysis aimed to the identification of gas flux anomalies due to variations in deep system (e.g. onset of volcanic crises) from those triggered by external conditions.

  1. siRNA对烟草原生质体中TMV RNA积累的干扰作用研究%siRNA-directed Interference of TMV RNA Accumulation in Tobacco Protoplast

    Institute of Scientific and Technical Information of China (English)

    赵明敏; 杨向东; 鲁红学; 张长青

    2007-01-01

    RNA interference(RNAi)is a mechanism of post-transcriptional gene silencing(PTGS)that has been described in plants.RNAi silences gene expression through small interfering RNA(siRNA)that guides mRNA degradation in a sequence-specific manner.Here,we report that siRNA inhibits virus accumulation by targeting TMV 126 kD protein gene in tobacco protoplasts.ELISA and Northern blot assays displayed that protoplasts electroporated with siRNA+TMV showed a sigmficant reduction of TMV RNA concentration.In hypersensitive host.leaves inoculated by protoplast transfected with siRNA+TMV displayed a few local lesions compared with the controls.These indicated that siRNA could interfere with TMV infection in tobacco protoplasts.Therefore,this protoplast system could facilitate rapid and quantitative analysis for siRNA-mediated interference on plant virus infection.%RNA干扰被认为是转录后基因沉默的一种机制.RNA干扰通过小干扰RNA特异性降解目标mRNA来沉默基因表达.本文以烟草花叶病毒126 kD蛋白为靶蛋白,在原生质体水平上研究了小干扰RNA对病毒侵染的干扰和抑制作用.ELISA和Northern杂交的实验结果表明,共转染小干扰RNA和TMV的原生质体内检测到较低的病毒含量.在枯斑寄主上,叶片接种小干扰RNA和TMV共转染原生质体后,与对照叶片相比,仅有很少量的病斑产生.这说明,小干扰RNA能够在原生质体水平对病毒起到干扰和抑制作用.因此认为,烟草原生质体系统有利于快速和定量分析小干扰RNA介导对植物病毒的抑制作用.

  2. Electrochemical monitoring of phytochelatin accumulation in Nicotiana tabacum cells exposed to sub-cytotoxic and cytotoxic levels of cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Fojta, Miroslav [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic)]. E-mail: fojta@ibp.cz; Fojtova, Miloslava [Laboratory of Molecular Epigenetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Havran, Ludek [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Pivonkova, Hana [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Dorcak, Vlastimil [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Sestakova, Ivana [J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejskova 3, 182 23 Prague 8 (Czech Republic)

    2006-02-03

    Cadmium belongs to the most dangerous environmental pollutants among the toxic heavy metals seriously affecting vital functions in both animal and plant cells. It has been previously shown that cadmium ions at 50-100 {mu}M concentrations caused tobacco BY-2 (TBY-2) cells to enter apoptosis within several days of exposure. Phytochelatins (PCs), the 'plant metallothioneins', are cysteine-rich peptides involved in detoxification of heavy metals in plants. The PCs are synthesized in response to the heavy metal exposure. In this paper, we utilized electrochemical analysis to monitor accumulation of PCs in the TBY-2 cells exposed to cadmium ions. Measurements of a characteristic PC signal at mercury electrode in the presence of cobalt ions made it possible to detect changes in the cellular PC levels during the time of cultivation, starting from 30 min after exposure. Upon TBY-2 cultivation in the presence of cytotoxic cadmium concentrations, the PC levels remarkably increased during the pre-apoptotic phase and reached a limiting value at cultivation times coinciding with apoptosis trigger. The PC level observed for a sub-cytotoxic cadmium concentration (10 {mu}M) was about three-times lower than that observed for the 50 or 100 {mu}M cadmium ions after 5 days of exposure. We show that using a simple electrochemical analysis, synthesis of PCs in plant cells can be easily followed in parallel with other tests of the cellular response to the toxic heavy metal stress.

  3. Nucleolar accumulation of RNA binding proteins induced by Actinomycin D is functional in Trypanosoma cruzi and Leishmania mexicana but not in T. brucei.

    Directory of Open Access Journals (Sweden)

    Ezequiel Názer

    Full Text Available We have recently shown in T. cruzi that a group of RNA Binding Proteins (RBPs, involved in mRNA metabolism, are accumulated into the nucleolus in response to Actinomycin D (ActD treatment. In this work, we have extended our analysis to other members of the trypanosomatid lineage. In agreement with our previous study, the mechanism seems to be conserved in L. mexicana, since both endogenous RBPs and a transgenic RBP were relocalized to the nucleolus in parasites exposed to ActD. In contrast, in T. brucei, neither endogenous RBPs (TbRRM1 and TbPABP2 nor a transgenic RBP from T. cruzi were accumulated into the nucleolus under such treatment. Interestingly, when a transgenic TbRRM1 was expressed in T. cruzi and the parasites exposed to ActD, TbRRM1 relocated to the nucleolus, suggesting that it contains the necessary sequence elements to be targeted to the nucleolus. Together, both experiments demonstrate that the mechanism behind nucleolar localization of RBPs, which is present in T. cruzi and L. mexicana, is not functional in T. brucei, suggesting that it has been lost or retained differentially during the evolution of the trypanosomatid lineage.

  4. AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

    Science.gov (United States)

    Hsu, Yi-Feng; Chen, Yun-Chu; Hsiao, Yu-Chun; Wang, Bing-Jyun; Lin, Shih-Yun; Cheng, Wan-Hsing; Jauh, Guang-Yuh; Harada, John J; Wang, Co-Shine

    2014-01-01

    The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth.

  5. Differential induction of chalcone synthase mRNA activity at the onset of phytoalexin accumulation in compatible and incompatible plant-pathogen interactions.

    Science.gov (United States)

    Bell, J N; Dixon, R A; Bailey, J A; Rowell, P M; Lamb, C J

    1984-06-01

    Changes in the mRNA activity of chalcone synthase, the first enzyme of phenylpropanoid metabolism specific to flavonoid/isoflavonoid biosynthesis, have been investigated in relation to expression of the phytoalexin defense response in race-cultivar specific interactions between hypocotyls of Phaseolus vulgaris and the partially biotrophic fungus Colletotrichum lindemuthianum, causal agent of anthracnose. In an incompatible interaction (host resistant) there is an early but localized increase in chalcone synthase mRNA activity prior to the onset of accumulation of the phenylpropanoid-derived phytoalexin phaseoflin and expression of hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there is no induction of mRNA activity in the early stages of infection but rather a delayed, widespread increase during attempted lesion limitation at the onset of symptom development. The data indicate that control of phytoalexin gene expression is a key early component in the defense responses of biologically stressed cells during a race-cultivar specific host-pathogen interaction.

  6. Monitoring mRNA and protein levels in bulk and in model vesicle-based artificial cells.

    Science.gov (United States)

    van Nies, Pauline; Canton, Alicia Soler; Nourian, Zohreh; Danelon, Christophe

    2015-01-01

    With rising interest in utilizing cell-free gene expression systems in bottom-up synthetic biology projects, novel labeling tools need to be developed to accurately report the dynamics and performance of the biosynthesis machinery operating in various reaction conditions. Monitoring the transcription activity has been simplified by the Spinach technology, an RNA aptamer that emits fluorescence upon binding to a small organic dye. Recently, we tracked the fluorescence of Spinach-tagged messenger RNA (mRNA) and its translation product the yellow fluorescent protein (YFP), both synthesized in the protein synthesis using recombinant elements system from a DNA template. Building on our previous study, we describe here an improved Spinach reporter with modified flanking sequences that confer higher propensity for aptamer folding and, thus, enhanced fluorescence brightness. Hence, the kinetics of mRNA and YFP production could be simultaneously monitored with unprecedented sensitivity. A combination of methodologies, comprising RNA gel analysis, real-time quantitative polymerase chain reaction, absorbance measurements, and fluorescence correlation spectroscopy, was used to convert fluorescence intensity units into absolute concentrations of transcript and YFP translational product. Furthermore, we demonstrated that the new Spinach construct greatly enhanced mRNA detection when gene expression was confined inside self-assembled lipid vesicles. Therefore, we argue that this assay could be used to evaluate systematically the performance of transcription and translation in model vesicle-based artificial cells.

  7. The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality.

    Science.gov (United States)

    Radhakrishnan, Aditya; Chen, Ying-Hsin; Martin, Sophie; Alhusaini, Najwa; Green, Rachel; Coller, Jeff

    2016-09-22

    A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. Here, we establish that the DEAD-box protein Dhh1p is a sensor of codon optimality that targets an mRNA for decay. First, we find mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. Biochemical experiments show Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find these effects on mRNA decay are sensitive to the number of slow-moving ribosomes on an mRNA. Moreover, we find Dhh1p overexpression leads to the accumulation of ribosomes specifically on mRNAs (and even codons) of low codon optimality. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay.

  8. Expression of Cucumber mosaic virus suppressor 2b alters FWA methylation and its siRNA accumulation in Arabidopsis thaliana.

    Science.gov (United States)

    Hamera, Sadia; Yan, Youngsheng; Song, Xiaoguang; Chaudhary, Safee Ullah; Murtaza, Iram; Su, Lei; Tariq, Muhammad; Chen, Xiaoying; Fang, Rongxiang

    2016-11-15

    The Cucumber mosaic virus (CMV) suppressor 2b co-localizes with AGO4 in cytoplasmic and nuclear fractions of Arabidopsis thaliana Biochemical fractionation of A. thaliana cellular extracts revealed that 2b and AGO4 coexist in multiple size exclusions. 2b transgenic A. thaliana exhibited an enhanced accumulation of 24nt siRNAs from flowering wageningen (FWA) and other heterochromatic loci. These plants also exhibited hypo-methylation of an endogenous- as well as transgene-FWA promoter at non-CG sites. In corroboration, both transgenic 2b and CMV infection affected the regulation of transposons which mimics the ago4 phenotype. In conclusion, 2b perturbs plant defense by interfering with AGO4-regulated transcriptional gene silencing.

  9. Expression of Cucumber mosaic virus suppressor 2b alters FWA methylation and its siRNA accumulation in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Sadia Hamera

    2016-11-01

    Full Text Available The Cucumber mosaic virus (CMV suppressor 2b co-localizes with AGO4 in cytoplasmic and nuclear fractions of Arabidopsis thaliana. Biochemical fractionation of A. thaliana cellular extracts revealed that 2b and AGO4 coexist in multiple size exclusions. 2b transgenic A. thaliana exhibited an enhanced accumulation of 24nt siRNAs from flowering wageningen (FWA and other heterochromatic loci. These plants also exhibited hypo-methylation of an endogenous- as well as transgene-FWA promoter at non-CG sites. In corroboration, both transgenic 2b and CMV infection affected the regulation of transposons which mimics the ago4 phenotype. In conclusion, 2b perturbs plant defense by interfering with AGO4-regulated transcriptional gene silencing.

  10. Comparison of Roche MONITOR and Organon Teknika NucliSens assays to quantify human immunodeficiency virus type 1 RNA in cerebrospinal fluid.

    Science.gov (United States)

    Spearman, P; Fiscus, S A; Smith, R M; Shepard, R; Johnson, B; Nicotera, J; Harris, V L; Clough, L A; McKinsey, J; Haas, D W

    2001-04-01

    We compared Roche MONITOR and Organon Teknika NucliSens assays for human immunodeficiency virus type 1 (HIV-1) RNA in cerebrospinal fluid (CSF). Results of 282 assays were highly correlated (r = 0.826), with MONITOR values being 0.29 +/- 0.4 log(10) copies/ml (mean +/- standard deviation) values. Both assays can reliably quantify HIV-1 RNA in CSF.

  11. Effects of EPA and DHA on lipid droplet accumulation and mRNA abundance of PAT proteins in caprine monocytes.

    Science.gov (United States)

    Lecchi, Cristina; Invernizzi, Guido; Agazzi, Alessandro; Modina, Silvia; Sartorelli, Paola; Savoini, Giovanni; Ceciliani, Fabrizio

    2013-04-01

    The present study investigated the in vitro effects on caprine monocytes of two ω-3 PUFAs, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on lipid droplet formation, an emerging process of fundamental importance in innate immunity regulation. The mRNA abundance of PAT protein family (PLIN1, PLIN2 and PLIN3), involved in the formation and trafficking of the droplets, was also assessed. The effects of EPA and DHA on monocyte apoptosis were studied as well. The number of lipid droplets per cell was found to be dependent on both type and concentration of fatty acid. ω-3 PUFAs upregulated PLIN3 and PLIN2 gene expression, as well as apoptosis rate. The present findings suggest that PUFA might modify innate immune functions of goat monocytes by interfering with the formation of lipid droplets and by upregulating proteins belonging to PAT protein family.

  12. Role of microRNA 1207-5P and its host gene, the long non-coding RNA Pvt1, as mediators of extracellular matrix accumulation in the kidney: implications for diabetic nephropathy.

    Science.gov (United States)

    Alvarez, M Lucrecia; Khosroheidari, Mahdieh; Eddy, Elena; Kiefer, Jeff

    2013-01-01

    Diabetic nephropathy is the most common cause of chronic kidney failure and end-stage renal disease in the Western World. One of the major characteristics of this disease is the excessive accumulation of extracellular matrix (ECM) in the kidney glomeruli. While both environmental and genetic determinants are recognized for their role in the development of diabetic nephropathy, epigenetic factors, such as DNA methylation, long non-coding RNAs, and microRNAs, have also recently been found to underlie some of the biological mechanisms, including ECM accumulation, leading to the disease. We previously found that a long non-coding RNA, the plasmacytoma variant translocation 1 (PVT1), increases plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor beta 1 (TGF-β1) in mesangial cells, the two main contributors to ECM accumulation in the glomeruli under hyperglycemic conditions, as well as fibronectin 1 (FN1), a major ECM component. Here, we report that miR-1207-5p, a PVT1-derived microRNA, is abundantly expressed in kidney cells, and is upregulated by glucose and TGF-β1. We also found that like PVT1, miR-1207-5p increases expression of TGF-β1, PAI-1, and FN1 but in a manner that is independent of its host gene. In addition, regulation of miR-1207-5p expression by glucose and TGFβ1 is independent of PVT1. These results provide evidence supporting important roles for miR-1207-5p and its host gene in the complex pathogenesis of diabetic nephropathy.

  13. Changes in Growth, Carbon and Nitrogen Enzyme Activity and mRNA Accumulation in the Halophilic Microalga Dunaliella viridisin Response to NaCl Stress

    Institute of Scientific and Technical Information of China (English)

    WANG Dongmei; WANG Weiwei; XU Nianjun; SUN Xue

    2016-01-01

    Manyspecies ofmicroalgaDunaliellaexhibit a remarkable tolerance to salinity and are therefore ideal for probing the effects of salinity. In this work, we assessed the effects of NaCl stress on the growth, activity and mRNA level of carbon and nitrogen metabolism enzymes ofD. viridis. The alga could grow over a salinity range of 0.44molL−1 to 3.00molL−1 NaCl, but the most rapid growth was observed at 1.00molL−1NaCl, followed by 2.00molL−1 NaCl. Paralleling these growth patterns, the highest initial and total Rubisco activities were detected in the presence of 1.00molL−1 NaCl, decreasing to 37.33% and 26.39% of those values, re-spectively, in the presence of 3.00molL−1 NaCl, respectively. However, the highest extracellular carbonic anhydrase (CA) activity was measured in the presence of 2.00molL−1 NaCl, followed by 1.00molL−1NaCl. Different from the two carbon enzymes, nitrate reductase (NR) activity showed a slight change under different NaCl concentrations. At the transcriptional level, the mRNAs of Rubisco large subunit (rbcL), and small subunit (rbcS), attained their highest abundances in the presence of 1.00 and 2.00molL−1 NaCl, respectively. The CA mRNA accumulation was induced from 0.44molL−1 to 3.00molL−1 NaCl, but the NR mRNA showed the decreasing tendency with the increasing salinity. In conclusion, the growth and carbon fixation enzyme of Rubisco displayed similar tendency in response to NaCl stress, CA was proved be salt-inducible within a certain salinity range and NR showed the least effect by NaCl inD. viridis.

  14. Changes in growth, carbon and nitrogen enzyme activity and mRNA accumulation in the halophilic microalga Dunaliella viridis in response to NaCl stress

    Science.gov (United States)

    Wang, Dongmei; Wang, Weiwei; Xu, Nianjun; Sun, Xue

    2016-12-01

    Many species of microalga Dunaliella exhibit a remarkable tolerance to salinity and are therefore ideal for probing the effects of salinity. In this work, we assessed the effects of NaCl stress on the growth, activity and mRNA level of carbon and nitrogen metabolism enzymes of D. viridis. The alga could grow over a salinity range of 0.44 mol L-1 to 3.00 mol L-1 NaCl, but the most rapid growth was observed at 1.00 mol L-1 NaCl, followed by 2.00 mol L-1 NaCl. Paralleling these growth patterns, the highest initial and total Rubisco activities were detected in the presence of 1.00 mol L-1 NaCl, decreasing to 37.33% and 26.39% of those values, respectively, in the presence of 3.00 mol L-1 NaCl, respectively. However, the highest extracellular carbonic anhydrase (CA) activity was measured in the presence of 2.00 mol L-1 NaCl, followed by 1.00 mol L-1 NaCl. Different from the two carbon enzymes, nitrate reductase (NR) activity showed a slight change under different NaCl concentrations. At the transcriptional level, the mRNAs of Rubisco large subunit ( rbcL), and small subunit ( rbcS), attained their highest abundances in the presence of 1.00 and 2.00 mol L-1 NaCl, respectively. The CA mRNA accumulation was induced from 0.44 mol L-1 to 3.00 mol L-1 NaCl, but the NR mRNA showed the decreasing tendency with the increasing salinity. In conclusion, the growth and carbon fixation enzyme of Rubisco displayed similar tendency in response to NaCl stress, CA was proved be salt-inducible within a certain salinity range and NR showed the least effect by NaCl in D. viridis.

  15. In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis.

    Science.gov (United States)

    Weiss, Victor U; Bliem, Christina; Gösler, Irene; Fedosyuk, Sofiya; Kratzmeier, Martin; Blaas, Dieter; Allmaier, Günter

    2016-06-01

    Liquid-phase electrophoresis either in the classical capillary format or miniaturized (chip CE) is a valuable tool for quality control of virus preparations and for targeting questions related to conformational changes of viruses during infection. We present an in vitro assay to follow the release of the RNA genome from a human rhinovirus (common cold virus) by using a molecular beacon (MB) and chip CE. The MB, a probe that becomes fluorescent upon hybridization to a complementary sequence, was designed to bind close to the 3' end of the viral genome. Addition of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known additive for reduction of bleaching and blinking of fluorophores in fluorescence microscopy, to the background electrolyte increased the sensitivity of our chip CE set-up. Hence, a fast, sensitive and straightforward method for the detection of viral RNA is introduced. Additionally, challenges of our assay will be discussed. In particular, we found that (i) desalting of virus preparations prior to analysis increased the recorded signal and (ii) the MB-RNA complex signal decreased with the time of virus storage at -70 °C. This suggests that 3'-proximal sequences of the viral RNA, if not the whole genome, underwent degradation during storage and/or freezing and thawing. In summary, we demonstrate, for two independent virus batches, that chip electrophoresis can be used to monitor MB hybridization to RNA released upon incubation of the native virus at 56 °C. Graphical Abstract Schematic of the study strategy: RNA released from HRV-A2 is detected by chip electrophoresis through the increase in fluorescence after genom complexation to a cognate molecular beacon.

  16. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  17. Monitoring the RNA distribution in human embryonic stem cells using Raman micro-spectroscopy and fluorescence imaging

    Science.gov (United States)

    Falamas, A.; Kalra, S.; Chis, V.; Notingher, I.

    2013-11-01

    The aim of this study was to monitor the intracellular distribution of nucleic acids in human embryonic stem cells. Raman micro-spectroscopy and fluorescence imaging investigations were employed to obtain high-spatial resolution maps of nucleic acids. The DNA Raman signal was identified based on the 782 cm-1 band, while the RNA characteristic signal was detected based on the 813 cm-1 fingerprint band assigned to O-P-O symmetric stretching vibrations. Additionally, principal components analysis was performed and nucleic acids characteristic Raman signals were identified in the data set, which were plotted at each position in the cells. In this manner, high intensity RNA signal was identified in the cells nucleolus and cytoplasm, while the nucleus presented a much lower signal.

  18. Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes

    DEFF Research Database (Denmark)

    Li, Bojun; Menzel, Ursula; Loebel, Claudia

    2016-01-01

    Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic...... differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can...... and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation....

  19. Liposomal nanocontainers as models for viral infection: monitoring viral genomic RNA transfer through lipid membranes.

    Science.gov (United States)

    Bilek, Gerhard; Matscheko, Nena M; Pickl-Herk, Angela; Weiss, Victor U; Subirats, Xavier; Kenndler, Ernst; Blaas, Dieter

    2011-08-01

    After uptake into target cells, many nonenveloped viruses undergo conformational changes in the low-pH environment of the endocytic compartment. This results in exposure of amphipathic viral peptides and/or hydrophobic protein domains that are inserted into and either disrupt or perforate the vesicular membranes. The viral nucleic acids thereby gain access to the cytosol and initiate replication. We here demonstrate the in vitro transfer of the single-stranded positive-sense RNA genome of human rhinovirus 2 into liposomes decorated with recombinant very-low-density lipoprotein receptor fragments. Membrane-attached virions were exposed to pH 5.4, mimicking the in vivo pH environment of late endosomes. This triggered the release of the RNA whose arrival in the liposomal lumen was detected via in situ cDNA synthesis by encapsulated reverse transcriptase. Subsequently, cDNA was PCR amplified. At a low ratio between virions and lipids, RNA transfer was positively correlated with virus concentration. However, membranes became leaky at higher virus concentrations, which resulted in decreased cDNA synthesis. In accordance with earlier in vivo data, the RNA passes through the lipid membrane without causing gross damage to vesicles at physiologically relevant virus concentrations.

  20. Target organs of the Manila clam Ruditapes philippinarum for studying metal accumulation and biomarkers in pollution monitoring: laboratory and in-situ transplantation experiments.

    Science.gov (United States)

    Won, Eun-Ji; Kim, Kyung-Tae; Choi, Jin-Young; Kim, Eun-Soo; Ra, Kongtae

    2016-08-01

    To characterize the target organs of the Manila clam Ruditapes philippinarum for use in environmental study, the accumulation of trace metals and three biomarkers was measured in different organs. Exposure with Cu and Pb carried out under laboratory conditions revealed a linear uptake of metals throughout the experimental period in each tissue. In particular, significant increase was observed in gills and mantle. The increase of intracellular reactive oxygen species showed the great potential of gills as a target tissue for both Cu and Pb exposure. The highest activity of glutathione S-transferase and their relative increase in activity were also observed in gills. Metallothionein-like protein levels, however, increased greatly in the digestive gland and mantle during Cu and Pb exposure, respectively, although all tissues, except the foot, showed significant changes after 24 h of metal exposure. In the field study, the highest concentration of metals was recorded in the gills and mantle, accounting for over 50 % of the total accumulated metal in all sites. Additionally, Cu and Pb increased significantly in these two organs, respectively. However, the order of accumulation rate in laboratory exposure was not concomitant with those of the lab-based study, suggesting that different routes of metal uptake and exposure duration induce distinct partitioning of metals and regulating system in R. philippinarum. These series of exposure studies demonstrated that gills, mantle, and digestive gland in R. philippinarum are potential target tissues in environmental monitoring study using metal concentrations and biomarkers.

  1. Environmental monitoring to the sources of atmospheric emission by the Trad-MCN bioassay and analysis of the accumulative potential for uranium and fluoride

    Energy Technology Data Exchange (ETDEWEB)

    Machado, Alessandra C.F.E., E-mail: alessandra@ctmsp.mar.mil.b [Centro Tecnologico da Marinha em Sao Paulo (CTMSP), SP (Brazil). Div. de Monitoracao Ambiental; Ramos, Monique M.B., E-mail: monique@ctmsp.mar.mil.b [Centro Tecnologico da Marinha em Sao Paulo (CTMSP), SP (Brazil). Div. de Analise de Seguranca; Alves, Edenise S., E-mail: ealves@ibot.sp.gov.b [Instituto de Botanica de Sao Paulo, Sao Paulo, SP (Brazil). Secao de Anatomia

    2009-07-01

    The biomonitoring of the atmospheric contamination constitutes important procedure for adoption of environmental control measures. Biological assays have been employed to evaluate genotoxic agents in the atmosphere. The Tradescantia-micronucleus (Trad-MCN) assay has been extensively used in environmental monitoring owing to its efficiency in the detection of chromosomic damages in cytological preparations of easy execution. In this study we tested the viability of use of Trad-MCN with Tradescantia pallida cv. Purpurea for environmental monitoring in the Experimental Center Aramar (CEA), in Ipero - SP and its leaf accumulation capacity. The plants were exposed in situ, in flower-beds or flowerpots, established close to the sources of atmospheric emission. The bioassay was accomplished according to the usual protocol. The micronucleus frequencies were compared using the variance Kruskal-Wallis test. The obtained results indicated that the biomonitoring model adopted was not the ideal for the CEA, considering that the plant suffered the influence of climatic condition. However the plant showed to have accumulative potential for uranium. (author)

  2. Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

    Science.gov (United States)

    Crocetti, Gregory R.; Hugenholtz, Philip; Bond, Philip L.; Schuler, Andrew; Keller, Jürg; Jenkins, David; Blackall, Linda L.

    2000-01-01

    Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were β-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the β-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing ≥98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the β-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR

  3. Monitoring of Chicken RNA Integrity as a Function of Prolonged Postmortem Duration

    Directory of Open Access Journals (Sweden)

    Yuwares Malila

    2015-11-01

    Full Text Available Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6 immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem. The delayed postmortem duration did not significantly affect A260/A280 and A260/A230 (p≥0.05, suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05, the value was not statistically different between 20 min and 6 h samples (p≥0.05, indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, hypoxanthine-guanine phosphoribosyltransferase (HPRT, peptidylprolylisomerase A (PPIA and TATA box-binding protein (TBP as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1, pyruvate dehydrogenase kinase isozyme 4 (PDK4, and peroxisome proliferator-activated receptor delta (PPARD were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05. Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05. The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken

  4. Monitoring the accumulated water soluble airborne compounds deposited on surfaces of showcases and walls in museums, archives and historical buildings

    DEFF Research Database (Denmark)

    Skytte, Lilian; Rasmussen, Kaare Lund; Svensmark, Bo

    2017-01-01

    themselves. This might make the compounds seem absent from analyses of indoor air samples. Context and purpose of the study: A new method of detecting water soluble pollutants without taking samples from the interior walls or from the CH objects themselves has been developed. The method involves sampling...... the pollutants accumulated on a surface near the CH object, e.g. a nearby wall or an interior glass surface of a showcase. The samples were obtained by gently flushing the surface with deionised water to collect the ions readily removed from the surface. The method was tested on a variety of surfaces. Results......-, and SO4 2-. The resulting concentrations were converted to μEq, providing the ionic balance and the relative amounts of the ions and elements present. Ionic balance was observed on smooth and inert surfaces, but in some cases the chemistry of the wall contributed to the flush water. Solid samples of some...

  5. Monitoring of the microbiota of fermented sausages by culture independent rRNA-based approaches.

    Science.gov (United States)

    Greppi, Anna; Ferrocino, Ilario; La Storia, Antonietta; Rantsiou, Kalliopi; Ercolini, Danilo; Cocolin, Luca

    2015-11-06

    In Italy, fermented sausages (called "salami") are consumed in large quantities. Salami samples from a local meat factory in the area of Torino were analyzed at 0, 3, 7, 30 and 45 days of ripening. Swab samples from the production environment were also collected at the beginning of the experiment. The diversity of metabolically active microbiota occurring during the natural fermentation of salami was evaluated by using RT-PCR-DGGE coupled with RNA-based pyrosequencing of the 16S rRNA gene. A culture-dependent approach was also applied to identify and characterize isolated Staphylococcaceae and LAB populations. Staphylococcus succinus, Staphylococcus xylosus and Lactobacillus sakei were the species most frequently isolated during the maturation time. Rep-PCR analysis showed that S. succinus and S. xylosus isolated from swabs and salami samples clustered together, suggesting possible contamination during the production process. RT-PCR-DGGE and rRNA-based pyrosequencing showed that the metabolically active populations were dominated by S. succinus, Lb. sakei and Leuconostoc carnosum. In this specific case study, only a few species belonging to Staphylococcaceae, Lactobacillaceae and Leuconostocaceae may be metabolically active and contribute to determine the final characteristics of the products.

  6. Spatio-temporal monitoring of deep-sea communities using metabarcoding of sediment DNA and RNA

    Directory of Open Access Journals (Sweden)

    Magdalena Guardiola

    2016-12-01

    Full Text Available We assessed spatio-temporal patterns of diversity in deep-sea sediment communities using metabarcoding. We chose a recently developed eukaryotic marker based on the v7 region of the 18S rRNA gene. Our study was performed in a submarine canyon and its adjacent slope in the Northwestern Mediterranean Sea, sampled along a depth gradient at two different seasons. We found a total of 5,569 molecular operational taxonomic units (MOTUs, dominated by Metazoa, Alveolata and Rhizaria. Among metazoans, Nematoda, Arthropoda and Annelida were the most diverse. We found a marked heterogeneity at all scales, with important differences between layers of sediment and significant changes in community composition with zone (canyon vs slope, depth, and season. We compared the information obtained from metabarcoding DNA and RNA and found more total MOTUs and more MOTUs per sample with DNA (ca. 20% and 40% increase, respectively. Both datasets showed overall similar spatial trends, but most groups had higher MOTU richness with the DNA template, while others, such as nematodes, were more diverse in the RNA dataset. We provide metabarcoding protocols and guidelines for biomonitoring of these key communities in order to generate information applicable to management efforts.

  7. Monitoring the biomass accumulation of recombinant yeast cultures: offline estimations of dry cell mass and cell counts.

    Science.gov (United States)

    Palmer, Shane M; Kunji, Edmund R S

    2012-01-01

    Biomass is one of the most important parameters for process optimization, scale-up and control in recombinant protein production experiments. However, a standard unit of biomass remains elusive. Methods of biomass monitoring have increasingly been developed towards online, in situ techniques in order to advance process analysis and control. Offline, ex situ methods, such as dry cell mass determination and direct cell counts, remain the reference for determining cell mass and number, respectively, but this type of analysis is time consuming. In this chapter, protocols are presented for determining these offline measures of the biomass yield of recombinant yeast cultures.

  8. Efficient siRNA delivery and tumor accumulation mediated by ionically cross-linked folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate nanoparticles: for the potential targeted ovarian cancer gene therapy.

    Science.gov (United States)

    Li, Tony Shing Chau; Yawata, Toshio; Honke, Koichi

    2014-02-14

    For effective ovarian cancer gene therapy, systemic administrated tumor-targeting siRNA/folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate (FA-PEG-COL) nanoparticles is vital for delivery to cancer site(s). siRNA/FA-PEG-COL nanoparticles were prepared by ionic gelation for effective FA receptor-expressing ovarian cancer cells transfection and in vivo accumulation. The chemical structure of FA-PEG-COL conjugate was characterized by MALDI-TOF-MS, FT-IR and (1)H NMR. The average size of siRNA/FA-PEG-COL nanoparticles was approximately 200 nm, and the surface charge was +8.4 mV compared to +30.5 mV with siRNA/COL nanoparticles. FA-PEG-COL nanoparticles demonstrated superior compatibility with erythrocytes in terms of degree of aggregation and haemolytic activity and also effects on cell viability was lower when compared with COL nanoparticles. FA grafting significantly facilitated the uptake of nanoparticles via receptor mediated endocytosis as demonstrated by flow cytometry. The in vitro transfection and gene knockdown efficiency of HIF-1α were superior to COL nanoparticles (76-62%, respectively) and was comparable to Lipofectamine 2000 (79%) as demonstrated by RT-qPCR and Western blot. Gene knockdown at the molecular level translated into effective inhibition of proliferation in vitro. Accumulation efficiency of FA-PEG-COL nanoparticles was investigated in BALB/c mice bearing OVK18 #2 tumor xenograft using in vivo imaging. The active targeting FA-PEG-COL nanoparticles showed significantly greater accumulation than the passive targeting COL nanoparticles. Based on the results obtained, siRNA/FA-PEG-COL nanoparticles show much potential for effective ovarian cancer treatment via gene therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. SU-E-I-10: Automatic Monitoring of Accumulated Dose Indices From DICOM RDSR to Improve Radiation Safety in X-Ray Angiography

    Energy Technology Data Exchange (ETDEWEB)

    Omar, A; Bujila, R; Nowik, P; Karambatsakidou, A [Karolinska University Hospital, Stockholm (Sweden)

    2014-06-01

    Purpose: To investigate the potential benefits of automatic monitoring of accumulated patient and staff dose indicators, i.e., CAK and KAP, from DICOM Radiation Dose Structured Reports (RDSR) in x-ray angiography (XA). Methods: Recently RDSR has enabled the convenient aggregation of dose indices and technique parameters for XA procedures. The information contained in RDSR objects for three XA systems, dedicated to different types of clinical procedures, has been collected and aggregated in a database for over one year using a system developed with open-source software at the Karolinska University Hospital. Patient weight was complemented to the RDSR data via an interface with the Hospital Information System (HIS). Results: The linearly approximated trend in KAP over a time period of a year for cerebrovascular, pelvic/peripheral vascular, and cardiovascular procedures showed a decrease of 12%, 20%, and 14%, respectively. The decrease was mainly due to hardware/software upgrades and new low-dose imaging protocols, and partially due to ongoing systematic radiation safety education of the clinical staff. The CAK was in excess of 3 Gy for 15 procedures, and exceeded 5 Gy for 3 procedures. The dose indices have also shown a significant dependence on patient weight for cardiovascular and pelvic/peripheral vascular procedures; a 10 kg shift in mean patient weight can result in a dose index increase of 25%. Conclusion: Automatic monitoring of accumulated dose indices can be utilized to notify the clinical staff and medical physicists when the dose index has exceeded a predetermined action level. This allows for convenient and systematic follow-up of patients in risk of developing deterministic skin injuries. Furthermore, trend analyses of dose indices over time is a valuable resource for the identification of potential positive or negative effects (dose increase/decrease) from changes in hardware, software, and clinical work habits.

  10. Mutagenesis of the catalytic and cleavage site residues of the hypovirus papain-like proteases p29 and p48 reveals alternative processing and contributions to optimal viral RNA accumulation.

    Science.gov (United States)

    Jensen, Kenneth S; Nuss, Donald L

    2014-10-01

    The positive-stranded RNA genome of the prototypic virulence-attenuating hypovirus CHV-1/EP713 contains two open reading frames (ORF), each encoding an autocatalytic papain-like leader protease. Protease p29, derived from the N-terminal portion of ORF A, functions as a suppressor of RNA silencing, while protease p48, derived from the N-terminal portion of ORF B, is required for viral RNA replication. The catalytic and cleavage site residues required for autoproteolytic processing have been functionally mapped in vitro for both proteases but not confirmed in the infected fungal host. We report here the mutagenesis of the CHV-1/EP713 infectious cDNA clone to define the requirements for p29 and p48 cleavage and the role of autoproteolysis in the context of hypovirus replication. Mutation of the catalytic cysteine and histidine residues for either p29 or p48 was tolerated but reduced viral RNA accumulation to ca. 20 to 50% of the wild-type level. Mutation of the p29 catalytic residues caused an accumulation of unprocessed ORF A product p69. Surprisingly, the release of p48 from the ORF B-encoded polyprotein was not prevented by mutation of the p48 catalytic and cleavage site residues and was independent of p29. The results show that, while dispensable for hypovirus replication, the autocatalytic processing of the leader proteases p29 and p48 contributes to optimal virus RNA accumulation. The role of the predicted catalytic residues in autoproteolytic processing of p29 was confirmed in the infected host, while p48 was found to also undergo alternative processing independent of the encoded papain-like protease activities. Importance: Hypoviruses are positive-strand RNA mycoviruses that attenuate virulence of their pathogenic fungal hosts. The prototypic hypovirus CHV-1/EP713, which infects the chestnut bight fungus Cryphonetria parasitica, encodes two papain-like autocatalytic leader proteases, p29 and p48, that also have important functions in suppressing the RNA

  11. Gene transcript accumulation and in situ mRNA hybridization of two putative glutamate dehydrogenase genes in etiolated Glycine max seedlings.

    Science.gov (United States)

    Dimou, M; Tsaniklidis, G; Aivalakis, G; Katinakis, P

    2015-01-01

    Glutamate dehydrogenase (EC 1.4.1.2) is a multimeric enzyme that catalyzes the reversible amination of α-ketoglutarate to form glutamate. We characterized cDNA clones of two Glycine max sequences, GmGDH1 and GmGDH2, that code for putative α- and β-subunits, respectively, of the NADH dependent enzyme. Temporal and spatial gene transcript accumulation studies using semiquantitative RT-PCR and in situ hybridization have shown an overlapping gene transcript accumulation pattern with differences in relative gene transcript accumulation in the organs examined. Detection of NADH-dependent glutamate dehydrogenase activity in situ using a histochemical method showed concordance with the spatial gene transcript accumulation patterns. Our findings suggest that although the two gene transcripts are co-localized in roots of etiolated soybean seedlings, the ratio of the two subunits of the active holoenzyme may vary among tissues.

  12. Combining flow cytometry and 16S rRNA gene pyrosequencing: a promising approach for drinking water monitoring and characterization.

    Science.gov (United States)

    Prest, E I; El-Chakhtoura, J; Hammes, F; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S

    2014-10-15

    The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Combining flow cytometry and 16S rRNA gene pyrosequencing: A promising approach for drinking water monitoring and characterization

    KAUST Repository

    Prest, Emmanuelle I E C

    2014-10-01

    The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5min intervals for 1h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345±15×103 to 425±35×103cellsmL-1) and in the percentage of intact bacterial cells (from 39±3.5% to 53±4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. © 2014 Elsevier Ltd.

  14. Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon.

    Science.gov (United States)

    van Beckhoven, J R C M; Stead, D E; van der Wolf, J M

    2002-01-01

    To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.

  15. The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters.

    Science.gov (United States)

    Li, Tianlu; De Clercq, Nikki; Medina, Daniel A; Garre, Elena; Sunnerhagen, Per; Pérez-Ortín, José E; Alepuz, Paula

    2016-02-01

    The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription.

  16. Monitoring the mycobiota during Greco di Tufo and Aglianico wine fermentation by 18S rRNA gene sequencing.

    Science.gov (United States)

    De Filippis, Francesca; La Storia, Antonietta; Blaiotta, Giuseppe

    2017-05-01

    Spontaneous alcoholic fermentation of grape must is a complex process, carried out by indigenous yeast populations arising from the vineyard or the winery environment and therefore representing an autochthonous microbial terroir of the production area. Microbial diversity at species and biotype level is extremely important in order to develop the composite and typical flavour profile of DOCG (Appellation of Controlled and Guaranteed Origin) wines. In this study, we monitored fungal populations involved in spontaneous fermentations of Aglianico and Greco di Tufo grape must by high-throughput sequencing (HTS) of 18S rRNA gene amplicons. We firstly proposed an alternative/addition to ITS as target gene in HTS studies and highlighted consistency between the culture-dependent and -independent approaches. A complex mycobiota was found at the beginning of the fermentation, mainly characterized by non-Saccharomyces yeasts and several moulds, with differences between the two types of grapes. Moreover, Interdelta patterns revealed a succession of several Saccharomyces cerevisiae biotypes and a high genetic diversity within this species.

  17. An Archaea 5S rRNA analog is stably expressed in Escherichia coli

    Science.gov (United States)

    Yang, Y.; Fox, G. E.

    1996-01-01

    Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

  18. Metabarcoding monitoring analysis: the pros and cons of using co-extracted environmental DNA and RNA data to assess offshore oil production impacts on benthic communities

    Directory of Open Access Journals (Sweden)

    Olivier Laroche

    2017-05-01

    Full Text Available Sequencing environmental DNA (eDNA is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene and eukaryotic (18S ribosomal RNA gene eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand. Macro-infauna (visual classification of benthic invertebrates and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs, by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from

  19. Metabarcoding monitoring analysis: the pros and cons of using co-extracted environmental DNA and RNA data to assess offshore oil production impacts on benthic communities.

    Science.gov (United States)

    Laroche, Olivier; Wood, Susanna A; Tremblay, Louis A; Lear, Gavin; Ellis, Joanne I; Pochon, Xavier

    2017-01-01

    Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil

  20. Metabarcoding monitoring analysis: the pros and cons of using co-extracted environmental DNA and RNA data to assess offshore oil production impacts on benthic communities

    KAUST Repository

    Laroche, Olivier

    2017-05-17

    Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil

  1. Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection.

    Science.gov (United States)

    Showalter, A M; Bell, J N; Cramer, C L; Bailey, J A; Varner, J E; Lamb, C J

    1985-10-01

    Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with (32)P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

  2. Radioactive cesium accumulation in seaweeds by the Fukushima 1 Nuclear Power Plant accident-two years' monitoring at Iwaki and its vicinity.

    Science.gov (United States)

    Kawai, Hiroshi; Kitamura, Akira; Mimura, Mari; Mimura, Tetsuro; Tahara, Tomoya; Aida, Daiki; Sato, Kenji; Sasaki, Hideaki

    2014-01-01

    Accumulations of radionuclides in marine macroalgae (seaweeds) resulting from the Fukushima 1 Nuclear Power Plant (F1NPP) accident in March 2011 have been monitored for two years using high-purity germanium detectors. Algal specimens were collected seasonally by snorkeling at Nagasaki, Iwaki, Fukushima Prefecture (Pref.), Japan, ca. 50 km perimeter from the F1NPP. Additional collections were done at Soma, Hironocho, Hisanohama and Shioyazaki in Fukushima Pref. as well as at Chiba Pref. and Hyogo Pref. as controls. In May 2011, specimens of most macroalgal species showed ¹³⁷Cs levels greater than 3,000 Bq kg⁻¹ at Shioyazaki and Nagasaki. The highest ¹³⁷Cs level recorded 7371.20 ± 173.95 Bq kg⁻¹ in Undaria pinnatifida (Harvey) Suringar on 2 May 2011, whereas seawater collected at the same time at Shioyazaki and Nagasaki measured 8.41 ± 3.21 and 9.74 ± 3.43 Bq L⁻¹, respectively. The concentration factor of marine macroalgae was estimated to be ca. 8-50, depending on taxa and considering a weight ratio of wet/dry samples of ca. 10. ¹³⁷Cs level declined remarkably during the following 5-6 months. In contrast, the ¹³⁷Cs level remained rather stable during the following 12-16 months, and maintained the range of 10-110 Bq kg⁻¹. Contamination was still detectable in many samples in March 2013, 24 months after the most significant pollution.

  3. Loss of PTB or negative regulation of Notch mRNA reveals distinct zones of Notch and actin protein accumulation in Drosophila embryo.

    Directory of Open Access Journals (Sweden)

    Cedric S Wesley

    Full Text Available Polypyrimidine Tract Binding (PTB protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1 the Notch mRNA is a potential target of PTB, (2 PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3 the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions.

  4. Spatiotemporal variations in metal accumulation, RNA/DNA ratio and energy reserve in Perna viridis transplanted along a marine pollution gradient in Hong Kong.

    Science.gov (United States)

    Yeung, Jamius W Y; Zhou, Guang-Jie; Leung, Kenneth M Y

    2017-01-21

    We examined spatiotemporal variations of metal levels and three growth related biomarkers, i.e., RNA/DNA ratio (RD), total energy reserve (Et) and condition index (CI), in green-lipped mussels Perna viridis transplanted into five locations along a pollution gradient in the marine environment of Hong Kong over 120days of deployment. There were significant differences in metal levels and biomarker responses among the five sites and six time points. Mussels in two clean sites displayed better CI and significantly lower levels of Ag, Cu, Pb and Zn in their tissues than the other sites. Temporal patterns of RD in P. viridis were found to be site-specific. Across all sites, Et decreased in P. viridis over the deployment period, though the rate of decrease varied significantly among the sites. Therefore, temporal variation of biomarkers should be taken to consideration in mussel-watch programs because such information can help discriminate pollution-induced change from natural variation.

  5. A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

    Science.gov (United States)

    Lacroix, C; Coquillé, V; Guyomarch, J; Auffret, M; Moraga, D

    2014-09-15

    mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs.

  6. Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

    Directory of Open Access Journals (Sweden)

    Yao Han

    Full Text Available MIGS (miRNA-induced gene silencing is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs. MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase and PDS (phytoene desaturase gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant

  7. Effects of insulin resistance and hepatic lipid accumulation on hepatic mRNA expression levels of apoB, MTP and L-FABP in non-alcoholic fatty liver disease.

    Science.gov (United States)

    Higuchi, Nobito; Kato, Masaki; Tanaka, Masatake; Miyazaki, Masayuki; Takao, Shinichiro; Kohjima, Motoyuki; Kotoh, Kazuhiro; Enjoji, Munechika; Nakamuta, Makoto; Takayanagi, Ryoichi

    2011-11-01

    Non-alcoholic fatty liver disease (NAFLD) is considered a hepatic manifestation of metabolic syndrome, which is known to be associated with insulin resistance (IR). NAFLD occurs when the rate of hepatic fatty acid uptake from plasma and de novo fatty acid synthesis is greater than the rate of fatty acid oxidation and excretion as very low-density lipoprotein (VLDL). To estimate the effects of IR on hepatic lipid excretion, mRNA expression levels of genes involved in VLDL assembly were analyzed in NAFLD liver. Twenty-two histologically proven NAFLD patients and 10 healthy control subjects were enrolled in this study. mRNA was extracted from liver biopsy samples and real-time PCR was performed to quantify the expression levels of apolipoprotein B (apoB), microsomal triglyceride transfer protein (MTP) and liver fatty-acid binding protein (L-FABP). Hepatic expression levels of the genes were compared between NAFLD patients and control subjects. In NAFLD patients, we also examined correlations between expression levels of the genes and metabolic factors, including IR, and the extent of obesity and hepatic lipid accumulation. Hepatic expression levels of apoB, MTP and L-FABP were significantly up-regulated in NAFLD patients compared to control subjects. The expression levels of MTP were correlated with those of apoB, but not with those of L-FABP. In the NAFLD liver, the expression levels of MTP were significantly reduced in patients with HOMA-IR >2.5. In addition, a significant reduction in MTP expression was observed in livers with advanced steatosis. Enhanced expression of genes involved in VLDL assembly may be promoted to release excess lipid from NAFLD livers. However, the progression of IR and hepatic steatosis may attenuate this compensatory process.

  8. Co-monitoring bacterial and dinoflagellates communities by denaturing gradient gel electrophoresis (DGGE) and SSU rRNA sequencing during a dinoflagellates bloom

    Institute of Scientific and Technical Information of China (English)

    KANJinjun; CHENFeng

    2004-01-01

    Dinoflagellates are unicellular eukaryotic protists that dominate in all coastal waters, and are also present in oceanic waters. Despite the central importance of dinoflagellates in global primary production, the relationship between dinoflagellates and bacteria are still poorly understood. In order to understand the ecological interaction between bacterial and dinoflageUates communities, denaturing gradient gel electrophoresis (DGGE) and SSU rRNA sequencing were applied to monitoring the population dynamics of bacteria and dinoflagellates from the onset to disappearance of a dinoflagellates bloom occurred in Baltimore Inner Harbor, from April 15 to 24, 2002. Although Prorocentrum minimum was the major bloom forming species under the light microscopy, DGGE method with dinoflagellate specific primers demonstrated that Prorocentrum micans, Gymnodinium galatheanum and Gyrodinium uncatenum were also present during the bloom. Population shifts among the minor dinoflagellate groups were observed. DGGE of PCR-amplified 16S rRNA gene fragments indicated that cyanobacteria, α, β, γ-proteobacteria, FlavobacteriumBacteroides-Cytophaga (FBC), and Planctomcetes were the major components of bacterial assemblages during the bloom. DGGE analysis showed that Cytophagales and α-proteobacteria played important roles at different stages of dinoflagellates bloom. DGGE can be used as a rapid tool to simultaneously monitor population dynamics of both bacterial and dinoflagellates communities in aquatic environments, which is demonstrated here.

  9. Monitoring p21 mRNA expression in living cell based on molecular beacon fluorescence increasing rate

    Institute of Scientific and Technical Information of China (English)

    TANG HongXing; YANG XiaoHai; WANG KeMin; TAN WeiHong; LIU Bin; HE LiFang; WANG Wei

    2008-01-01

    Studying the expression level of mRNA in living cells will offer tremendous opportunities for ad-vancement in cell biology research, disease diagnostics, and drug discovery. In this paper, a molecular beacon (MB) specific for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyn-geal carcinoma cells. After injecting the p21MB into nasopharyngeal carcinoma cell and p33-trans-fected nasopharyngeal carcinoma cell, the consistent increase of fluorescent signal intensity was de-tected in both cell lines, and maximum fluorescence intensity achieved in about 15 min. In about 4 min following microinjection, the fluorescence increasing rate was significantly different between these two cell lines, which indicate the different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can effi-ciently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.

  10. Light inhibits gravity-regulated peg formation and asymmetric mRNA accumulation of auxin-inducible CsIAA1 in the cortex of the transition zone in cucumber seedlings

    Science.gov (United States)

    Fujii, Nobuharu; Saito, Yuko; Miyazawa, Yutaka; Takahashi, Hideyuki

    When cucumber seedlings are grown horizontally, a specialized protuberance, termed the peg, develops on the lower side of the transition zone between the hypocotyl and the root. Gravimorphogenesis regulates the lateral positioning of the peg in the transition zone and it has been suggested that auxin plays an important role in peg formation in cucumber seedlings. Here, we found that light inhibited auxin-regulated peg formation. In the transition zone of horizontally positioned cucumber seedlings grown in the dark, we detected an asymmetric accumulation of mRNA from the auxin-inducible gene CsIAA1 in the epidermis and cortex. However, in seedlings grown under illumination, this asymmetry was greatly reduced. In dark- and light-grown seedlings, application of 10 -3 M indole-3-acetic acid induced peg formation on both the lower and upper sides of the transition zone. These results suggest that light inhibits peg formation via modification of auxin distribution and/or levels in the transition zone of cucumber seedlings.

  11. Moss monitoring as a mirror of land use? Nitrogen and metal accumulation in mosses of two regions in middle Europe; Moosmonitoring als Spiegel der Landnutzung? Stickstoff- und Metallakkumulation in Moosen zweier Regionen Mitteleuropas

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, W.; Hornsmann, I.; Pesch, R.; Schmidt, G. [Hochschule Vechta (Germany). Lehrstuhl fuer Landschaftsoekologie; Fraenzle, S.; Wuenschmann, S.; Heidenreich, H.; Markert, B. [Internationales Hochschulinstitut, Zittau (Germany)

    2008-02-15

    Goal, Scope and Background. The study was conducted to test the hypothesis that the regional variability of nitrogen (N) and metal accumulations in terrestrial ecosystems are due to historical and recent ways of land use. To this end, in two regions of Central Europe the metal and N accumulations in both regions should be examined by comparative moss analysis. The regions should be of quantitatively specified representativity for selected ecological characteristics of Europe. Within both regions these characteristics should be covered by the sites where the moss samples were collected. The number of samples should allow for geostatistical estimation of the measured nitrogen and metal loads. Results. By use of the ecological regionalisation of Europe the Weser-Ems Region (WER) and the Euro Region Nissa (ERN) were selected for investigation. The sampling sites represent quite well the natural landscapes and the land use categories of both regions. The measurement values corroborate the decline of metal accumulation observed since the beginning of the European Mosses Monitoring Survey in 1990. The metal loads of the mosses in the ERN exceed those in the WER significantly. The opposite holds true for the N concentrations: those in the WER are significantly higher than those in the ERN. (orig.)

  12. Comparative study of the validity of three regions of the 18S-rRNA gene for massively parallel sequencing-based monitoring of the planktonic eukaryote community.

    Science.gov (United States)

    Tanabe, Akifumi S; Nagai, Satoshi; Hida, Kohsuke; Yasuike, Motoshige; Fujiwara, Atushi; Nakamura, Yoji; Takano, Yoshihito; Katakura, Seiji

    2016-03-01

    The nuclear 18S-rRNA gene has been used as a metabarcoding marker in massively parallel sequencing (MPS)-based environmental surveys for plankton biodiversity research. However, different hypervariable regions have been used in different studies, and their utility has been debated among researchers. In this study, detailed investigations into 18S-rRNA were carried out; we investigated the effective number of sequences deposited in international nucleotide sequence databases (INSDs), the amplification bias, and the amplicon sequence variability among the three variable regions, V1-3, V4-5 and V7-9, using in silico polymerase chain reaction (PCR) amplification based on INSDs. We also examined the primer universality and the taxonomic identification power, using MPS-based environmental surveys in the Sea of Okhotsk, to determine which region is more useful for MPS-based monitoring. The primer universality was not significantly different among the three regions, but the number of sequences deposited in INSDs was markedly larger for the V4-5 region than for the other two regions. The sequence variability was significantly different, with the highest variability in the V1-3 region, followed by the V7-9 region, and the lowest variability in the V4-5 region. The results of the MPS-based environmental surveys showed significantly higher identification power in the V1-3 and V7-9 regions than in the V4-5 region, but no significant difference was detected between the V1-3 and V7-9 regions. We therefore conclude that the V1-3 region will be the most suitable for future MPS-based monitoring of natural eukaryote communities, as the number of sequences deposited in INSDs increases.

  13. HIF-1α inhibition by siRNA or chetomin in human malignant glioma cells: effects on hypoxic radioresistance and monitoring via CA9 expression

    Directory of Open Access Journals (Sweden)

    Bache Matthias

    2010-11-01

    Full Text Available Abstract Background Hypoxia induces activation of the HIF-1 pathway and is an essential characteristic of malignant gliomas. Hypoxia has been linked to tumor progression, therapy resistance and poor prognosis. However, little is known about the impact of HIF-1α inhibition on radioresistance of malignant glioma. Methods In this study, we investigated the effects of the inhibition of HIF-1α on cell survival and radiosensitivity in U251MG and U343MG glioma cells, using two different strategies. HIF-1α inhibition was achieved by siRNA targeting of HIF-1α or via chetomin, a disruptor of interactions between HIF-1α and p300. The inhibition of the HIF-1 pathway was monitored by quantitative real-time PCR and Western blot analyses of the expression levels of HIF-1α and CA9. CA9 expression was investigated as a potential indicator of the efficacy of HIF-1 inhibition and the resulting radiosensitivity of malignant glioma cell lines was determined by clonogenic assay after irradiation under normoxic (2-10 Gy or hypoxic (2-15 Gy conditions. Results Although siRNA and chetomin show distinct modes of action, both attenuated the hypoxia-induced radioresistance of malignant glioma cell lines U251MG (DMF10: 1.35 and 1.18 and U343MG (DMF10: 1.78 and 1.48. However, siRNA and chetomin showed diverse effects on radiosensitivity under normoxic conditions in U251MG (DMF10: 0.86 and 1.35 and U343MG (DMF10: 1.33 and 1.02 cells. Conclusions Results from this in vitro study suggest that inhibition of HIF-1α is a promising strategy to sensitize human malignant gliomas to radiotherapy and that CA9 could serve as an indicator of effective HIF-1-related radiosensitization.

  14. Monitoring of the antiviral potential of bee venom and wax extracts against Adeno-7 (DNA) and Rift Valley fever virus (RNA) viruses models.

    Science.gov (United States)

    Hassan, Mostafa I; Mohamed, Aly F; Amer, Moner A; Hammad, Kotb M; Riad, Saber A

    2015-04-01

    This study monitored the antiviral potential of bee venom and four wax extracts, ethanol white and black beeswax (EWW/EBW) and acetone white and black beeswax (AWW/ABW) extracts. Two different virus models namely Adeno-7 as DNA model and RVFV as RNA virus models. End point calculation assay was used to calculate virus depletion titer. The depletion of viral infectivity titer of ABW to Adeno-7 virus showed strong antiviral activity recorded a depletion of viral infectivity titer (1.66 log (10)/ ml) that gave equal action with bee venom and more than interferon IFN (1 log (10)/ ml). On the other hand, antiviral activity of EBW showed a moderate potential, while AWW showed no antiviral activity. Finally EWW showed synergetic activity against Adeno-7 virus activity. Thus, activity of wax extracts to RVFV was arranged in order of IFN bee venom > AWW & EBW > EWW and ABW recorded 3.34, 0.65, 0.5, 0.34 respectively. It is the first time to study the beeswax effect against DNA and RNA virus' models; acetone black beeswax recorded a depletion titer 1.66 log (10)/ml.

  15. Circulating cell-free microRNA as biomarkers for screening, diagnosis and monitoring of neurodegenerative diseases and other neurologic pathologies

    Directory of Open Access Journals (Sweden)

    Kira S Sheinerman

    2013-09-01

    Full Text Available Many neurodegenerative diseases, such as Alzheimer’s disease, Parkinson disease, vascular and frontotemporal dementias, as well as other chronic neurological pathologies, are characterized by slow development with a long asymptomatic period followed by a stage with mild clinical symptoms. As a consequence, these serious pathologies are diagnosed late in the course of a disease, when massive death of neurons has already occurred and effective therapeutic intervention is problematic. Thus, the development of screening tests capable of detecting neurodegenerative diseases during early, preferably asymptomatic, stages is a high unmet need. Since such tests are to be used for screening of large populations, they should be non-invasive and relatively inexpensive. Further, while subjects identified by screening tests can be further tested with more invasive and expensive methods, e.g. analysis of cerebrospinal fluid or imaging techniques, to be of practical utility screening tests should have high sensitivity and specificity. In this review, we discuss advantages and disadvantages of various approaches to developing screening tests based on analysis of circulating cell-free miRNA. Applications of circulating miRNA-based tests for diagnosis of acute and chronic brain pathologies, for research of normal brain aging, and for disease and treatment monitoring are also discussed.

  16. 累积式放射性气溶胶连续监测仪的实验运行数据处理%Processing Methods for Operation Test Data of Radioactive Aerosols Monitor Based on Accumulation Techniques

    Institute of Scientific and Technical Information of China (English)

    傅翠明; 席萍萍; 马英豪; 谭玲龙; 沈福

    2011-01-01

    This article introduces a radioactive aerosol continuous monitor based on accumulation sampling and measuring and three methods for processing the operation data. The monitoring results are processed by the 3 methods which are applied both under the conditions of natural background and at workplaces of a nuclear facility. How the monitoring results are assessed and how to calculate the detection limit when using the 3 different methods are explained. Moreover, the advantages and disadvantages of the 3 methods are discussed.%介绍了一种按累积式采样和计数测量方式运行的α/β放射性气溶胶连续监测仪及其实验运行数据的3种处理方法。用这3种方法分别给出了在只有氡(和Th)子体的天然本底和在核设施工作场所的条件下的实验数据计算结果,并对相关的数据处理结果做出了评价,给出了3种不同数据处理方法的监测仪探测下限的计算公式,并且讨论了3种数据处理方法的优缺点以及与此有关的问题。

  17. Evaluation of damage accumulation behavior and strength anisotropy of NITE SiC/SiC composites by acoustic emission, digital image correlation and electrical resistivity monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Nozawa, Takashi, E-mail: nozawa.takashi67@jaea.go.jp [Japan Atomic Energy Agency, Rokkasho, Aomori (Japan); Ozawa, Kazumi [Japan Atomic Energy Agency, Rokkasho, Aomori (Japan); Asakura, Yuuki; Kohyama, Akira [Muroran Institute of Technology, Muroran, Hokkaido (Japan); Tanigawa, Hiroyasu [Japan Atomic Energy Agency, Rokkasho, Aomori (Japan)

    2014-12-15

    Understanding the cracking process of the composites is essential to establish the design basis for practical applications. This study aims to investigate the damage accumulation process and its anisotropy for nano-infiltration transient eutectic sintered (NITE) SiC/SiC composites by various characterization techniques such as the acoustic emission (AE), digital image correlation (DIC) and electrical resistivity (ER) measurements. Cracking behavior below the proportional limit stress (PLS) was specifically addressed. Similar to the other generic SiC/SiC composites, the 1st AE event was identified below the PLS for NITE SiC/SiC composites with a dependency of fabric orientation. The DIC results support that the primary failure mode depending on fiber orientation affected more than the other minor modes did. Detailed AE waveform analysis by wavelet shows a potential to classify the failure behavior depending on architecture. Cracking below the PLS is a potential concern in component deign but the preliminary ER measurements imply that the impact of cracking below the PLS on composite function was limited.

  18. Monitoring of contaminated toxic and heavy metals, from mine tailings through age accumulation, in soil and some wild plants at Southeast Egypt.

    Science.gov (United States)

    Rashed, M N

    2010-06-15

    This study includes tailing from gold mine, at Allaqi Wadi Aswan, Egypt, used by incident Egyptian and after by some English companies. Tailings, soils and wild plants (Acia Raddiena and Aerva Javanica) were sampled and analysed for toxic metals (Hg, Cd, Pb and As) and associated heavy metals (Cr, Ag, Ni, Au, Mo, Zn, Mn and Cu) using ICP-MS, ICP-AES, CVAAS and FAAS techniques. The present work concerns the distribution and mobility of these metals from tailing to the surrounding soils and wild flora. The results reveal that Cr, Cu, Zn, Ni, Ag, Au, Mn, Hg, As, Ag, Au and Pb in soil decreased as faraway from the tailing, after then irregular trends as a result of input from surrounding rocks. Acia Raddiena plant accumulated As, Cd and Pb in higher levels than Aerva Javanica. Quantification of soil and plant pollution was studied using enrichment factors, contamination factor, pollution index and bioaccumulation factors and show good interpretations of the results. The overall results of this study show that the soil and plants near the gold mine tailing were highly toxic, and the plants and soil must not be uses for grazing or agriculture. Copyright 2010 Elsevier B.V. All rights reserved.

  19. Multicenter comparison of Roche COBAS AMPLICOR MONITOR version 1.5, Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex version 3.0 for quantification of human immunodeficiency virus type 1 RNA in plasma.

    Science.gov (United States)

    Murphy, D G; Côté, L; Fauvel, M; René, P; Vincelette, J

    2000-11-01

    The performance and characteristics of Roche COBAS AMPLICOR HIV-1 MONITOR version 1.5 (CA MONITOR 1.5) UltraSensitive (usCA MONITOR 1. 5) and Standard (stCA MONITOR 1.5) procedures, Organon Teknika NucliSens HIV-1 RNA QT with Extractor (NucliSens), and Bayer Quantiplex HIV RNA version 3.0 (bDNA 3.0) were compared in a multicenter trial. Samples used in this study included 460 plasma specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected persons, 100 plasma specimens from HIV antibody (anti-HIV)-negative persons, and culture supernatants of HIV-1 subtype A to E isolates diluted in anti-HIV-negative plasma. Overall, bDNA 3.0 showed the least variation in RNA measures upon repeat testing. For the Roche assays, usCA MONITOR 1.5 displayed less variation in RNA measures than stCA MONITOR 1.5. NucliSens, at an input volume of 2 ml, showed the best sensitivity. Deming regression analysis indicated that the results of all three assays were significantly correlated (P < 0.0001). However, the mean difference in values between CA MONITOR 1.5 and bDNA 3.0 (0.274 log(10) RNA copies/ml; 95% confidence interval, 0.192 to 0.356) was significantly different from 0, indicating that CA MONITOR 1.5 values were regularly higher than bDNA 3.0 values. Upon testing of 100 anti-HIV-negative plasma specimens, usCA MONITOR 1.5 and NucliSens displayed 100% specificity, while bDNA 3.0 showed 98% specificity. NucliSens quantified 2 of 10 non-subtype B viral isolates at 1 log(10) lower than both CA MONITOR 1.5 and bDNA 3.0. For NucliSens, testing of specimens with greater than 1,000 RNA copies/ml at input volumes of 0.1, 0.2, and 2.0 ml did not affect the quality of results. Additional factors differing between assays included specimen throughput and volume requirements, limit of detection, ease of execution, instrument work space, and costs of disposal. These characteristics, along with assay performance, should be considered when one is selecting a viral load assay.

  20. Primitive red alga Cyanidioschyzon merolae accumulates storage glucan and triacylglycerol under nitrogen depletion.

    Science.gov (United States)

    Takusagawa, Mari; Nakajima, Yohei; Saito, Takafumi; Misumi, Osami

    2016-07-14

    Most microalgae accumulate neutral lipids, including triacylglycerol (TAG), into spherical structures called lipid bodies (LBs) under environmental stress conditions such as nutrient depletion. In green algae, starch accumulation precedes TAG accumulation, and the starch is thought to be a substrate for TAG synthesis. However, the relationship between TAG synthesis and the starch content in red algae, as well as how TAG accumulation is regulated, is unclear. In this study, we cultured the primitive red alga Cyanidioschyzon merolae under nitrogen-depleted conditions, and monitored the formation of starch granules (SGs) and LBs using microscopy. SGs stained with potassium iodide were observed at 24 h; however, LBs stained specifically with BODIPY 493/503 were observed after 48 h. Quantitative analysis of neutral sugar and cytomorphological semi-quantitative analysis of TAG accumulation also supported these results. Thus, the accumulation of starch occurred and preceded the accumulation of TAG in cells of C. merolae. However, TAG accumulation was not accompanied by a decrease in the starch content, suggesting that the starch is a major carbon storage sink, at least under nitrogen-depleted conditions. Quantitative real-time PCR revealed that the mRNA levels of genes involved in starch and TAG synthesis rarely changed during the culture period, suggesting that starch and TAG synthesis in C. merolae are not controlled through gene transcription but at other stages, such as translation and/or enzymatic activity.

  1. RNA extracted from blood samples with a rapid automated procedure is fit for molecular diagnosis or minimal residual disease monitoring in patients with a variety of malignant blood disorders.

    Science.gov (United States)

    Bechlian, Didier; Honstettre, Amélie; Terrier, Michèle; Brest, Christelle; Malenfant, Carine; Mozziconacci, Marie-Joëlle; Chabannon, Christian

    2009-06-01

    Scientific studies in oncology, cancer diagnosis, and monitoring tumor response to therapeutics currently rely on a growing number of clinico-pathological information. These often include molecular analyses. The quality of these analyses depends on both pre-analytical and analytical information and often includes the extraction of DNA and/or RNA from human tissues and cells. The quality and quantity of obtained nucleic acids are of utmost importance. The use of automated techniques presents several advantages over manual techniques, such as reducing technical time and thus cost, and facilitating standardization. The purpose of this study was to validate an automated technique for RNA extraction from cells of patients treated for various malignant blood diseases. A well-established manual technique was compared to an automated technique, in order to extract RNA from blood samples drawn for the molecular diagnosis of a variety of leukemic diseases or monitoring of minimal residual disease. The quality of the RNA was evaluated by real-time quantitative RT-PCR (RQ-PCR) analyses of the Abelson gene transcript. The results show that both techniques produce RNA with comparable quality and quantity, thus suggesting that an automated technique can be substituted for the reference and manual technique used in the daily routine of a molecular pathology laboratory involved in minimal residual disease monitoring. Increased costs of reagents and disposables used for automated techniques can be compensated by a decrease in human resource.

  2. Symptom severity and viral protein or RNA accumulation in lettuce affected by big-vein disease Severidad de síntomas y acumulación de proteínas o ARN virales en lechugas afectadas por la enfermedad de las venas grandes

    OpenAIRE

    Carolina Araya; Elizabeth Peña; Erika Salazar; Lisset Román; Claudia Medina; Roxana Mora; Agustín Aljaro; Inés-Marlene Rosales

    2011-01-01

    Big-vein disease (BVD) is a widespread and economically damaging disease in lettuce (Lactuca sativa L.). Typical symptoms are chlorotic clearing around leaf veins, leaf deformations, and impaired head development. In this research, we studied the relationship between symptom intensity and protein and viral RNA accumulation in infected plants. Naturally infected lettuce plants, from the field or greenhouse, were classified according to their symptomatology: mild, moderate, severe, and symptoml...

  3. A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

    Science.gov (United States)

    Pitulle, C.; Hedenstierna, K. O.; Fox, G. E.

    1995-01-01

    Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed. Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences. In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes. This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted. Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette. The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E. coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes. These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.

  4. Functional Assays for Specific Targeting and Delivery of RNA Nanoparticles to Brain Tumor

    Science.gov (United States)

    Lee, Tae Jin; Haque, Farzin; Vieweger, Mario; Yoo, Ji Young; Kaur, Balveen; Guo, Peixuan; Croce, Carlo M.

    2017-01-01

    Cumulative progress in nanoparticle development has opened a new era of targeted delivery of therapeutics to cancer cells and tissue. However, developing proper detection methods has lagged behind resulting in the lack of precise evaluation and monitoring of the systemically administered nanoparticles. RNA nanoparticles derived from the bacteriophage phi29 DNA packaging motor pRNA have emerged as a new generation of drugs for cancer therapy. Multifunctional RNA nanoparticles can be fabricated by bottom-up self-assembly of engineered RNA fragments harboring targeting (RNA aptamer or chemical ligand), therapeutic (siRNA, miRNA, ribozymes, and small molecule drugs), and imaging (fluorophore, radiolabels) modules. We have recently demonstrated that RNA nanoparticles can reach and target intracranial brain tumors in mice upon systemic injection with little or no accumulation in adjacent healthy brain tissues or in major healthy internal organs. Herein, we describe various functional imaging methods (fluorescence confocal microscopy, flow cytometry, fluorescence whole body imaging, and magnetic resonance imaging) to evaluate and monitor RNA nanoparticle targeting to intracranial brain tumors in mice. Such imaging techniques will allow in-depth evaluation of specifically delivered RNA therapeutics to brain tumors. PMID:25896001

  5. RNA helicases

    OpenAIRE

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In...

  6. RNA topology

    OpenAIRE

    Frank-Kamenetskii, Maxim D.

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases.

  7. Defects in 18 S or 28 S rRNA processing activate the p53 pathway.

    Science.gov (United States)

    Hölzel, Michael; Orban, Mathias; Hochstatter, Julia; Rohrmoser, Michaela; Harasim, Thomas; Malamoussi, Anastassia; Kremmer, Elisabeth; Längst, Gernot; Eick, Dirk

    2010-02-26

    The p53 tumor suppressor pathway is activated by defective ribosome synthesis. Ribosomal proteins are released from the nucleolus and block human double minute-2 (Hdm2) that targets p53 for degradation. However, it remained elusive how abrogation of individual rRNA processing pathways contributes to p53 stabilization. Here, we show that selective inhibition of 18 S rRNA processing provokes accumulation of p53 as efficiently as abrogated 28 S rRNA maturation. We describe hUTP18 as a novel mammalian rRNA processing factor that is specifically involved in 18 S rRNA production. hUTP18 was essential for the cleavage of the 5'-external transcribed spacer leader sequence from the primary polymerase I transcript, but was dispensable for rRNA transcription. Because maturation of the 28 S rRNA was unaffected in hUTP18-depleted cells, our results suggest that the integrity of both the 18 S and 28 S rRNA synthesis pathways can be monitored independently by the p53 pathway. Interestingly, accumulation of p53 after hUTP18 knock down required the ribosomal protein L11. Therefore, cells survey the maturation of the small and large ribosomal subunits by separate molecular routes, which may merge in an L11-dependent signaling pathway for p53 stabilization.

  8. The monitoring of gene functions on a cell-defined siRNA microarray in human bone marrow stromal and U2OS cells

    Directory of Open Access Journals (Sweden)

    Hi Chul Kim

    2016-06-01

    Full Text Available Here, we developed a cell defined siRNA microarray (CDSM for human bone marrow stromal cells (hBMSCs designed to control the culture of cells inside the spot area without reducing the efficiency of siRNA silencing, “Development of a cell-defined siRNA microarray for analysis of gene functionin human bone marrow stromal cells” (Kim et al., 2016 [1]. First, we confirmed that p65 protein inhibition efficiency was maintained when hBMSCs were culture for 7 days on the siRNA spot, and siRNA spot activity remained in spite of long term storage (10 days and 2 months. Additionally, we confirmed p65 protein inhibition in U2OS cells after 48 h reverse transfection.

  9. Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells - Liquid biopsies for monitoring complications of pregnancy.

    Science.gov (United States)

    Truong, Grace; Guanzon, Dominic; Kinhal, Vyjayanthi; Elfeky, Omar; Lai, Andrew; Longo, Sherri; Nuzhat, Zarin; Palma, Carlos; Scholz-Romero, Katherin; Menon, Ramkumar; Mol, Ben W; Rice, Gregory E; Salomon, Carlos

    2017-01-01

    Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.

  10. Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells – Liquid biopsies for monitoring complications of pregnancy

    Science.gov (United States)

    Truong, Grace; Guanzon, Dominic; Kinhal, Vyjayanthi; Elfeky, Omar; Lai, Andrew; Longo, Sherri; Nuzhat, Zarin; Palma, Carlos; Scholz-Romero, Katherin; Menon, Ramkumar; Mol, Ben W.; Rice, Gregory E.; Salomon, Carlos

    2017-01-01

    Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications. PMID:28350871

  11. The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients.

    Science.gov (United States)

    Collins, Mahlon; Riascos, David; Kovalik, Tina; An, Jiyan; Krupa, Kelly; Krupa, Kristin; Hood, Brian L; Conrads, Thomas P; Renton, Alan E; Traynor, Bryan J; Bowser, Robert

    2012-11-01

    RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

  12. The non-coding RNA Ncr0700/PmgR1 is required for photomixotrophic growth and the regulation of glycogen accumulation in the cyanobacterium Synechocystis sp. PCC 6803

    DEFF Research Database (Denmark)

    de Porcellinis, Alice Jara; Klähn, Stephan; Rosgaard, Lisa

    2016-01-01

    activity and possible factors acting downstream of PmgA are unknown. Here, a genome-wide microarray analysis of a ΔpmgA strain identified the expression of 36 protein-coding genes and 42 non-coding transcripts as significantly altered. From these, the non-coding RNA Ncr0700 was identified as the transcript...... most strongly reduced in abundance. Ncr0700 is widely conserved among cyanobacteria. In Synechocystis its expression is inversely correlated with light intensity. Similarly to a ΔpmgA mutant, a Δncr0700 deletion strain showed an approximately 2-fold increase in glycogen content under photoautotrophic...

  13. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  14. Recombination monitor

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, S. Y. [Brookhaven National Lab. (BNL), Upton, NY (United States); Blaskiewicz, M. [Brookhaven National Lab. (BNL), Upton, NY (United States)

    2017-02-03

    This is a brief report on LEReC recombination monitor design considerations. The recombination produced Au78+ ion rate is reviewed. Based on this two designs are discussed. One is to use the large dispersion lattice. It is shown that even with the large separation of the Au78+ beam from the Au79+ beam, the continued monitoring of the recombination is not possible. Accumulation of Au78+ ions is needed, plus collimation of the Au79+ beam. In another design, it is shown that the recombination monitor can be built based on the proposed scheme with the nominal lattice. From machine operation point of view, this design is preferable. Finally, possible studies and the alternative strategies with the basic goal of the monitor are discussed.

  15. Tumor fluorescence detection technique researches on accumulation of protoporphyrin-Ⅸ in cancer cells induced by FECH-siRNA%亚铁螯合酶抑制诱导细胞内原卟啉Ⅸ积聚的肿瘤荧光成像

    Institute of Scientific and Technical Information of China (English)

    姚前尹; 林绍强; 李囡; 陶良阔; 孙见薇; 温嘉耀; 王晓玉

    2011-01-01

    Aim:To explore the utilization of inhibitor FECH-siRNA in cancer cells. Methods: A siRNA targeting FECH was designed and synthesized chemically, and then loaded with dye Lipofectamine 2000, transferred into HO8910PM cells. Semi-quantitative RT-PCR was used to examine the FECH mR-NA expression. Fluorescence microscopy and confocal laser icroscopy were used to detect the fluorescence intensity in HO891OPM cells in vitro. Results: The expression of FECH mRNA in siRNA-transfectedcells was significantly lower than that in non-transfected cells, with a knocking-down efficiency of 70.1% ( vs the transfer agent group). The intensity of red fluorescence from high level ALA groups is significantly higher than that from low level ALA groups under fluorescent microscope. After interference with siR-NA in the'same concentration of ALA, the intensity of red fluorescence in siRNA-cells is significantlyhigher. Conclusion:The chemically synthesized siRNA targeting FECH can effectively downregulate the FECH expression in HO891 OPM cells which further cause the decline of FECH, increase of PplX accumulation , and plays a synergic and additive effect on PplX accumulation to reduce the of systemic toxicity of ALA and improve the detection of tumor cells.%目的:探讨靶向FECH的siRNA在肿瘤荧光成像中的作用.方法:设计并合成靶向FECH的siRNA,用脂质体转染剂Lipofectamine2000携带后转染H08910PM细胞,用RT-PCR半定量检测细胞中FECHmRNA的表达用荧光显微镜和激光共聚焦显微镜在体外检测H08910PM细胞的荧光强度.结果:siRNA转染后H08910PM细胞的FECHmRNA的表达水平低于未转染组,与转染剂组比敲减效率为70.1%.荧光显微镜成像图显示:随氨基酮戊酸(ALA)浓度的增高,红色荧光逐渐增强;同一ALA浓度下siRNA干扰后,其红色荧光增强.结论:化学合成的靶向FECH的siRNA能够下调H08910PM细胞中FECH基因的表达,增加肿瘤细胞内的PplX积聚,siRNA与小浓

  16. Symptom severity and viral protein or RNA accumulation in lettuce affected by big-vein disease Severidad de síntomas y acumulación de proteínas o ARN virales en lechugas afectadas por la enfermedad de las venas grandes

    Directory of Open Access Journals (Sweden)

    Carolina Araya

    2011-03-01

    Full Text Available Big-vein disease (BVD is a widespread and economically damaging disease in lettuce (Lactuca sativa L.. Typical symptoms are chlorotic clearing around leaf veins, leaf deformations, and impaired head development. In this research, we studied the relationship between symptom intensity and protein and viral RNA accumulation in infected plants. Naturally infected lettuce plants, from the field or greenhouse, were classified according to their symptomatology: mild, moderate, severe, and symptomless. Coat protein accumulation was evaluated by a double antibody sandwich/enzyme-linked immunosorbent assay (DAS-ELISA, and RNA levels were studied by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR and quantitative RT-PCR. Virus coat protein accumulation did not differ for the two viruses associated with this disease among lettuce plants showing different symptom severity. Similarly, abundance of Mirafiori lettuce big-vein virus (MLBVV-RNA3 or Lettuce big-vein associated virus (LBVaV-RNA-2 were not different (P > 0.05 for diverse big vein disease severity rating scales. This suggests that symptom severity expressed by big-vein diseased lettuce plants did not necessarily reflect the accumulation of viruses associated with this disease in the host. Therefore, lettuce plants showing mild symptoms of BVD do not necessarily present lower virus levels than plants showing more severe symptomatology.La enfermedad de las venas grandes de la lechuga (Lactuca sativa L. es de origen viral, está ampliamente distribuida en el mundo, y provoca graves daños económicos en este cultivo. Los síntomas típicos de la enfermedad son clorosis alrededor de las venas, deformación de hojas y ausencia de formación de cabezas. En este trabajo se estudió la relación entre la intensidad de síntomas y la acumulación de proteínas y ARNs de origen viral en plantas afectadas por esta enfermedad. Lechugas infectadas naturalmente, provenientes de campo y de

  17. When to Monitor CD4 Cell Count and HIV RNA to Reduce Mortality and AIDS-Defining Illness in Virologically Suppressed HIV-Positive Persons on Antiretroviral Therapy in High-Income Countries: A Prospective Observational Study

    Science.gov (United States)

    Caniglia, Ellen C.; Sabin, Caroline; Robins, James M.; Logan, Roger; Cain, Lauren E.; Abgrall, Sophie; Mugavero, Michael J.; Hernandez-Diaz, Sonia; Meyer, Laurence; Seng, Remonie; Drozd, Daniel R.; Seage, George R.; Bonnet, Fabrice; Dabis, Francois; Moore, Richard R.; Reiss, Peter; van Sighem, Ard; Mathews, William C.; del Amo, Julia; Moreno, Santiago; Deeks, Steven G.; Muga, Roberto; Boswell, Stephen L.; Ferrer, Elena; Eron, Joseph J.; Napravnik, Sonia; Jose, Sophie; Phillips, Andrew; Olson, Ashley; Justice, Amy C.; Tate, Janet P.; Bucher, Heiner C.; Egger, Matthias; Touloumi, Giota; Sterne, Jonathan A.; Costagliola, Dominique; Saag, Michael; Hernán, Miguel A.

    2016-01-01

    Objective: To illustrate an approach to compare CD4 cell count and HIV-RNA monitoring strategies in HIV-positive individuals on antiretroviral therapy (ART). Design: Prospective studies of HIV-positive individuals in Europe and the USA in the HIV-CAUSAL Collaboration and The Center for AIDS Research Network of Integrated Clinical Systems. Methods: Antiretroviral-naive individuals who initiated ART and became virologically suppressed within 12 months were followed from the date of suppression. We compared 3 CD4 cell count and HIV-RNA monitoring strategies: once every (1) 3 ± 1 months, (2) 6 ± 1 months, and (3) 9–12 ± 1 months. We used inverse-probability weighted models to compare these strategies with respect to clinical, immunologic, and virologic outcomes. Results: In 39,029 eligible individuals, there were 265 deaths and 690 AIDS-defining illnesses or deaths. Compared with the 3-month strategy, the mortality hazard ratios (95% CIs) were 0.86 (0.42 to 1.78) for the 6 months and 0.82 (0.46 to 1.47) for the 9–12 month strategy. The respective 18-month risk ratios (95% CIs) of virologic failure (RNA >200) were 0.74 (0.46 to 1.19) and 2.35 (1.56 to 3.54) and 18-month mean CD4 differences (95% CIs) were −5.3 (−18.6 to 7.9) and −31.7 (−52.0 to −11.3). The estimates for the 2-year risk of AIDS-defining illness or death were similar across strategies. Conclusions: Our findings suggest that monitoring frequency of virologically suppressed individuals can be decreased from every 3 months to every 6, 9, or 12 months with respect to clinical outcomes. Because effects of different monitoring strategies could take years to materialize, longer follow-up is needed to fully evaluate this question. PMID:26895294

  18. A low molecular weight artificial RNA of unique size with multiple probe target regions

    Science.gov (United States)

    Pitulle, C.; Dsouza, L.; Fox, G. E.

    1997-01-01

    Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E. coli. These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring. Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs. This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence. This aRNA is 160 nucleotides in length and again accumulates to high levels in the E. coli cytoplasm without incorporating into ribosomes. The 3xPen aRNA illustrates two improvements in detection. First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs. Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization. These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences. It thus may be possible to detect them directly. An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided.

  19. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    2017-01-01

    for biosensorer,  der kan spore enten microRNA’er eller små molekyler, eksemplificeret ved S-adenosylmethionin (SAM). Slutteligt indikerer foreløbige resultater, at apta-FRET SAM sensoren kan udtrykkes i Escherichia coli-celler, hvilket viser, at RNA-origami arkitekturen muliggør cotransskriptionel foldning af......RNA-nanoteknologi feltet har demonstreret alsidigheden af RNA som byggemateriale, og rationelt designede RNA-nanostrukturer er blevet brugt i udviklingen af strukturelle platforme og dynamiske anordninger med anvendelser både in vitro og in vivo. Naturlige RNA-strukturer foldes cotransskriptionelt...... fra en enkelt RNA-streng, og udfører en lang række komplekse cellulære funktioner. Mange af funktionerne er blevet udnyttet til at skabe funktionelle RNA-baserede nanoapparater, men den nuværende litteratur giver kun få eksempler på cotranskriptionel produktion af RNA-nanostrukturer. I 2014...

  20. RNA granules

    OpenAIRE

    Anderson, Paul; Kedersha, Nancy

    2006-01-01

    Cytoplasmic RNA granules in germ cells (polar and germinal granules), somatic cells (stress granules and processing bodies), and neurons (neuronal granules) have emerged as important players in the posttranscriptional regulation of gene expression. RNA granules contain various ribosomal subunits, translation factors, decay enzymes, helicases, scaffold proteins, and RNA-binding proteins, and they control the localization, stability, and translation of their RNA cargo. We review the relationshi...

  1. A Viral Noncoding RNA Complements a Weakened Viral RNA Silencing Suppressor and Promotes Efficient Systemic Host Infection

    Science.gov (United States)

    Flobinus, Alyssa; Hleibieh, Kamal; Klein, Elodie; Ratti, Claudio; Bouzoubaa, Salah; Gilmer, David

    2016-01-01

    Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long-distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3 in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA-dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3. PMID:27782046

  2. RNA genetics

    Energy Technology Data Exchange (ETDEWEB)

    Domingo, E. (Instituto de Biologia Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, Canto Blanco, Madrid (ES)); Holland, J.J. (California Univ., San Diego, La Jolla, CA (USA). Dept. of Biology); Ahlquist, P. (Wisconsin Univ., Madison, WI (USA). Dept. of Plant Pathology)

    1988-01-01

    This book contains the proceedings on RNA genetics: RNA-directed virus replication Volume 1. Topics covered include: Replication of the poliovirus genome; Influenza viral RNA transcription and replication; and Relication of the reoviridal: Information derived from gene cloning and expression.

  3. RNA epigenetics

    OpenAIRE

    Liu, Nian; Pan, Tao

    2014-01-01

    Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modifi...

  4. 基于高光谱的冬油菜植株氮素积累量监测模型%Monitoring models of plant N accumulation of winter oilseed rape based on hyperspectral technology

    Institute of Scientific and Technical Information of China (English)

    李岚涛; 马驿; 魏全全; 汪善勤; 任涛; 李小坤; 丛日环; 王振; 王少华

    2015-01-01

    Quick, non-destructive and accurate monitoring and diagnosis of plant nitrogen accumulation (PNA) is important for site-specific N management in winter oilseed rape production. To develop a method for determining PNA of winter oilseed rape (Brassica napusL.) with the hyperspectral techniques, field experiments were carried out for two growing seasons (2013-2014 and 2014-2015) at Meichuan town (30°06′47′′ N, 115°35′35′′ E), Hubei province, China. Rapeseed cultivar of Huayouza No. 9 (with low glucosinolate and erucic acid concentrations) was chosen as the test cultivar. Five N (as urea) fertilization rates were applied in the 2013-2014 growing season, i.e., 0 (N0), 90 (N90), 180 (N180), 270(N270) and 360 kg/hm2(N360). Additionally, for further examining the effects of N status on crop growth and spectral reflectance characteristics, three additional N rates, 45 (N45), 135 (N135) and 225 kg/hm2 (N225) were applied in the 2014-2015 growing season. Canopy hyperspectral reflectance and PNA under different N application rates at seedling, budding and flowering stage during the two growing seasons were measured separately using a Field Spec Pro spectrometer (Analytical Spectral Devices Inc. (ASD), Boulder, CO, USA) and chemical assays in the laboratory. Using linear and nonlinear regression methods, the estimate model for PNA of winter oilseed rape was built on the basis of the experiment data in 2013-2014 acted as training data set, and its precision had been evaluated and tested based on the experiment data in 2014-2015 acted as testing data set. The coefficient of determination (R2), relative root mean square error (RRMSE) and mean relative error (MRE) were used to evaluate the fitness between observed and predicted PNA values. The following sensitivity analysis method, Noise Equivalent (NE) model was calculated to assess the sensitivity of the optimal spectral parameters for detecting changes in PNA across different growth stages. The results indicated

  5. Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique.

    Science.gov (United States)

    Jani, Ilesh V; Meggi, Bindiya; Vubil, Adolfo; Sitoe, Nádia E; Bhatt, Nilesh; Tobaiwa, Ocean; Quevedo, Jorge I; Loquiha, Osvaldo; Lehe, Jonathan D; Vojnov, Lara; Peter, Trevor F

    2016-08-01

    Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted.

  6. Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique

    Science.gov (United States)

    Meggi, Bindiya; Vubil, Adolfo; Sitoe, Nádia E.; Bhatt, Nilesh; Tobaiwa, Ocean; Quevedo, Jorge I.; Loquiha, Osvaldo; Lehe, Jonathan D.; Vojnov, Lara; Peter, Trevor F.

    2016-01-01

    Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted. PMID:27252459

  7. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    in diabetes resulting from the diabetic state, a dysfunction that includes increased production of hydrogen peroxide. We suggest that the intracellular RNA oxidation is compartmentalized from the traditional biomarkers in the extracellular compartment, and therefore provides independent prognostic value...... diabetes. In agreement with our previous finding, DNA oxidation did not show any prognostic value. RNA oxidation represents oxidative stress intracellularly, presumably predominantly in the cytosol. The mechanism of RNA oxidation is not clear, but hypothesized to result from mitochondrial dysfunction...

  8. Dye sublimation as a measure of accumulated heat exposure.

    Science.gov (United States)

    Shi, Xiaoju; Ying, Xiaofang; Deng, Zongwu

    2013-10-21

    Heat history monitor: Combination of the sublimation and adsorption processes of specific dyes can be used as a measure of accumulated heat exposure. Mass transfer from the sublimation layer to the adsorption layer strongly depends on temperature and results in a gradual color change in the adsorption layer. The total color change reflects the accumulated heat exposure.

  9. Epstein-Barr virus (EBV) DNA in whole blood as a superior prognostic and monitoring factor than EBV-encoded small RNA in situ hybridization in diffuse large B-cell lymphoma.

    Science.gov (United States)

    Liang, J-H; Lu, T-X; Tian, T; Wang, L; Fan, L; Xu, J; Zhang, R; Gong, Q-X; Zhang, Z-H; Li, J-Y; Xu, W

    2015-06-01

    Epstein-Barr virus (EBV) status was retrospectively analysed by the use of EBV-encoded small RNA (EBER) in situ hybridization (ISH) and EBV DNA analysis in whole blood with diffuse large B-cell lymphoma, to assess the clinical significance for diagnosis, prognostication, and monitoring of tumour burden. Three hundred and twenty-nine patients were retrospectively enrolled, with 232 patients being available for EBER ISH analysis, 189 patients for EBV DNA analysis, and 138 patients for both analyses. EBER was positive in 24 (10.3%) patients, and EBV DNA was positive in 18 (9.5%) patients; the two analyses had 92.8% concordance. Patients with pretreatment EBER positivity had worse overall survival (OS) than those without EBER positivity (p 0.03); the same pattern was observed for EBV DNA (p EBV DNA were combined (p EBV DNA (hazard ratio 3.71, 95% CI 1.78-7.74, p EBV DNA, the transformation from positive to negative after cycle 3 with chemotherapy may have the most capacity to distinguish a superior from an inferior outcome. These findings suggest that EBV DNA in whole blood has good concordance with EBER ISH, and that it may be a better prognostic and monitoring biomarker than EBER.

  10. Accumulation by Conservation

    NARCIS (Netherlands)

    Büscher, Bram; Fletcher, Robert

    2014-01-01

    Following the financial crisis and its aftermath, it is clear that the inherent contradictions of capitalist accumulation have become even more intense and plunged the global economy into unprecedented turmoil and urgency. Governments, business leaders and other elite agents are frantically searchin

  11. RNA. Introduction.

    Science.gov (United States)

    Bao, Marie Z; Kruger, Robert P; Rivas, Fabiola; Smith, Orla; Szewczak, Lara

    2009-02-20

    Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells.

  12. rRNA-based monitoring of the microbiota involved in Fontina PDO cheese production in relation to different stages of cow lactation.

    Science.gov (United States)

    Dolci, Paola; De Filippis, Francesca; La Storia, Antonietta; Ercolini, Danilo; Cocolin, Luca

    2014-08-18

    Fontina Protected Denomination of Origin (PDO) cheese is a full-fat semi-cooked cheese traditionally made in Northwest Italy (Aosta Valley) and manufactured from raw cow's milk. The management of cattle farms in Aosta Valley calls for seasonal migration to high pastures during the summer and the concentration of calving during the autumn and the beginning of the winter. Based on cattle physiology and given to calving seasonality, three cow lactation phases i.e. post-partum, oestrus and early gestation, can be identified and an effect could be hypothesized on average milk composition and on cheese quality. The aim of the present paper was to investigate the bacterial dynamics during Fontina PDO cheese manufacturing and ripening, in relation to the different lactation stages, in order to evaluate a possible correlation between microbiota and phase of lactation. For this purpose, microbial RNA analysis was carried out by RT-PCR coupled with DGGE and high-throughput sequencing. A good performance of the starter cultures was highlighted throughout Fontina PDO manufacturing and ripening; in fact, the starter prevailed against the autochthonous microbiota. Thus, the microbial activity, which was supposed to affect the final quality of Fontina PDO cheese, appeared to be strictly associated to the presence of the starter, which did not show any difference in its performance according to the different stages of cow lactation. Therefore, the results of this research highlighted a negligible correlation between the microbiota of raw milk and the organoleptic quality and typicity of Fontina cheese in relation to lactation seasonality.

  13. Antiproton Accumulator (AA)

    CERN Multimedia

    Photographic Service

    1980-01-01

    The AA in its final stage of construction, before it disappeared from view under concrete shielding. Antiprotons were first injected, stochastically cooled and accumulated in July 1980. From 1981 on, the AA provided antiprotons for collisions with protons, first in the ISR, then in the SPS Collider. From 1983 on, it also sent antiprotons, via the PS, to the Low-Energy Antiproton Ring (LEAR). The AA was dismantled in 1997 and shipped to Japan.

  14. Molecular Beacons: Powerful Tools for Imaging RNA in Living Cells

    OpenAIRE

    Ricardo Monroy-Contreras; Luis Vaca

    2011-01-01

    Recent advances in RNA functional studies highlights the pivotal role of these molecules in cell physiology. Diverse methods have been implemented to measure the expression levels of various RNA species, using either purified RNA or fixed cells. Despite the fact that fixed cells offer the possibility to observe the spatial distribution of RNA, assays with capability to real-time monitoring RNA transport into living cells are needed to further understand the role of RNA dynamics in cellular fu...

  15. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation.

  16. Ice slurry accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Christensen, K.G.; Kauffeld, M.

    1998-06-01

    More and more refrigeration systems are designed with secondary loops, thus reducing the refrigerant charge of the primary refrigeration plant. In order not to increase energy consumption by introducing a secondary refrigerant, alternatives to the well established single phase coolants (brines) and different concepts of the cooling plant have to be evaluated. Combining the use of ice-slurry - mixture of water, a freezing point depressing agent (antifreeze) and ice particles - as melting secondary refrigerant and the use of a cool storage makes it possible to build plants with secondary loops without increasing the energy consumption and investment. At the same time the operating costs can be kept at a lower level. The accumulation of ice-slurry is compared with other and more traditional storage systems. The method is evaluated and the potential in different applications is estimated. Aspects of practically use of ice-slurry has been examined in the laboratory at the Danish Technological Institute (DTI). This paper will include the final conclusions from this work concerning tank construction, agitator system, inlet, outlet and control. The work at DTI indicates that in some applications systems with ice-slurry and accumulation tanks have a great future. These applications are described by a varying load profile and a process temperature suiting the temperature of ice-slurry (-3 - -8/deg. C). (au)

  17. DDX3 DEAD-Box RNA Helicase Is Required for Hepatitis C Virus RNA Replication▿

    OpenAIRE

    2007-01-01

    DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supern...

  18. MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.

    Science.gov (United States)

    Xu, Kai; Lin, Jing; Zandi, Roza; Roth, Jack A; Ji, Lin

    2016-07-21

    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

  19. Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

    Science.gov (United States)

    Raghavan, V.

    1991-01-01

    Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.

  20. The Antiproton Accumulator (AA)

    CERN Multimedia

    1980-01-01

    Section 06 - 08*) of the AA where the dispersion (and hence the horizontal beam size) is large. One can distinguish (left to right): A vacuum-tank, two bending magnets (BST06 and BST07 in blue) with a quadrupole (QDN07, in red) in between, another vacuum-tank, a wide quadrupole (QFW08) and a further tank . The tanks are covered with heating tape for bake-out. The tank left of BST06 contained the stack core pickup for stochastic cooling (see 7906193, 7906190, 8005051), the two other tanks served mainly as vacuum chambers in the region where the beam was large. Peter Zettwoch works on BST06. *) see: H. Koziol, Antiproton Accumulator Parameter List, PS/AA/Note 84-2 (1984)

  1. Solids Accumulation Scouting Studies

    Energy Technology Data Exchange (ETDEWEB)

    Duignan, M. R.; Steeper, T. J.; Steimke, J. L.

    2012-09-26

    The objective of Solids Accumulation activities was to perform scaled testing to understand the behavior of remaining solids in a Double Shell Tank (DST), specifically AW-105, at Hanford during multiple fill, mix, and transfer operations. It is important to know if fissionable materials can concentrate when waste is transferred from staging tanks prior to feeding waste treatment plants. Specifically, there is a concern that large, dense particles containing plutonium could accumulate in poorly mixed regions of a blend tank heel for tanks that employ mixing jet pumps. At the request of the DOE Hanford Tank Operations Contractor, Washington River Protection Solutions, the Engineering Development Laboratory of the Savannah River National Laboratory performed a scouting study in a 1/22-scale model of a waste staging tank to investigate this concern and to develop measurement techniques that could be applied in a more extensive study at a larger scale. Simulated waste tank solids: Gibbsite, Zirconia, Sand, and Stainless Steel, with stainless steel particles representing the heavier particles, e.g., plutonium, and supernatant were charged to the test tank and rotating liquid jets were used to mix most of the solids while the simulant was pumped out. Subsequently, the volume and shape of the mounds of residual solids and the spatial concentration profiles for the surrogate for heavier particles were measured. Several techniques were developed and equipment designed to accomplish the measurements needed and they included: 1. Magnetic particle separator to remove simulant stainless steel solids. A device was designed and built to capture these solids, which represent the heavier solids during a waste transfer from a staging tank. 2. Photographic equipment to determine the volume of the solids mounds. The mounds were photographed as they were exposed at different tank waste levels to develop a composite of topographical areas. 3. Laser rangefinders to determine the volume of

  2. Mutation rates among RNA viruses

    OpenAIRE

    Drake, John W.; Holland, John J.

    1999-01-01

    The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viru...

  3. Monitoring Biomass and N Accumulation at Jointing Stage in Winter Wheat Based on SPOT-5 Images%基于SPOT-5影像的冬小麦拔节期生物量及氮积累量监测

    Institute of Scientific and Technical Information of China (English)

    王备战; 冯晓; 温暖; 郑涛; 杨武德

    2012-01-01

    [Objective] The monitoring equations to determine the above-ground biomass weight and N accumulation of winter wheat at jointing stage were established based on SPOT-5 images to provide reference for prediction of productivity and nitrogen regulation in mass production of wheat. [Method] Using SPOT-5 images and test data, the relationships were analyzed between spectral parameter and above-ground biomass weight, N accumulation of winter wheat at jointing stage, then the monitoring equations were established. [Result] The correlation between reflectance of green, red, near-infrared band and above-ground biomass weight, N accumulation reached 0.01 significant level. Reflectance of near-infrared band showed the most sensitive response for above-ground biomass weight, and reflectance of red band showed the most sensitive response to above-ground N accumulation. The exponential function model based on NDVI inversed plant above-ground biomass weight was the best, and R2 was 0.696** and that of RMSE was 258.92 kg·hm-2. The exponential function model based on the reflectance ratio of near-infrared band to red band inversed plant above-ground N accumulation was the best, and R2 was 0.717** and that of RMSE was 19.24 kg·hm-2. Based on the monitoring models, the thematic maps were produced to monitor above-ground biomass and N accumulation at jointing stage. [Conclusion] Using SPOT-5 images can achieve high precision in growth monitoring of winter wheat at jointing stage and it has a wide application prospect.%[目的]建立基于SPOT-5遥感信息的冬小麦拔节期地上部生物量和氮积累量的遥感监测模型,为利用SPOT-5影像进行大面积小麦生产力预测和氮素调控提供依据.[方法]分析冬小麦拔节期地上部生物量和氮积累量与同期SPOT-5光谱参数之间的定量关系,筛选出适宜的光谱参数,建立并评价冬小麦拔节期地上部生物量和氮积累量的遥感监测模型.[结果]绿光、红光和近红外波

  4. The use of 16S rRNA gene metagenetic monitoring of refrigerated food products for understanding the kinetics of microbial subpopulations at different storage temperatures: the example of white pudding.

    Science.gov (United States)

    Cauchie, Emilie; Gand, Mathieu; Kergourlay, Gilles; Taminiau, Bernard; Delhalle, Laurent; Korsak, Nicolas; Daube, Georges

    2017-04-17

    In order to control food losses and wastage, monitoring the microbial diversity of food products, during processing and storage is important, as studies have highlighted the metabolic activities of some microorganisms which can lead to spoilage. Knowledge of this diversity can be greatly improved by using a metagenetic approach based on high throughput 16S rRNA gene sequencing, which enables a much higher resolution than culture-based methods. Moreover, the Jameson effect, a phenomenon described by Jameson in 1962, is often used to classify bacterial strains within an ecosystem. According to this, we have studied the bacterial microbiota of Belgian white pudding during storage at different temperatures using culture-dependent and independent methods. The product was inoculated with a mix of dominant strains previously isolated from this foodstuff at the end of its shelf life (Carnobacterium maltaromaticum, Lactobacillus fuchuensis, Lactobacillus graminis, Lactobacillus oligofermentans, Lactococcus lactis, Leuconostoc mesenteroides, Raoultella terrigena and Serratia sp.). Daily during 16days, the absolute abundance of inoculated strain was monitored by combining total count on plate agar and metagenetic analysis. The results were confirmed by qPCR analysis. The growth of each species was modelled for each temperature conditions, representative of good or bad storage practices. These data allowed the bacterial strains subdivision into three classes based on criteria of growth parameters for the studied temperature: the "dominant", the "subdominant" and the "inhibited" bacterial species, according to their maximal concentration (Nmax, log CFU/g), growth rate (μmax, 1/h) and time to reach the stationary phase (TRSP, days). Thereby, depending on the storage conditions, these data have permitted to follow intrinsically the evolution of each strain on the bacterial ecosystem of Belgian white pudding. Interestingly, it has shown that the reliability of the Jameson effect

  5. The Antiproton Accumulator (AA)

    CERN Multimedia

    1980-01-01

    A section of the AA where the dispersion (and hence the horizontal beam size) is large. One can distinguish (left to right): A large vacuum-tank, a quadrupole (QDN09*), a bending magnet (BST08), another vacuum-tank, a wide quadrupole (QFW08) and (in the background) a further bending magnet (BST08). The tanks are covered with heating tape for bake-out. The tank left of QDN09 contained the kickers for stochastic pre-cooling (see 790621, 8002234, 8002637X), the other one served mainly as vacuum chamber in the region where the beam was large. Peter Zettwoch works on QFW08. * see: H. Koziol, Antiproton Accumulator Parameter List, PS/AA/Note 84-2 (1984) See under 7911303, 7911597X, 8004261 and 8202324. For photos of the AA in different phases of completion (between 1979 and 1982) see: 7911303, 7911597X, 8004261, 8004608X, 8005563X, 8005565X, 8006716X, 8006722X, 8010939X, 8010941X, 8202324, 8202658X, 8203628X .

  6. ITER helium ash accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Hogan, J.T.; Hillis, D.L.; Galambos, J.; Uckan, N.A. (Oak Ridge National Lab., TN (USA)); Dippel, K.H.; Finken, K.H. (Forschungszentrum Juelich GmbH (Germany, F.R.). Inst. fuer Plasmaphysik); Hulse, R.A.; Budny, R.V. (Princeton Univ., NJ (USA). Plasma Physics Lab.)

    1990-01-01

    Many studies have shown the importance of the ratio {upsilon}{sub He}/{upsilon}{sub E} in determining the level of He ash accumulation in future reactor systems. Results of the first tokamak He removal experiments have been analysed, and a first estimate of the ratio {upsilon}{sub He}/{upsilon}{sub E} to be expected for future reactor systems has been made. The experiments were carried out for neutral beam heated plasmas in the TEXTOR tokamak, at KFA/Julich. Helium was injected both as a short puff and continuously, and subsequently extracted with the Advanced Limiter Test-II pump limiter. The rate at which the He density decays has been determined with absolutely calibrated charge exchange spectroscopy, and compared with theoretical models, using the Multiple Impurity Species Transport (MIST) code. An analysis of energy confinement has been made with PPPL TRANSP code, to distinguish beam from thermal confinement, especially for low density cases. The ALT-II pump limiter system is found to exhaust the He with maximum exhaust efficiency (8 pumps) of {approximately}8%. We find 1<{upsilon}{sub He}/{upsilon}{sub E}<3.3 for the database of cases analysed to date. Analysis with the ITER TETRA systems code shows that these values would be adequate to achieve the required He concentration with the present ITER divertor He extraction system.

  7. Engineering Structurally Interacting RNA (sxRNA)

    Science.gov (United States)

    Doyle, Francis; Lapsia, Sameer; Spadaro, Salvatore; Wurz, Zachary E.; Bhaduri-McIntosh, Sumita; Tenenbaum, Scott A.

    2017-01-01

    RNA-based three-way junctions (3WJs) are naturally occurring structures found in many functional RNA molecules including rRNA, tRNA, snRNA and ribozymes. 3WJs are typically characterized as resulting from an RNA molecule folding back on itself in cis but could also form in trans when one RNA, for instance a microRNA binds to a second structured RNA, such as a mRNA. Trans-3WJs can influence the final shape of one or both of the RNA molecules and can thus provide a means for modulating the availability of regulatory motifs including potential protein or microRNA binding sites. Regulatory 3WJs generated in trans represent a newly identified regulatory category that we call structurally interacting RNA or sxRNA for convenience. Here we show that they can be rationally designed using familiar cis-3WJ examples as a guide. We demonstrate that an sxRNA “bait” sequence can be designed to interact with a specific microRNA “trigger” sequence, creating a regulatable RNA-binding protein motif that retains its functional activity. Further, we show that when placed downstream of a coding sequence, sxRNA can be used to switch “ON” translation of that sequence in the presence of the trigger microRNA and the amount of translation corresponded with the amount of microRNA present. PMID:28350000

  8. Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

    Science.gov (United States)

    Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

    2017-08-01

    Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export.IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

  9. Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution.

    Science.gov (United States)

    Chursov, Andrey; Frishman, Dmitrij; Shneider, Alexander

    2013-09-01

    Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the 'long-term evolution experiment'. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins.

  10. Integrated optical fiber lattice accumulators

    OpenAIRE

    Atherton, Adam F

    1997-01-01

    Approved for public release; distribution is unlimited. Sigma-delta modulators track a signal by accumulating the error between an input signal and a feedback signal. The accumulated energy is amplitude analyzed by a comparator. The comparator output signal is fed back and subtracted from the input signal. This thesis is primarily concerned with designing accumulators for inclusion in an optical sigma-delta modulator. Fiber lattice structures with optical amplifiers are used to perform the...

  11. RNA-DNA Chimeras in the Context of an RNA World Transition to an RNA/DNA World.

    Science.gov (United States)

    Gavette, Jesse V; Stoop, Matthias; Hud, Nicholas V; Krishnamurthy, Ramanarayanan

    2016-10-10

    The RNA world hypothesis posits that DNA and proteins were later inventions of early life, or the chemistry that gave rise to life. Most scenarios put forth for the emergence of DNA assume a clean separation of RNA and DNA polymer, and a smooth transition between RNA and DNA. However, based on the reality of "clutter" and lack of sophisticated separation/discrimination mechanisms in a protobiological (and/or prebiological) world, heterogeneous RNA-DNA backbone containing chimeric sequences could have been common-and have not been fully considered in models transitioning from an RNA world to an RNA-DNA world. Herein we show that there is a significant decrease in Watson-Crick duplex stability of the heterogeneous backbone chimeric duplexes that would impede base-pair mediated interactions (and functions). These results point to the difficulties for the transition from one homogeneous system (RNA) to another (RNA/DNA) in an RNA world with a heterogeneous mixture of ribo- and deoxyribonucleotides and sequences, while suggesting an alternative scenario of prebiological accumulation and co-evolution of homogeneous systems (RNA and DNA).

  12. Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model.

    Science.gov (United States)

    Herrero-Fresno, Ana; Martínez, Noelia; Sánchez-Llana, Esther; Díaz, María; Fernández, María; Martin, Maria Cruz; Ladero, Victor; Alvarez, Miguel A

    2012-07-02

    Tyramine and histamine are the biogenic amines (BAs) most commonly found in cheese, in which they appear as a result of the microbial enzymatic decarboxylation of tyrosine and histidine respectively. Given their toxic effects, their presence in high concentrations in foods should be avoided. In this work, samples of three cheeses (Zamorano, Cabrales and Emmental) with long ripening periods, and that often have high BA concentrations, were screened for the presence of BA-degrading lactic acid bacteria (LAB). Seventeen isolates were found that were able to degrade tyramine and histamine in broth culture. All 17 isolates were identified by 16S rRNA sequencing as belonging to Lactobacillus casei. They were typed by plasmid S1-PFGE and genomic macrorestriction-PFGE analysis. Two strains (L. casei 4a and 5b) associated with high degradation rates for both BAs were selected to test how this ability might affect histamine and tyramine accumulation in a Cabrales-like mini-cheese manufacturing model. The quantification of BAs and the monitoring of the strains' growth over ripening were undertaken by RP-HPLC and qPCR respectively. Both strains were found to reduce histamine and tyramine accumulation. These two strains might be suitable for use as adjunct cultures for reducing the presence of BAs in cheese.

  13. Inducible control of subcellular RNA localization using a synthetic protein-RNA aptamer interaction.

    Directory of Open Access Journals (Sweden)

    Brian J Belmont

    Full Text Available Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis-elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs, which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically-inducible RNA-protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i the Tet Repressor protein (TetR genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii a target transcript containing genetically encoded TetR-binding RNA aptamers. TetR-fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

  14. Cyclin-dependent kinase 9 links RNA polymerase II transcription to processing of ribosomal RNA.

    Science.gov (United States)

    Burger, Kaspar; Mühl, Bastian; Rohrmoser, Michaela; Coordes, Britta; Heidemann, Martin; Kellner, Markus; Gruber-Eber, Anita; Heissmeyer, Vigo; Strässer, Katja; Eick, Dirk

    2013-07-19

    Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

  15. Monitoring nitrogen accumulation in mosses in central European forests.

    Science.gov (United States)

    Pesch, Roland; Schröder, Winfried; Schmidt, Gunther; Genssler, Lutz

    2008-10-01

    In order to assess whether nitrogen (N) loads in mosses reflect different land uses, 143 sites in North Rhine-Westphalia, the Weser-Ems Region and the Euro Region Nissa were sampled between 2000 and 2005. The data were analysed statistically with available surface information on land use and forest conditions. N bioaccumulation in mosses in the Weser-Ems Region with high densities of agricultural land use and livestock exceeded the concentrations in the more industrialised Euro Region Nissa. In all three study areas agricultural and livestock spatial densities were found to be positively correlated with N bioaccumulation in mosses. In North Rhine-Westphalia, the N concentrations in mosses was also moderately correlated with N concentrations in leaves and needles of forest trees. The moss method proved useful to assess the spatial patterns of N bioaccumulation due to land use.

  16. Glia to axon RNA transfer.

    Science.gov (United States)

    Sotelo, José Roberto; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José Roberto; Calliari, Aldo; Cal, Karina; Bresque, Mariana; Dipaolo, Andrés; Farias, Joaquina; Mercer, John A

    2014-03-01

    The existence of RNA in axons has been a matter of dispute for decades. Evidence for RNA and ribosomes has now accumulated to a point at which it is difficult to question, much of the disputes turned to the origin of these axonal RNAs. In this review, we focus on studies addressing the origin of axonal RNAs and ribosomes. The neuronal soma as the source of most axonal RNAs has been demonstrated and is indisputable. However, the surrounding glial cells may be a supplemental source of axonal RNAs, a matter scarcely investigated in the literature. Here, we review the few papers that have demonstrated that glial-to-axon RNA transfer is not only feasible, but likely. We describe this process in both invertebrate axons and vertebrate axons. Schwann cell to axon ribosomes transfer was conclusively demonstrated (Court et al. [2008]: J. Neurosci 28:11024-11029; Court et al. [2011]: Glia 59:1529-1539). However, mRNA transfer still remains to be demonstrated in a conclusive way. The intercellular transport of mRNA has interesting implications, particularly with respect to the integration of glial and axonal function. This evolving field is likely to impact our understanding of the cell biology of the axon in both normal and pathological conditions. Most importantly, if the synthesis of proteins in the axon can be controlled by interacting glia, the possibilities for clinical interventions in injury and neurodegeneration are greatly increased.

  17. Use of De Novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols.

    Directory of Open Access Journals (Sweden)

    Cresten B Mansfeldt

    Full Text Available Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596 of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.

  18. Use of De Novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols.

    Science.gov (United States)

    Mansfeldt, Cresten B; Richter, Lubna V; Ahner, Beth A; Cochlan, William P; Richardson, Ruth E

    2016-01-01

    Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596) of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.

  19. Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage.

    Directory of Open Access Journals (Sweden)

    Konstantin Axt

    Full Text Available During yeast ribosome synthesis, three early cleavages generate the 20S precursor to the 18S rRNA component of the 40S subunits. These cleavages can occur either on the nascent transcript (nascent transcript cleavage; NTC or on the 35S pre-rRNA that has been fully transcribed and released from the rDNA (released transcript cleavage; RTC. These alternative pathways cannot be assessed by conventional RNA analyses, since the pre-rRNA products of NTC and RTC are identical. They can, however, be distinguished kinetically by metabolic labeling and quantified by modeling of the kinetic data. The aim of this work was to use these approaches as a practical tool to identify factors that mediate the decision between utilization of NTC and RTC. The maturation pathways of the 40S and 60S ribosomal subunits are largely distinct. However, depletion of some early-acting 60S synthesis factors, including the 5'-exonuclease Rat1, leads to accumulation of the 35S pre-rRNA and delayed 20S pre-rRNA synthesis. We speculated that this might reflect the loss of NTC. Rat1 acts catalytically in 5.8S and 25S rRNA processing but binds to the pre-rRNA prior to these activities. Kinetic data for strains depleted of Rat1 match well with the modeled effects of strongly reduced NTC. This was confirmed by EM visualization of "Miller" chromatin spreads of nascent pre-rRNA transcripts. Modeling further indicates that NTC takes place in a limited time window, when the polymerase has transcribed ∼ 1.5 Kb past the A2 cleavage site. We speculate that assembly of early-acting 60S synthesis factors is monitored as a quality control system prior to NTC.

  20. Accumulation chamber as monitoring system for biogas emission from solid waste land filling: preliminary experimental results and elaborations on italian provincial scale; Camera di accumulo portatile per il monitoraggio di emissioni di biogas da discarica: risultati sperimentali ed elaborazioni preliminari a scala provinciale

    Energy Technology Data Exchange (ETDEWEB)

    Capaccioni, B.; Pirillo, L. [Urbino Univ. Carlo Bo, Urbino (Italy). Istituto di Vulcanologia e Geochimica; Didero, M. [Urbino Univ. Carlo Bo, Urbino (Italy). Istituto di Geologia Applicata; Lucci, P.; Scartoni, P. [Area Territorio e Ambiente Provincia di Arezzo, Arezzo (Italy). Servizio Ecologia; Tatano, F. [Urbino Univ. Carlo Bo, Urbino (Italy). Facolta' di Scienze Ambientali

    2005-04-01

    On site CO{sub 2} flux measurements with the static, not stationary accumulation chamber system were experimentally carried out in no. 5 MSW (active and closed) landfills located in the territory of the Province of Arezzo (Tuscany Region). Corresponding CO{sub 2} emission flux maps were contoured and analysed, revealing a possible, preliminary geometrical classification of biogas dispersion: diffuse dispersion (internal), lateral/angular dispersion (internal), and external dispersion. Also specific (volume and surface) biogas emission parameters were calculated and graphically compared for the monitored inactive facilities. [Italian] La metodologia strumentale portatile della camera di accumulo, statica non stazionaria, e' stata adoperata sperimentalmente per la misura on site di flussi puntuali di emissione di CO{sub 2} in cinque discariche (attive e non) per RSU ed assimilabili localizzate nel territorio della Provincia di Arezzo. Si sono elaborate, ed analizzate criticamente, le corrispondenti mappe areali di flusso, che hanno consentito di delineare una possibile classificazione geometrica - ancorche' preliminare - di dispersioni di biogas generabili da discarica: diffuse interne, laterali/angolari interne, esterne. Con riferimento alle discariche inattive d'indagine, si sono altresi' determinati, e rappresentati graficamente, i valori di possibili parametri specifici (volumetrico, superficiale) di dispersione di biogas.

  1. Probing the MicroRNA and Small Interfering RNA Pathways with Virus-Encoded Suppressors of RNA SilencingW⃞

    Science.gov (United States)

    Dunoyer, Patrice; Lecellier, Charles-Henri; Parizotto, Eneida Abreu; Himber, Christophe; Voinnet, Olivier

    2004-01-01

    In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing. PMID:15084715

  2. Emerging connections between RNA and autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lubas, Michal; Lund, Anders H

    2017-01-01

    of studies have focused on protein, lipid and carbohydrate catabolism via autophagy, accumulating data supports the view that several types of RNA and associated ribonucleoprotein complexes are specifically recruited to phagophores (precursors to autophagosomes) and subsequently degraded in the lysosome/vacuole...

  3. Triggering of RNA interference with RNA-RNA, RNA-DNA, and DNA-RNA nanoparticles.

    Science.gov (United States)

    Afonin, Kirill A; Viard, Mathias; Kagiampakis, Ioannis; Case, Christopher L; Dobrovolskaia, Marina A; Hofmann, Jen; Vrzak, Ashlee; Kireeva, Maria; Kasprzak, Wojciech K; KewalRamani, Vineet N; Shapiro, Bruce A

    2015-01-27

    Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use.

  4. Manganese As a Metal Accumulator

    Science.gov (United States)

    Manganese deposits in water distribution systems accumulate metals, radionuclides and oxyanions by a combination of surface complexation, adsorption and solid substitution, as well as a combination of oxidation followed by manganese reduction and sorption of the oxidized constitu...

  5. Manganese As a Metal Accumulator

    Science.gov (United States)

    Manganese deposits in water distribution systems accumulate metals, radionuclides and oxyanions by a combination of surface complexation, adsorption and solid substitution, as well as a combination of oxidation followed by manganese reduction and sorption of the oxidized constitu...

  6. DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication.

    Science.gov (United States)

    Ariumi, Yasuo; Kuroki, Misao; Abe, Ken-ichi; Dansako, Hiromichi; Ikeda, Masanori; Wakita, Takaji; Kato, Nobuyuki

    2007-12-01

    DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supernatants were suppressed in DDX3 knockdown cells after inoculation of the cell culture-generated HCVcc. Thus, DDX3 is required for HCV RNA replication.

  7. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaofei [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036 (China); Deng, Ping; Cui, Hongguang [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); Wang, Aiming, E-mail: aiming.wang@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada)

    2015-11-15

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  8. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity.

    Science.gov (United States)

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Kazadi, David; Sun, Jianbo; Federation, Alexander; Chao, Jaime; Elliott, Oliver; Liu, Zhi-Ping; Economides, Aris N; Bradner, James E; Rabadan, Raul; Basu, Uttiya

    2015-05-01

    We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.

  9. Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase.

    Science.gov (United States)

    Nohales, María-Ángeles; Flores, Ricardo; Daròs, José-Antonio

    2012-08-21

    Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95-G96 and containing 5'-phosphomonoester and 3'-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA ligase to act as RNA-dependent RNA polymerase and RNA ligase, respectively.

  10. Modeling IRA Accumulation and Withdrawals

    OpenAIRE

    Sabelhaus, John

    2000-01-01

    Empirical analysis of IRA accumulation and withdrawal patterns is limited because information about IRA balances and flows is not available for a sample of taxpayers. This paper combines survey data on IRA balances with individual tax return data on IRA flows to study IRA accumulation and withdrawal patterns across cohorts. The analysis shows that IRA rules such as penalties for early withdrawals and minimum distribution requirements have predictable effects on IRA flows. The estimated propen...

  11. RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

    Science.gov (United States)

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-07-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network.

  12. RNA chaperones encoded by RNA viruses

    Institute of Scientific and Technical Information of China (English)

    Jie Yang; Hongjie Xia; Qi Qian; Xi Zhou

    2015-01-01

    RNAs are functionally diverse macromolecules whose proper functions rely strictly upon their correct tertiary structures. However, because of their high structural flexibility, correct folding of RNAs is challenging and slow. Therefore, cells and viruses encode a variety of RNA remodeling proteins, including helicases and RNA chaperones. In RNA viruses, these proteins are believed to play pivotal roles in all the processes involving viral RNAs during the life cycle. RNA helicases have been studied extensively for decades, whereas RNA chaperones, particularly virus-encoded RNA chaperones, are often overlooked. This review describes the activities of RNA chaperones encoded by RNA viruses, particularly the ones identified and characterized in recent years, and the functions of these proteins in different steps of viral life cycles, and presents an overview of this unique group of proteins.

  13. Extracellular RNA Communication (ExRNA)

    Data.gov (United States)

    Federal Laboratory Consortium — Until recently, scientists believed RNA worked mostly inside the cell that produced it. Some types of RNA help translate genes into proteins that are necessary for...

  14. Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing

    Science.gov (United States)

    Nguyen, Anh H.; Lee, Jong Uk; Sim, Sang Jun

    2016-02-01

    RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ~29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

  15. Reducing arsenic accumulation in rice grain through iron oxide amendment.

    Science.gov (United States)

    Farrow, Eric M; Wang, Jianmin; Burken, Joel G; Shi, Honglan; Yan, Wengui; Yang, John; Hua, Bin; Deng, Baolin

    2015-08-01

    Effects of soil-arsenic (As), phosphorus and iron oxide on As accumulation in rice grain were investigated. Cultivars that have significantly different sensitivity to As, straighthead-resistant Zhe 733 and straighthead-susceptible Cocodrie, were used to represent different cultivar varieties. The grain accumulation of other elements of concern, selenium (Se), molybdenum (Mo), and cadmium (Cd) was also monitored. Results demonstrated that high soil-As not only resulted in high grain-As, but could also result in high grain-Se, and Zhe 733 had significantly less grain-As than Cocodrie did. However, soil-As did not impact grain-Mo and Cd. Among all elements monitored, iron oxide amendment significantly reduced grain-As for both cultivars, while the phosphate application only reduced grain-Se for Zhe 733. Results also indicated that cultivar type significantly impacted grain accumulation of all monitored trace elements. Therefore, applying iron oxide to As-contaminated land, in addition to choosing appropriate rice cultivar, can effectively reduce the grain accumulation of As.

  16. Accumulation of contaminants from urban rainfall runoff in blue crabs: A pilot study

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — The objective of this pilot study was to determine the feasibility of using caged blue crabs Callinectes sapidus to monitor accumulation of contaminants in urban...

  17. Tat RNA silencing suppressor activity contributes to perturbation of lymphocyte miRNA by HIV-1

    Directory of Open Access Journals (Sweden)

    Yu Lianbo

    2011-05-01

    Full Text Available Abstract Background MicroRNA (miRNA-mediated RNA silencing is integral to virtually every cellular process including cell cycle progression and response to virus infection. The interplay between RNA silencing and HIV-1 is multifaceted, and accumulating evidence posits a strike-counterstrike interface that alters the cellular environment to favor virus replication. For instance, miRNA-mediated RNA silencing of HIV-1 translation is antagonized by HIV-1 Tat RNA silencing suppressor activity. The activity of HIV-1 accessory proteins Vpr/Vif delays cell cycle progression, which is a process prominently modulated by miRNA. The expression profile of cellular miRNA is altered by HIV-1 infection in both cultured cells and clinical samples. The open question stands of what, if any, is the contribution of Tat RNA silencing suppressor activity or Vpr/Vif activity to the perturbation of cellular miRNA by HIV-1. Results Herein, we compared the perturbation of miRNA expression profiles of lymphocytes infected with HIV-1NL4-3 or derivative strains that are deficient in Tat RNA silencing suppressor activity (Tat K51A substitution or ablated of the vpr/vif open reading frames. Microarrays recapitulated the perturbation of the cellular miRNA profile by HIV-1 infection. The miRNA expression trends overlapped ~50% with published microarray results on clinical samples from HIV-1 infected patients. Moreover, the number of miRNA perturbed by HIV-1 was largely similar despite ablation of Tat RSS activity and Vpr/Vif; however, the Tat RSS mutation lessened HIV-1 downregulation of twenty-two miRNAs. Conclusions Our study identified miRNA expression changes attributable to Tat RSS activity in HIV-1NL4-3. The results accomplish a necessary step in the process to understand the interface of HIV-1 with host RNA silencing activity. The overlap in miRNA expression trends observed between HIV-1 infected CEMx174 lymphocytes and primary cells supports the utility of cultured

  18. A complex genome-microRNA interplay in human mitochondria.

    Science.gov (United States)

    Shinde, Santosh; Bhadra, Utpal

    2015-01-01

    Small noncoding regulatory RNA exist in wide spectrum of organisms ranging from prokaryote bacteria to humans. In human, a systematic search for noncoding RNA is mainly limited to the nuclear and cytosolic compartments. To investigate whether endogenous small regulatory RNA are present in cell organelles, human mitochondrial genome was also explored for prediction of precursor microRNA (pre-miRNA) and mature miRNA (miRNA) sequences. Six novel miRNA were predicted from the organelle genome by bioinformatics analysis. The structures are conserved in other five mammals including chimp, orangutan, mouse, rat, and rhesus genome. Experimentally, six human miRNA are well accumulated or deposited in human mitochondria. Three of them are expressed less prominently in Northern analysis. To ascertain their presence in human skeletal muscles, total RNA was extracted from enriched mitochondria by an immunomagnetic method. The expression of six novel pre-miRNA and miRNA was confirmed by Northern blot analysis; however, low level of remaining miRNA was found by sensitive Northern analysis. Their presence is further confirmed by real time RT-PCR. The six miRNA find their multiple targets throughout the human genome in three different types of software. The luciferase assay was used to confirm that MT-RNR2 gene was the potential target of hsa-miR-mit3 and hsa-miR-mit4.

  19. RNA-CODE: a noncoding RNA classification tool for short reads in NGS data lacking reference genomes.

    Directory of Open Access Journals (Sweden)

    Cheng Yuan

    Full Text Available The number of transcriptomic sequencing projects of various non-model organisms is still accumulating rapidly. As non-coding RNAs (ncRNAs are highly abundant in living organism and play important roles in many biological processes, identifying fragmentary members of ncRNAs in small RNA-seq data is an important step in post-NGS analysis. However, the state-of-the-art ncRNA search tools are not optimized for next-generation sequencing (NGS data, especially for very short reads. In this work, we propose and implement a comprehensive ncRNA classification tool (RNA-CODE for very short reads. RNA-CODE is specifically designed for ncRNA identification in NGS data that lack quality reference genomes. Given a set of short reads, our tool classifies the reads into different types of ncRNA families. The classification results can be used to quantify the expression levels of different types of ncRNAs in RNA-seq data and ncRNA composition profiles in metagenomic data, respectively. The experimental results of applying RNA-CODE to RNA-seq of Arabidopsis and a metagenomic data set sampled from human guts demonstrate that RNA-CODE competes favorably in both sensitivity and specificity with other tools. The source codes of RNA-CODE can be downloaded at http://www.cse.msu.edu/~chengy/RNA_CODE.

  20. Comparison of methods for miRNA extraction from plasma and quantitative recovery of RNA from plasma and cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Melissa A McAlexander

    2013-05-01

    Full Text Available Interest in extracellular RNA has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific extracellular RNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Towards these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and partially or fully quantitative recovery of RNA is observed from increasing volumes of plasma and cerebrospinal fluid.

  1. Review of miRNA Technology

    Institute of Scientific and Technical Information of China (English)

    Zhang Tangyao

    2015-01-01

    miRNAs are non-coding RNA molecules exist in eukaryotic with 22 nucleotides. The abnormal expression of miRNAs are also lead to somedisease. The monitoring of cancer related miRNAs, oncomiRs, will help diagnose caners. The main methods to analyzing the profile of miRNA expression fordiagnosing cancer are microarray test and real-time PCR. The the studies on miRomics will bring revolutionary breakthrough to medicine and carcinobiology.

  2. CLP1 as a novel player in linking tRNA splicing to neurodegenerative disorders.

    Science.gov (United States)

    Weitzer, Stefan; Hanada, Toshikatsu; Penninger, Josef M; Martinez, Javier

    2015-01-01

    Defects in RNA metabolic pathways are well-established causes for neurodegenerative disorders. Several mutations in genes involved in pre-messenger RNA (pre-mRNA) and tRNA metabolism, RNA stability and protein translation have been linked to motor neuron diseases. Our study on a mouse carrying a catalytically inactive version of the RNA kinase CLP1, a component of the tRNA splicing endonuclease complex, revealed a neurological disorder characterized by progressive loss of lower spinal motor neurons. Surprisingly, mutant mice accumulate a novel class of tRNA-derived fragments. In addition, patients with homozygous missense mutations in CLP1 (R140H) were recently identified who suffer from severe motor-sensory defects, cortical dysgenesis and microcephaly, and exhibit alterations in transfer RNA (tRNA) splicing. Here, we review functions of CLP1 in different RNA pathways and provide hypotheses on the role of the tRNA splicing machinery in the generation of tRNA fragments and the molecular links to neurodegenerative disorders. We further immerse the biology of tRNA splicing into topics of (t)RNA metabolism and oxidative stress, putting forward the idea that defects in tRNA processing leading to tRNA fragment accumulation might trigger the development of neurodegenerative diseases.

  3. Completeness of the Accumulation Calculus

    Institute of Scientific and Technical Information of China (English)

    虞慧群; 孙永强; 等

    1998-01-01

    The accumulation calculs(AC for short)is an interval based temporal logic to specify and reason about hybrid real-time systems.This paper presents a formal proof system for AC,and proves that the system is complete relative to that of Interval Temporal Logic(ITL for short)on real domain.

  4. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  5. Apolipoprotein E promotes lipid accumulation and differentiation in human adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lasrich, Dorothee; Bartelt, Alexander [Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg (Germany); Grewal, Thomas, E-mail: thomas.grewal@sydney.edu.au [Faculty of Pharmacy A15, The University of Sydney, Sydney, NSW 2006 (Australia); Heeren, Joerg, E-mail: heeren@uke.de [Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg (Germany)

    2015-09-10

    Several studies in mice indicate a role for apolipoprotein E (APOE) in lipid accumulation and adipogenic differentiation in adipose tissue. However, little is yet known if APOE functions in a similar manner in human adipocytes. This prompted us to compare lipid loading and expression of adipocyte differentiation markers in APOE-deficient and control adipocytes using the differentiated human mesenchymal stem cell line hMSC-Tert as well as primary human and mouse adipocytes as model systems. Differentiated hMSC-Tert were stably transduced with or without siRNA targeting APOE while murine adipocytes were isolated from wild type and Apoe knockout mice. Human APOE knockdown hMSC-Tert adipocytes accumulated markedly less triglycerides compared to control cells. This correlated with strongly decreased gene expression levels of adipocyte markers such as adiponectin (ADIPOQ) and fatty acid binding protein 4 (FABP4) as well as the key transcription factor driving adipocyte differentiation, peroxisome proliferator activator receptor gamma (PPARG), in particular the PPARG2 isoform. Similarly, differentiation of murine Apoe-deficient adipocytes was characterized by reduced gene expression of Adipoq, Fabp4 and Pparg. Interestingly, incubation of APOE-deficient hMSC-Tert adipocytes with conditioned media from APOE3-overexpressing adipocytes or APOE-containing Very Low Density Lipoprotein (VLDL) partially restored triglyceride accumulation, but were unable to induce adipocyte differentiation, as judged by expression of adipocyte markers. Taken together, depletion of endogenous APOE in human adipocytes severely impairs lipid accumulation, which is associated with an inability to initiate differentiation. - Highlights: • Immortalized human mesenchymal stem cells were used to study adipocyte development. • Knockdown of endogenous APOE lead to impaired lipid accumulation and adipogenesis. • APOE supplementation partially restored lipid accumulation but not differentiation.

  6. Dandelion (Taraxacum officinale) - monitor for environmental pollution

    Energy Technology Data Exchange (ETDEWEB)

    Kuleff, I.; Djingova, R.

    1984-01-01

    Neutron activation analysis has been used to determine the amounts of As, Cd, Cr, Hg, Sb, Se, and Zn in the leaves of Taraxacum officinale and show that it accumulates these elements. The accumulation corresponds to the extent of environmental pollution. Since Taraxacum officinale is widely distributed it may be successfully used for monitoring metal pollution.

  7. Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis.

    Science.gov (United States)

    Boswell, Sarah A; Snavely, Andrew; Landry, Heather M; Churchman, L Stirling; Gray, Jesse M; Springer, Michael

    2017-03-06

    Pharmacological perturbation is a powerful tool for understanding mRNA synthesis, but identification of the specific steps of this multi-step process that are targeted by small molecules remains challenging. Here we applied strand-specific total RNA sequencing (RNA-seq) to identify and distinguish specific pharmacological effects on transcription and pre-mRNA processing in human cells. We found unexpectedly that the natural product isoginkgetin, previously described as a splicing inhibitor, inhibits transcription elongation. Compared to well-characterized elongation inhibitors that target CDK9, isoginkgetin caused RNA polymerase accumulation within a broader promoter-proximal band, indicating that elongation inhibition by isoginkgetin occurs after release from promoter-proximal pause. RNA-seq distinguished isoginkgetin and CDK9 inhibitors from topoisomerase I inhibition, which alters elongation across gene bodies. We were able to detect these and other specific defects in mRNA synthesis at low sequencing depth using simple metagene-based metrics. These metrics now enable total-RNA-seq-based screening for high-throughput identification of pharmacological effects on individual stages of mRNA synthesis.

  8. Human Adipose-Derived Stem Cells Expanded Under Ambient Oxygen Concentration Accumulate Oxidative DNA Lesions and Experience Procarcinogenic DNA Replication Stress.

    Science.gov (United States)

    Bétous, Rémy; Renoud, Marie-Laure; Hoede, Claire; Gonzalez, Ignacio; Jones, Natalie; Longy, Michel; Sensebé, Luc; Cazaux, Christophe; Hoffmann, Jean-Sébastien

    2017-01-01

    Adipose-derived stem cells (ADSCs) have led to growing interest in cell-based therapy because they can be easily harvested from an abundant tissue. ADSCs must be expanded in vitro before transplantation. This essential step causes concerns about the safety of adult stem cells in terms of potential transformation. Tumorigenesis is driven in its earliest step by DNA replication stress, which is characterized by the accumulation of stalled DNA replication forks and activation of the DNA damage response. Thus, to evaluate the safety of ADSCs during ex vivo expansion, we monitored DNA replication under atmospheric (21%) or physiologic (1%) oxygen concentration. Here, by combining immunofluorescence and DNA combing, we show that ADSCs cultured under 21% oxygen accumulate endogenous oxidative DNA lesions, which interfere with DNA replication by increasing fork stalling events, thereby leading to incomplete DNA replication and fork collapse. Moreover, we found by RNA sequencing (RNA-seq) that culture of ADSCs under atmospheric oxygen concentration leads to misexpression of cell cycle and DNA replication genes, which could contribute to DNA replication stress. Finally, analysis of acquired small nucleotide polymorphism shows that expansion of ADSCs under 21% oxygen induces a mutational bias toward deleterious transversions. Overall, our results suggest that expanding ADSCs at a low oxygen concentration could reduce the risk for DNA replication stress-associated transformation, as occurs in neoplastic tissues. Stem Cells Translational Medicine 2017;6:68-76.

  9. Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum

    Directory of Open Access Journals (Sweden)

    Valenzuela Jacob

    2012-06-01

    Full Text Available Abstract Background Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P and whole-genome transcripts were monitored over time via RNA-sequence determination. Results The specific Nile Red (NR fluorescence (NR fluorescence per cell increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases. Conclusions The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous

  10. A Systematic Analysis of Coal Accumulation Process

    Institute of Scientific and Technical Information of China (English)

    CHENG Aiguo

    2008-01-01

    Formation of coal seam and coal-rich zone is an integrated result of a series of factors in coal accumulation process. The coal accumulation system is an architectural aggregation of coal accumulation factors. It can be classified into 4 levels: the global coal accumulation super-system, the coal accumulation domain mega.system, the coal accumulation basin system, and the coal seam or coal seam set sub-system. The coal accumulation process is an open, dynamic, and grey system, and is meanwhile a system with such natures as aggregation, relevance, entirety, purpose-orientated, hierarchy, and environment adaptability. In this paper, we take coal accumulation process as a system to study origin of coal seam and coal-rich zone; and we will discuss a methodology of the systematic analysis of coal accumulation process. As an example, the Ordos coal basin was investigated to elucidate the application of the method of the coal accumulation system analysis.

  11. Combinatorics of RNA-RNA interaction

    DEFF Research Database (Denmark)

    Li, Thomas J X; Reidys, Christian

    2012-01-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure...... means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including...

  12. Metal accumulating plants: Medium's role

    Science.gov (United States)

    Rabier, J.; Prudent, P.; Szymanska, B.; Mevy, J.-P.

    2003-05-01

    To evaluate phytoremediation potentialities by metal accumulation in tolerant plants, trials are carried out using in vitro cultures. Organie compounds influence on metal accumulation is studied with metals supplemented media. The tested compounds on zinc and lead absorption by Brassica juncea, are chelating agents (EDTA, citric acid) and soluble organic fractions of compost. EDTA seems to enhance the transfer of lead in plant but it is the opposite in the case of zinc. Citric acid stimulates root absorption for both zinc and lead. For the aqueous extracts of compost, variable effects are obtained according to the origin of compost (green wastes and food wastes). In'all tested conditions of cultures, zinc is mainly exported towards shoot while lead is stored in root.

  13. Bacterial accumulation in viscosity gradients

    Science.gov (United States)

    Waisbord, Nicolas; Guasto, Jeffrey

    2016-11-01

    Cell motility is greatly modified by fluid rheology. In particular, the physical environments in which cells function, are often characterized by gradients of viscous biopolymers, such as mucus and extracellular matrix, which impact processes ranging from reproduction to digestion to biofilm formation. To understand how spatial heterogeneity of fluid rheology affects the motility and transport of swimming cells, we use hydrogel microfluidic devices to generate viscosity gradients in a simple, polymeric, Newtonian fluid. Using video microscopy, we characterize the random walk motility patterns of model bacteria (Bacillus subtilis), showing that both wild-type ('run-and-tumble') cells and smooth-swimming mutants accumulate in the viscous region of the fluid. Through statistical analysis of individual cell trajectories and body kinematics in both homogeneous and heterogeneous viscous environments, we discriminate passive, physical effects from active sensing processes to explain the observed cell accumulation at the ensemble level.

  14. Mechanisms of intrahepatic triglyceride accumulation

    OpenAIRE

    Ress, Claudia; Kaser, Susanne

    2016-01-01

    Hepatic steatosis defined as lipid accumulation in hepatocytes is very frequently found in adults and obese adolescents in the Western World. Etiologically, obesity and associated insulin resistance or excess alcohol intake are the most frequent causes of hepatic steatosis. However, steatosis also often occurs with chronic hepatitis C virus (HCV) infection and is also found in rare but potentially life-threatening liver diseases of pregnancy. Clinical significance and outcome of hepatic trigl...

  15. Mechanisms of intrahepatic triglyceride accumulation.

    Science.gov (United States)

    Ress, Claudia; Kaser, Susanne

    2016-01-28

    Hepatic steatosis defined as lipid accumulation in hepatocytes is very frequently found in adults and obese adolescents in the Western World. Etiologically, obesity and associated insulin resistance or excess alcohol intake are the most frequent causes of hepatic steatosis. However, steatosis also often occurs with chronic hepatitis C virus (HCV) infection and is also found in rare but potentially life-threatening liver diseases of pregnancy. Clinical significance and outcome of hepatic triglyceride accumulation are highly dependent on etiology and histological pattern of steatosis. This review summarizes current concepts of pathophysiology of common causes of hepatic steatosis, including non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, chronic HCV infections, drug-induced forms of hepatic steatosis, and acute fatty liver of pregnancy. Regarding the pathophysiology of NAFLD, this work focuses on the close correlation between insulin resistance and hepatic triglyceride accumulation, highlighting the potential harmful effects of systemic insulin resistance on hepatic metabolism of fatty acids on the one side and the role of lipid intermediates on insulin signalling on the other side. Current studies on lipid droplet morphogenesis have identified novel candidate proteins and enzymes in NAFLD.

  16. Short hairpin RNA targeting NP mRNA inhibiting Newcastle disease virus production and other viral structural mRNA transcription.

    Science.gov (United States)

    Yue, Hua; Deng, Shu; Yang, Fa-Long; Li, Ding-Fei; Fu, An-Jing; Yang, Fan; Tang, Cheng

    2009-02-01

    Newcastle disease virus (NDV), formally recognized as avian paramyxovirus 1 (APMV-1), is the etiological agent of Newcastle disease (ND), an affliction which can cause severe losses in the poultry industry. Better understanding of the molecular basis of viral structural genes involved with production should contribute significantly toward the development of improved prophylactic and therapeutic reagents to control the infection. Here we show that a short hairpin RNA (shRNA) eukaryotic expression vector targeting nucleocapsid (NP) gene of NDV can potently inhibit NDV production in both primary cells and embryonated chicken eggs. Moreover, shRNA specific for NP abolished the accumulation of not only the corresponding mRNA but also P, HN, F, M gene mRNA. The findings reveal that newly synthesized NP mRNA is essential for NDV transcription and replication, and provide a basis for the development of shRNAs as a prophylaxis and therapy for NDV infection in poultry.

  17. The influence of RNA-dependent RNA polymerase 1 on potato virus Y infection and on other antiviral response genes.

    Science.gov (United States)

    Rakhshandehroo, Farshad; Takeshita, Minoru; Squires, Julie; Palukaitis, Peter

    2009-10-01

    The gene encoding RNA-dependent RNA polymerase 1 (RDR1) is involved in basal resistance to several viruses. Expression of the RDR1 gene also is induced in resistance to Tobacco mosaic virus (TMV) mediated by the N gene in tobacco (Nicotiana tabacum cv. Samsun NN) in an incompatible hypersensitive response, as well as in a compatible response against Potato virus Y (PVY). Reducing the accumulation of NtRDR1 transcripts by RNA inhibition mediated by transgenic expression of a double-stranded RNA hairpin corresponding to part of the RDR1 gene resulted in little or no induction of accumulation of RDR1 transcripts after infection by PVY. Plants with lower accumulation of RDR1 transcripts showed much higher accumulation levels of PVY. Reduced accumulation of NtRDR1 transcripts also resulted in lower or no induced expression of three other antiviral, defense-related genes after infection by PVY. These genes encoded a mitochondrial alternative oxidase, an inhibitor of virus replication (IVR), and a transcription factor, ERF5, all involved in resistance to infection by TMV, as well as RDR6, involved in RNA silencing. The extent of the effect on the induced NtIVR and NtERF5 genes correlated with the extent of suppression of the NtRDR1 gene.

  18. Coronatine Gene Expression In Vitro and In Planta, and Protein Accumulation During Temperature Downshift in Pseudomonas syringae

    Directory of Open Access Journals (Sweden)

    Alexander Schenk

    2009-06-01

    Full Text Available The plant pathogenic bacterium Pseudomonas syringae PG4180 synthesizes high levels of the phytotoxin coronatine (COR at the virulence-promoting temperature of 18 °C, but negligible amounts at 28 °C. Temperature-dependent COR gene expression is regulated by a modified two-component system, consisting of a response regulator, CorR, the histidine protein kinase CorS, and a third component, termed CorP. We analyzed at transcriptional and translational levels the expression of corS and the cma operon involved in COR biosynthesis after a temperature downshift from 28 to 18 °C. Expression of cma was induced within 20 min and increased steadily whereas corS expression was only slightly temperature-dependent. Accumulation of CmaB correlated with accumulation of cma mRNA. However, cma transcription was suppressed by inhibition of de novo protein biosynthesis. A transcriptional fusion of the cma promoter to a promoterless egfp gene was used to monitor the cma expression in vitro and in planta. A steady induction of cma::egfp by temperature downshift was observed in both environments. The results indicate that PG4180 responds to a temperature decrease with COR gene expression. However, COR gene expression and protein biosynthesis increased steadily, possibly reflecting adaptation to long-term rather than rapid temperature changes.

  19. Assessing the 5S ribosomal RNA heterogeneity in Arabidopsis thaliana using short RNA next generation sequencing data.

    Science.gov (United States)

    Szymanski, Maciej; Karlowski, Wojciech M

    2016-01-01

    In eukaryotes, ribosomal 5S rRNAs are products of multigene families organized within clusters of tandemly repeated units. Accumulation of genomic data obtained from a variety of organisms demonstrated that the potential 5S rRNA coding sequences show a large number of variants, often incompatible with folding into a correct secondary structure. Here, we present results of an analysis of a large set of short RNA sequences generated by the next generation sequencing techniques, to address the problem of heterogeneity of the 5S rRNA transcripts in Arabidopsis and identification of potentially functional rRNA-derived fragments.

  20. Alternative applications for distinct RNA sequencing strategies

    Science.gov (United States)

    Han, Leng; Vickers, Kasey C.; Samuels, David C.

    2015-01-01

    Recent advances in RNA library preparation methods, platform accessibility and cost efficiency have allowed high-throughput RNA sequencing (RNAseq) to replace conventional hybridization microarray platforms as the method of choice for mRNA profiling and transcriptome analyses. RNAseq is a powerful technique to profile both long and short RNA expression, and the depth of information gained from distinct RNAseq methods is striking and facilitates discovery. In addition to expression analysis, distinct RNAseq approaches also allow investigators the ability to assess transcriptional elongation, DNA variance and exogenous RNA content. Here we review the current state of the art in transcriptome sequencing and address epigenetic regulation, quantification of transcription activation, RNAseq output and a diverse set of applications for RNAseq data. We detail how RNAseq can be used to identify allele-specific expression, single-nucleotide polymorphisms and somatic mutations and discuss the benefits and limitations of using RNAseq to monitor DNA characteristics. Moreover, we highlight the power of combining RNA- and DNAseq methods for genomic analysis. In summary, RNAseq provides the opportunity to gain greater insight into transcriptional regulation and output than simply miRNA and mRNA profiling. PMID:25246237

  1. Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs.

    Science.gov (United States)

    Fischer, Martin A; Güllert, Simon; Neulinger, Sven C; Streit, Wolfgang R; Schmitz, Ruth A

    2016-01-01

    The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis

  2. Abscisic Acid accumulates at positive turgor potential in excised soybean seedling growing zones.

    Science.gov (United States)

    Creelman, R A; Mullet, J E

    1991-04-01

    Abscisic acid (ABA) accumulated in soybean (Glycine max [L.] Merr. cv Williams) hypocotyl elongating regions when seedlings were transferred to low water potential vermiculite (Psi = -0.3 megapascals) even though positive turgor is retained in this tissue. Accumulation of ABA in growing zones could occur from de novo biosynthesis within this tissue or transport from adjacent nongrowing zones. Both growing and nongrowing hypocotyl and root tissues accumulated significant levels of ABA when excised and dehydrated to reduce turgor. Surprisingly, excised growing zones (which experienced no water loss) also accumulated ABA when incubated in darkness for 4 hours at 100% relative humidity and 29 degrees C. Induction of ABA accumulation in the excised elongating region of the hypocotyl was not caused by disruption of root pressure or wounding. While excision of hypocotyl elongating regions induced ABA accumulation, no change in either extensin or p33 mRNA levels was observed. Accumulation of extensin or p33 mRNA required more severe wounding. This suggests that ABA is not involved in the response of these genes in wounded tissue and that wound signals are not causing ABA accumulation in excised tissue. Accumulation of ABA in excised elongating regions was correlated with growth inhibition and a decline in turgor to the yield threshold (Psi;(p) = 0.37 megapascals; R Matyssek, S Maruyama, JS Boyer [1988] Plant Physiol 86: 1163-1167). Inhibiting hypocotyl growth by transferring seedlings to lower temperatures or light did not cause ABA accumulation. We conclude that induction of ABA accumulation in growing zones is more sensitive to changes in turgor than the induction which occurs in mature tissues.

  3. Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

    Science.gov (United States)

    Yoon, Ju-Yeon; Han, Kyoung-Sik; Park, Han-Yong; Choi, Seung-Kook

    2012-06-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

  4. The Vienna RNA Websuite

    Science.gov (United States)

    Gruber, Andreas R.; Lorenz, Ronny; Bernhart, Stephan H.; Neuböck, Richard; Hofacker, Ivo L.

    2008-01-01

    The Vienna RNA Websuite is a comprehensive collection of tools for folding, design and analysis of RNA sequences. It provides a web interface to the most commonly used programs of the Vienna RNA package. Among them, we find folding of single and aligned sequences, prediction of RNA–RNA interactions, and design of sequences with a given structure. Additionally, we provide analysis of folding landscapes using the barriers program and structural RNA alignments using LocARNA. The web server together with software packages for download is freely accessible at http://rna.tbi.univie.ac.at/. PMID:18424795

  5. Potential Accumulative Effect of the Herbicide Glyphosate on Glyphosate-Tolerant Maize Rhizobacterial Communities over a Three-Year Cultivation Period

    Science.gov (United States)

    Barriuso, Jorge; Marín, Silvia; Mellado, Rafael P.

    2011-01-01

    Background Glyphosate is a herbicide that is liable to be used in the extensive cultivation of glyphosate-tolerant cultivars. The potential accumulation of the relative effect of glyphosate on the rhizobacterial communities of glyphosate-tolerant maize has been monitored over a period of three years. Methodology/Principal Findings The composition of rhizobacterial communities is known to vary with soil texture, hence, the analyses have been performed in two agricultural fields with a different soil texture. The accumulative effects of glyphosate have been monitored by means of high throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from rhizobacterial communities. The relative composition of the rhizobacterial communities does vary in each field over the three-year period. The overall distribution of the bacterial phyla seems to change from one year to the next similarly in the untreated and glyphosate-treated soils in both fields. The two methods used to estimate bacterial diversity offered consistent results and are equally suitable for diversity assessment. Conclusions/Significance The glyphosate treatment during the three-year period of seasonal cultivation in two different fields did not seem to significantly change the maize rhizobacterial communities when compared to those of the untreated soil. This may be particularly relevant with respect to a potential authorisation to cultivate glyphosate-tolerant maize in the European Union. PMID:22096595

  6. Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

    Directory of Open Access Journals (Sweden)

    Kiwamu Hyodo

    2015-05-01

    Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

  7. Skeletal muscle apolipoprotein B expression reduces muscular triglyceride accumulation

    DEFF Research Database (Denmark)

    Bartels, Emil D; Ploug, Thorkil; Størling, Joachim

    2014-01-01

    . In this study, we investigated whether expression of a human apoB transgene affects triglyceride accumulation and insulin sensitivity in skeletal muscle in fat fed obese mice. Results. Expression of apoB and MTP mRNA and the human apoB transgene was seen in skeletal muscle of the transgene mice. Human apo...... insulin levels after 9 and 12 months, respectively, improved intra peritoneal glucose tolerance after 6 months, and a trend towards increased insulin-stimulated glucose uptake in isolated skeletal muscle. Conclusions. The data suggests that overexpression of apoB decreases skeletal muscle lipid......Abstract Background. Lipid accumulation in skeletal muscle is associated with impaired insulin sensitivity in type 2 diabetes. In cardiac myocytes, lipoprotein secretion controlled by apolipoproteinB (apoB) and microsomal triglyceride transfer protein (MTP) affects lipid homeostasis. Design...

  8. Lentivirus-expressed siRNA vectors against Alzheimer disease.

    Science.gov (United States)

    Peng, Kevin A; Masliah, Eliezer

    2010-01-01

    Amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease, and the accumulation of APP products ultimately leads to the familiar histopathological and clinical manifestations associated with this most common form of dementia. A protein that has been shown to promote APP accumulation is beta-secretase (beta-site APP cleaving enzyme 1, or BACE1), which is increased in the cerebrospinal fluid in those affected with Alzheimer disease. Through in vivo studies using APP transgenic mice, we demonstrated that decreasing the expression of BACE1 via lentiviral vector delivery of BACE1 siRNA has the potential for significantly reducing the cleavage of APP, accumulation of these products, and consequent neurodegeneration. As such, lentiviral-expressed siRNA against BACE1 is a therapeutic possibility in the treatment of Alzheimer disease. We detail the use of lentivirus-expressed siRNA as a method to ameliorate Alzheimer disease neuropathology in APP transgenic mice.

  9. Iron accumulation with age, oxidative stress and functional decline.

    Directory of Open Access Journals (Sweden)

    Jinze Xu

    Full Text Available Identification of biological mediators in sarcopenia is pertinent to the development of targeted interventions to alleviate this condition. Iron is recognized as a potent pro-oxidant and a catalyst for the formation of reactive oxygen species in biological systems. It is well accepted that iron accumulates with senescence in several organs, but little is known about iron accumulation in muscle and how it may affect muscle function. In addition, it is unclear if interventions which reduced age-related loss of muscle quality, such as calorie restriction, impact iron accumulation. We investigated non-heme iron concentration, oxidative stress to nucleic acids in gastrocnemius muscle and key indices of sarcopenia (muscle mass and grip strength in male Fischer 344 X Brown Norway rats fed ad libitum (AL or a calorie restricted diet (60% of ad libitum food intake starting at 4 months of age at 8, 18, 29 and 37 months of age. Total non-heme iron levels in the gastrocnemius muscle of AL rats increased progressively with age. Between 29 and 37 months of age, the non-heme iron concentration increased by approximately 200% in AL-fed rats. Most importantly, the levels of oxidized RNA in gastrocnemius muscle of AL rats were significantly increased as well. The striking age-associated increase in non-heme iron and oxidized RNA levels and decrease in sarcopenia indices were all attenuated in the calorie restriction (CR rats. These findings strongly suggest that the age-related iron accumulation in muscle contributes to increased oxidative damage and sarcopenia, and that CR effectively attenuates these negative effects.

  10. Dietary Niacin Supplementation Suppressed Hepatic Lipid Accumulation in Rabbits.

    Science.gov (United States)

    Liu, Lei; Li, Chunyan; Fu, Chunyan; Li, Fuchang

    2016-12-01

    An experiment was conducted to investigate the effect of niacin supplementation on hepatic lipid metabolism in rabbits. Rex Rabbits (90 d, n = 32) were allocated to two equal treatment groups: Fed basal diet (control) or fed basal diet with additional 200 mg/kg niacin supplementation (niacin). The results show that niacin significantly increased the levels of plasma adiponectin, hepatic apoprotein B and hepatic leptin receptors mRNA (p0.05). However, niacin treatment significantly inhibited the hepatocytes lipid accumulation compared with the control group (prabbits.

  11. Accumulated damage process of thermal sprayed coating under rolling contact by acoustic emission technique

    Science.gov (United States)

    Xu, Jia; Zhou, Zhen-yu; Piao, Zhong-yu

    2016-09-01

    The accumulated damage process of rolling contact fatigue (RCF) of plasma-sprayed coatings was investigated. The influences of surface roughness, loading condition, and stress cycle frequency on the accumulated damage status of the coatings were discussed. A ball-ondisc machine was employed to conduct RCF experiments. Acoustic emission (AE) technique was introduced to monitor the RCF process of the coatings. AE signal characteristics were investigated to reveal the accumulated damage process. Result showed that the polished coating would resist the asperity contact and remit accumulated damage. The RCF lifetime would then extend. Heavy load would aggravate the accumulated damage status and induce surface fracture. Wear became the main failure mode that reduced the RCF lifetime. Frequent stress cycle would aggravate the accumulated damage status and induce interface fracture. Fatigue then became the main failure mode that also reduced the RCF lifetime.

  12. RNA modifications by oxidation

    DEFF Research Database (Denmark)

    Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

    2012-01-01

    The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found...... to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

  13. Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology.

    Science.gov (United States)

    Shu, Dan; Li, Hui; Shu, Yi; Xiong, Gaofeng; Carson, William E; Haque, Farzin; Xu, Ren; Guo, Peixuan

    2015-10-27

    MicroRNAs play important roles in regulating the gene expression and life cycle of cancer cells. In particular, miR-21, an oncogenic miRNA is a major player involved in tumor initiation, progression, invasion and metastasis in several cancers, including triple negative breast cancer (TNBC). However, delivery of therapeutic miRNA or anti-miRNA specifically into cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miR-21 to block the growth of TNBC in orthotopic mouse models. The 15 nm therapeutic RNA nanoparticles contains the 58-nucleotide (nt) phi29 pRNA-3WJ as a core, a 8-nt sequence complementary to the seed region of miR-21, and a 39-nt epidermal growth factor receptor (EGFR) targeting aptamer for internalizing RNA nanoparticles into cancer cells via receptor mediated endocytosis. The RNase resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection into mice and strongly bound to tumors with little or no accumulation in healthy organs 8 h postinjection, and subsequently repressed tumor growth at low doses. The observed specific cancer targeting and tumor regression is a result of several key attributes of RNA nanoparticles: anionic charge which disallows nonspecific passage across negatively charged cell membrane; "active" targeting using RNA aptamers which increases the homing of RNA nanoparticles to cancer cells; nanoscale size and shape which avoids rapid renal clearance and engulfment by lung macrophages and liver Kupffer cells; favorable biodistribution profiles with little accumulation in healthy organs, which minimizes nonspecific side effects; and favorable pharmacokinetic profiles with extended in vivo half-life. The results demonstrate the clinical potentials of RNA nanotechnology based platform to deliver miRNA based therapeutics for cancer treatment.

  14. Targeting RNA-splicing for SMA treatment.

    Science.gov (United States)

    Zhou, Jianhua; Zheng, Xuexiu; Shen, Haihong

    2012-03-01

    The central dogma of DNA-RNA-protein was established more than 40 years ago. However, important biological processes have been identified since the central dogma was developed. For example, methylation is important in the regulation of transcription. In contrast, proteins, are more complex due to modifications such as phosphorylation, glycosylation, ubiquitination, or cleavage. RNA is the mediator between DNA and protein, but it can also be modulated at several levels. Among the most profound discoveries of RNA regulation is RNA splicing. It has been estimated that 80% of pre-mRNA undergo alternative splicing, which exponentially increases biological information flow in cellular processes. However, an increased number of regulated steps inevitably accompanies an increased number of errors. Abnormal splicing is often found in cells, resulting in protein dysfunction that causes disease. Splicing of the survival motor neuron (SMN) gene has been extensively studied during the last two decades. Accumulating knowledge on SMN splicing has led to speculation and search for spinal muscular atrophy (SMA) treatment by stimulating the inclusion of exon 7 into SMN mRNA. This mini-review summaries the latest progress on SMN splicing research as a potential treatment for SMA disease.

  15. Working with RNA

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when po...

  16. New aspects of RNA processing in prokaryotes.

    Science.gov (United States)

    Evguenieva-Hackenberg, Elena; Klug, Gabriele

    2011-10-01

    The pivotal role of posttranscriptional gene regulation is strongly underlined by genome-wide analyses showing strikingly low correlation between mRNA and protein levels in bacterial and archaeal cells. The stability of an mRNA and its availability for translation contribute to posttranscriptional gene regulation, and are determined by the following factors: i) the cell-specific set of ribonucleases and related proteins, ii) regulatory RNAs, and iii) the sequence and structural features of the RNA molecule itself. High-resolution analyses of whole prokaryotic transcriptomes allow comprehensive mapping of processed transcripts, detection of essentially all expressed regulatory RNAs, and monitoring of the global impact of ribonucleases and other processing factors. This opens new perspectives for the understanding of the molecular mechanisms responsible for mRNA decay in prokaryotes. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. RNA self-assembly and RNA nanotechnology.

    Science.gov (United States)

    Grabow, Wade W; Jaeger, Luc

    2014-06-17

    CONSPECTUS: Nanotechnology's central goal involves the direct control of matter at the molecular nanometer scale to build nanofactories, nanomachines, and other devices for potential applications including electronics, alternative fuels, and medicine. In this regard, the nascent use of nucleic acids as a material to coordinate the precise arrangements of specific molecules marked an important milestone in the relatively recent history of nanotechnology. While DNA served as the pioneer building material in nucleic acid nanotechnology, RNA continues to emerge as viable alternative material with its own distinct advantages for nanoconstruction. Several complementary assembly strategies have been used to build a diverse set of RNA nanostructures having unique structural attributes and the ability to self-assemble in a highly programmable and controlled manner. Of the different strategies, the architectonics approach uniquely endeavors to understand integrated structural RNA architectures through the arrangement of their characteristic structural building blocks. Viewed through this lens, it becomes apparent that nature routinely uses thermodynamically stable, recurrent modular motifs from natural RNA molecules to generate unique and more complex programmable structures. With the design principles found in natural structures, a number of synthetic RNAs have been constructed. The synthetic nanostructures constructed to date have provided, in addition to affording essential insights into RNA design, important platforms to characterize and validate the structural self-folding and assembly properties of RNA modules or building blocks. Furthermore, RNA nanoparticles have shown great promise for applications in nanomedicine and RNA-based therapeutics. Nevertheless, the synthetic RNA architectures achieved thus far consist largely of static, rigid particles that are still far from matching the structural and functional complexity of natural responsive structural elements such

  18. Chitosan/siRNA functionalized titanium surface via a layer-by-layer approach for in vitro sustained gene silencing and osteogenic promotion

    Directory of Open Access Journals (Sweden)

    Song W

    2015-03-01

    Full Text Available Wen Song,1,* Xin Song,2,* Chuanxu Yang,2 Shan Gao,2 Lasse Hyldgaard Klausen,2 Yumei Zhang,1 Mingdong Dong,2 Jørgen Kjems21State Key Laboratory of Military Stomatology, Department of Prosthetic Dentistry, School of Stomatology, The Fourth Military Medical University, Xi’an, People’s Republic of China; 2Interdisciplinary Nanoscience Center (iNANO, Aarhus University, Aarhus, Denmark*These authors contributed equally to this workAbstract: Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA for sustained gene silencing in nearby cells, the layer-by-layer (LbL approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP. The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more

  19. Research of Mosses Accumulation Properties Used for Assessment of Regional and Local Atmospheric Pollution

    Directory of Open Access Journals (Sweden)

    Nadezhda K. Ryzhakova

    2014-10-01

    Full Text Available The monitoring of atmospheric heavy metal and other toxic element depositions by using widespread bryophytes as biomonitors has been widely used. Choosing most appropriate moss species in relation to their accumulation properties is the main goal of this research. The accumulation of heavy metals and other toxic elements by widespread mosses of midland have been studied. The research is focused on assessing accumulation properties of 4 species of terrestrial moss, 4 species of paludal moss and 2 species of epiphytic moss. The concentrations of 32 elements have been determined in moss samples by neutron activation analysis (NAA and atom emission spectrometry (AES. Interspecies and intraspecies comparison revealed significant differences in accumulation properties. The accumulation capacity of Dicranum polysetum was higher than other terrestrial mosses and Aulacomnium palustre had higher accumulation capacity than other paludal mosses. These moss species have been used for monitoring atmospheric pollutants in an immense territory, particularly for research of transboundary transfer of heavy metal pollution. The accumulation property of epiphytic moss was higher than others. The epiphytic moss could be found on the bark of old trees (aspens, poplars, birch that are of frequent occurrence in urban areas. Therefore, epiphytic moss can be used for monitoring atmospheric pollutants in an immense territory and for research of atmospheric pollution in industrial centers, inhabited locations as well as assessment of atmospheric contamination in local pollution source. DOI: http://dx.doi.org/10.5755/j01.erem.69.3.5566

  20. Biota-Sediment Accumulation Factor Data

    Data.gov (United States)

    U.S. Environmental Protection Agency — The Biota-Sediment Accumulation Factor contains approximately 20,000 biota-sediment accumulation factors (BSAFs) from 20 locations (mostly Superfund sites) for...

  1. Technical Benefits of Tram Vehicle Energy Accumulator

    Directory of Open Access Journals (Sweden)

    Zbigniew Drazek

    2004-01-01

    Full Text Available Energy saving effects connected with use of energy accumulator on board of tram vehicle instead of substation are presented. Differences in results regarding weak and strong power supply system when taking into account energy losses and energy recuperation are pointed out. Running mode and energy changed from substation by a tram vehicle with accumulator is compared to a tram vehicle without on-board accumulator but supplied from substation equipped with energy accumulator.

  2. Combinatorics of RNA-RNA interaction.

    Science.gov (United States)

    Li, Thomas J X; Reidys, Christian M

    2012-02-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including their generating function, singularity analysis as well as explicit recurrence relations. In particular, our results imply simple asymptotic formulas for the number of joint structures.

  3. Combinatorics of RNA-RNA interaction

    CERN Document Server

    Li, Thomas J X

    2010-01-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called ``zig-zag'' configuration. This paper presents the combinatorics of RNA interaction structures including their generating function, singularity analysis as well as explicit recurrence relations. In particular, our results imply simple asymptotic formulas for the number of joint structures.

  4. The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability.

    Science.gov (United States)

    Copela, Laura A; Chakshusmathi, Ghadiyaram; Sherrer, R Lynn; Wolin, Sandra L

    2006-04-01

    Although the La protein stabilizes nascent pre-tRNAs from nucleases, influences the pathway of pre-tRNA maturation, and assists correct folding of certain pre-tRNAs, it is dispensable for growth in both budding and fission yeast. Here we show that the Saccharomyces cerevisiae La shares functional redundancy with both tRNA modification enzymes and other proteins that contact tRNAs during their biogenesis. La is important for growth in the presence of mutations in either the arginyl tRNA synthetase or the tRNA modification enzyme Trm1p. In addition, two pseudouridine synthases, PUS3 and PUS4, are important for growth in strains carrying a mutation in tRNA(Arg)(CCG) and are essential when La is deleted in these strains. Depletion of Pus3p results in accumulation of the aminoacylated mutant tRNA(Arg)(CCG) in nuclei, while depletion of Pus4p results in decreased stability of the mutant tRNA. Interestingly, the degradation of mutant unstable forms of tRNA(Arg)(CCG) does not require the Trf4p poly(A) polymerase, suggesting that yeast cells possess multiple pathways for tRNA decay. These data demonstrate that La functions redundantly with both tRNA modifications and proteins that associate with tRNAs to achieve tRNA structural stability and efficient biogenesis.

  5. 47 CFR 32.3100 - Accumulated depreciation.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Accumulated depreciation. 32.3100 Section 32... Accumulated depreciation. (a) This account shall include the accumulated depreciation associated with the... with depreciation amounts concurrently charged to Account 6561, Depreciation...

  6. Cytoplasmic Z-RNA

    Energy Technology Data Exchange (ETDEWEB)

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-09-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

  7. Raman crystallography of RNA.

    Science.gov (United States)

    Gong, Bo; Chen, Jui-Hui; Yajima, Rieko; Chen, Yuanyuan; Chase, Elaine; Chadalavada, Durga M; Golden, Barbara L; Carey, Paul R; Bevilacqua, Philip C

    2009-10-01

    Raman crystallography is the application of Raman spectroscopy to single crystals. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. Here, we describe the application of Raman crystallography to functional RNA molecules. RNA represents unique opportunities and challenges for Raman crystallography. One issue that confounds studies of RNA is its tendency to adopt multiple non-functional folds. Raman crystallography has the advantage that it isolates a single state of the RNA within the crystal and can evaluate its fold, metal ion binding properties (ligand identity, stoichiometry, and affinity), proton binding properties (identity, stoichiometry, and affinity), and catalytic potential. In particular, base-specific stretches can be identified and then associated with the binding of metal ions and protons. Because measurements are carried out in the hanging drop at ambient, rather than cryo, conditions and because RNA crystals tend to be approximately 70% solvent, RNA dynamics and conformational changes become experimentally accessible. This review focuses on experimental setup and procedures, acquisition and interpretation of Raman data, and determination of physicochemical properties of the RNA. Raman crystallographic and solution biochemical experiments on the HDV RNA enzyme are summarized and found to be in excellent agreement. Remarkably, characterization of the crystalline state has proven to help rather than hinder functional characterization of functional RNA, most likely because the tendency of RNA to fold heterogeneously is limited in a crystalline environment. Future applications of Raman crystallography to RNA are briefly discussed.

  8. Methods for RNA Analysis

    DEFF Research Database (Denmark)

    Olivarius, Signe

    of the transcriptome, 5’ end capture of RNA is combined with next-generation sequencing for high-throughput quantitative assessment of transcription start sites by two different methods. The methods presented here allow for functional investigation of coding as well as noncoding RNA and contribute to future......While increasing evidence appoints diverse types of RNA as key players in the regulatory networks underlying cellular differentiation and metabolism, the potential functions of thousands of conserved RNA structures encoded in mammalian genomes remain to be determined. Since the functions of most...... RNAs rely on interactions with proteins, the establishment of protein-binding profiles is essential for the characterization of RNAs. Aiming to facilitate RNA analysis, this thesis introduces proteomics- as well as transcriptomics-based methods for the functional characterization of RNA. First, RNA...

  9. Markov models for accumulating mutations

    CERN Document Server

    Beerenwinkel, Niko

    2007-01-01

    We introduce and analyze a waiting time model for the accumulation of genetic changes. The continuous time conjunctive Bayesian network is defined by a partially ordered set of mutations and by the rate of fixation of each mutation. The partial order encodes constraints on the order in which mutations can fixate in the population, shedding light on the mutational pathways underlying the evolutionary process. We study a censored version of the model and derive equations for an EM algorithm to perform maximum likelihood estimation of the model parameters. We also show how to select the maximum likelihood poset. The model is applied to genetic data from different cancers and from drug resistant HIV samples, indicating implications for diagnosis and treatment.

  10. Ectoine accumulation in Brevibacterium epidermis.

    Science.gov (United States)

    Onraedt, Annelies; De Muynck, Cassandra; Walcarius, Bart; Soetaert, Wim; Vandamme, Erick

    2004-10-01

    As a halotolerant bacterial species, Brevibacterium epidermis DSM 20659 can grow at relatively high salinity, tolerating up to 2 M NaCl. It synthesizes ectoine and the intracellular content increases with the medium salinity, with a maximum of 0.14 g ectoine/g CDW at 1 M NaCl. Sugar-stressed cells do not synthesize ectoine. Ectoine synthesis is also affected by the presence of external osmolytes. Added betaine is taken up and completely replaced ectoine, while L-proline is only temporarily accumulated after which ectoine is synthesized. The strain can metabolize ectoine; L-glutamate is a better carbon source for ectoine synthesis than L-aspartate.

  11. Electron-Positron Accumulator (EPA)

    CERN Document Server

    Photographic Service

    1986-01-01

    After acceleration in the low-current linac LIL-W, the electrons and positrons are accumulated in EPA to obtain a sufficient intensity and a suitable time-structure, before being passed on to the PS for further acceleration to 3.5 GeV. Electrons circulate from right to left, positrons in the other direction. Dipole bending magnets are red, focusing quadrupoles blue, sextupoles for chromaticity-control orange. The vertical tube at the left of the picture belongs to an optical transport system carrying the synchrotron radiation to detectors for beam size measurement. Construction of EPA was completed in spring 1986. LIL-W and EPA were conceived for an energy of 600 MeV, but operation was limited to 500 MeV.

  12. Energy Accumulation by Hydrogen Technologies

    Directory of Open Access Journals (Sweden)

    Jiřina Čermáková

    2012-01-01

    Full Text Available Photovoltaic power plants as a renewable energy source have been receiving rapidly growing attention in the Czech Republic and in the other EU countries. This rapid development of photovoltaic sources is having a negative effect on the electricity power system control, because they depend on the weather conditions and provide a variable and unreliable supply of electric power. One way to reduce this effect is by accumulating electricity in hydrogen. The aim of this paper is to introduce hydrogen as a tool for regulating photovoltaic energy in island mode. A configuration has been designed for connecting households with the photovoltaic hybrid system, and a simulation model has been made in order to check the validity of this system. The simulation results provide energy flows and have been used for optimal sizing of real devices. An appropriate system can deliver energy in a stand-alone installation.

  13. Rotator cuff tear reduces muscle fiber specific force production and induces macrophage accumulation and autophagy.

    Science.gov (United States)

    Gumucio, Jonathan P; Davis, Max E; Bradley, Joshua R; Stafford, Patrick L; Schiffman, Corey J; Lynch, Evan B; Claflin, Dennis R; Bedi, Asheesh; Mendias, Christopher L

    2012-12-01

    Full-thickness tears to the rotator cuff can cause severe pain and disability. Untreated tears progress in size and are associated with muscle atrophy and an infiltration of fat to the area, a condition known as "fatty degeneration." To improve the treatment of rotator cuff tears, a greater understanding of the changes in the contractile properties of muscle fibers and the molecular regulation of fatty degeneration is essential. Using a rat model of rotator cuff injury, we measured the force generating capacity of individual muscle fibers and determined changes in muscle fiber type distribution that develop after a full thickness rotator cuff tear. We also measured the expression of mRNA and miRNA transcripts involved in muscle atrophy, lipid accumulation, and matrix synthesis. We hypothesized that a decrease in specific force of rotator cuff muscle fibers, an accumulation of type IIb fibers, and an upregulation in fibrogenic, adipogenic, and inflammatory gene expression occur in torn rotator cuff muscles. Thirty days following rotator cuff tear, we observed a reduction in muscle fiber force production, an induction of fibrogenic, adipogenic, and autophagocytic mRNA and miRNA molecules, and a dramatic accumulation of macrophages in areas of fat accumulation. Copyright © 2012 Orthopaedic Research Society.

  14. Baicalein inhibits lipid accumulation by regulating early adipogenesis and m-TOR signaling.

    Science.gov (United States)

    Seo, Min-Jung; Choi, Hyeon-Son; Jeon, Hui-Jeon; Woo, Mi-Seon; Lee, Boo-Yong

    2014-05-01

    Baicalein is a type of flavonoid that originates from Scutellaria baicalensis. In this study, we examined how baicalein inhibits lipid accumulation during adipogenesis in 3T3-L1 cells. Our data show that baicalein inhibited lipid accumulation during adipogenesis in a dose-dependent manner. Baicalein inhibition was limited to the early adipogenic stage. Cell cycle analysis showed that baicalein induced cell cycle arrest in the G0/G1 phase through cyclin downregulation. In addition, baicalein suppressed the mRNA expression of early adipogenic factors leading to downregulation of late adipogenic factors at mRNA and protein levels. Inhibition of adipogenic factors by baicalein was correlated with downregulation of lipid synthetic enzymes. Additionally, baicalein negatively regulated the m-TOR signaling pathway involved in lipid accumulation during adipogenesis, thus inhibiting phosphorylation of m-TOR and p70S6K. In a zebrafish study, baicalein significantly reduced lipid accumulation in Nile Red staining. Consistent with a report using cell lines, mRNA expression of adipogenic factors was decreased in a dose-dependent manner by baicalein. This result reflects a reduction in total triglyceride levels based on a triglyceride assay. Our data suggest that baicalein inhibits lipid accumulation by controlling the cell cycle and m-TOR signaling in 3T3-L1 cells, and its anti-adipogenic effect was found in a zebrafish model.

  15. Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability.

    Science.gov (United States)

    Ruiz, Oscar N; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

    2011-06-01

    Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183,000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 μm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioneins in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation.

  16. Monitoring XAF1 mRNA levels in CML patients before and after HSCT by RT-PCR%CML患者造血干细胞移植前后XAF1 mRNA的表达

    Institute of Scientific and Technical Information of China (English)

    李治国; 张继红

    2011-01-01

    Objective To establish a SYBR Green Ⅰ real-time quantitative RT-PCR method for investigating the correlation between XAF1 mRNA expression levels and relapse of leukemia after allo-bematopoietic stem cell transplantation( HSCT). Methods Serial monitorting of XAFlmRNA levels by SYBR Green Ⅰ real-time quantitative RT-PCR technique was performed in 12 chronic myelogenous leukemia (CML) patients. Results XAF1 levels in newly diagnosed group, 1 month after HSCT group and 3 months after HSCT group were 0.01 ( 0 ~ 0.03 ) ,0. 04 ( 0.01 ~ 0.05 ) and 0.11 (0.05 ~ 0. 16). In the patients without XAF1, the symptoms of fatigue,night sweats and fever were obvious, peripheral leukocyte levels were all above 50 × 109/L. The symptoms of fatigue, night sweats and fever were alleviative in the patients with XAF1 after transplantation and peripheral leukocyte levels were all less than 25 ×109/L. In 6 patients without XAF1 ,XAF1 could be detected 3 months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BP. Three of them with high level survived without relapse. XAF1 level increased rapidly after HSCT. Conclusion Quantification of XAF1 mRNA levels is necessary for HSCT recipients.%目的 建立检测XAF1 mRNA的SYBR Green I实时定量PCR(RQ-PCR)的方法,并探讨XAF1表达水平与异基因造血干细胞移植(Allo-HSCT)后白血病复发之间的相关性.方法 选择12例慢性髓细胞性白血病(CML)白血病患者,应用ABI7500RT-PCR仪动态监测患者移植前后不同时间段的36份骨髓单个核细胞中XAF1表达水平.结果 移植前XAF1阴性CML患者乏力及盗汗症状明显、发热,外周血白细胞均>50×109/L;移植后XAF1阳性患者乏力及盗汗症状减轻、无发热,外周血白细胞水平均<25×109/L;移植前、移植后1月组及移植后3月及以后组XAF1相对表达水平为0.01(0~0.03)、0.04(0.01~0.05)及0.11(0.05~0.16).定期随访表明,6例XAF1阴性患者中,4例CML-AP或 BP患者在移植3

  17. Using miRNA-Analyzer for the Analysis of miRNA Data

    Science.gov (United States)

    Guzzi, Pietro Hiram; Tradigo, Giuseppe; Veltri, Pierangelo

    2016-01-01

    MicroRNAs (miRNAs) are small biological molecules that play an important role during the mechanisms of protein formation. Recent findings have demonstrated that they act as both positive and negative regulators of protein formation. Thus, the investigation of miRNAs, i.e., the determination of their level of expression, has developed a huge interest in the scientific community. One of the leading technologies for extracting miRNA data from biological samples is the miRNA Affymetrix platform. It provides the quantification of the level of expression of the miRNA in a sample, thus enabling the accumulation of data and allowing the determination of relationships among miRNA, genes, and diseases. Unfortunately, there is a lack of a comprehensive platform able to provide all the functions needed for the extraction of information from miRNA data. We here present miRNA-Analyzer, a complete software tool providing primary functionalities for miRNA data analysis. The current version of miRNA-Analyzer wraps the Affymetrix QCTool for the preprocessing of binary data files, and then provides feature selection (the filtering by species and by the associated p-value of preprocessed files). Finally, preprocessed and filtered data are analyzed by the Multiple Experiment Viewer (T-MEV) and Short Time Series Expression Miner (STEM) tools, which are also wrapped into miRNA-Analyzer, thus providing a unique environment for miRNA data analysis. The tool offers a plug-in interface so it is easily extensible by adding other algorithms as plug-ins. Users may download the tool freely for academic use at https://sites.google.com/site/mirnaanalyserproject/d. PMID:27983673

  18. Self-circularizing 5'-ETS RNAs accumulate along with unprocessed pre ribosomal RNAs in growth-stressed Entamoeba histolytica.

    Science.gov (United States)

    Gupta, Abhishek Kumar; Panigrahi, Sunil Kumar; Bhattacharya, Alok; Bhattacharya, Sudha

    2012-01-01

    The primary transcript of rRNA genes is a large pre rRNA which is precisely processed to release the mature rRNAs. The 5'-external transcribed spacer (ETS) of rRNA genes contains important sites for pre rRNA processing. Once the processing is accomplished the ETS is rapidly degraded. We show that in growth-stressed cells of the human parasitic protist Entamoeba histolytica the A'-A(0) sub-fragment of the 5'-ETS accumulates to high levels as a family of RNA molecules of size 666 to 912 nt. These etsRNAs are circular in vivo and can spontaneously self-circularize in vitro. The accumulation of etsRNAs is accompanied by accumulation of unprocessed pre rRNA, indicating a possible role of etsRNAs in inhibition of processing during growth stress. Our data shows for the first time that processed etsRNA is not a mere by-product destined for degradation but is stabilized by circularization and could play a regulatory role as noncoding RNA.

  19. Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation.

    Science.gov (United States)

    Verrier, S B; Jean-Jean, O

    2000-04-01

    In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.

  20. TMV induces RNA decay pathways to modulate gene silencing and disease symptoms.

    Science.gov (United States)

    Conti, Gabriela; Zavallo, Diego; Venturuzzi, Andrea L; Rodriguez, Maria C; Crespi, Martin; Asurmendi, Sebastian

    2017-01-01

    RNA decay pathways comprise a combination of RNA degradation mechanisms that are implicated in gene expression, development and defense responses in eukaryotes. These mechanisms are known as the RNA Quality Control or RQC pathways. In plants, another important RNA degradation mechanism is the post-transcriptional gene silencing (PTGS) mediated by small RNAs (siRNAs). Notably, the RQC pathway antagonizes PTGS by preventing the entry of dysfunctional mRNAs into the silencing pathway to avoid global degradation of mRNA by siRNAs. Viral transcripts must evade RNA degrading mechanisms, thus viruses encode PTGS suppressor proteins to counteract viral RNA silencing. Here, we demonstrate that tobacco plants infected with TMV and transgenic lines expressing TMV MP and CP (coat protein) proteins (which are not linked to the suppression of silencing) display increased transcriptional levels of RNA decay genes. These plants also showed accumulation of cytoplasmic RNA granules with altered structure, increased rates of RNA decay for transgenes and defective transgene PTGS amplification. Furthermore, knockdown of RRP41 or RRP43 RNA exosome components led to lower levels of TMV accumulation with milder symptoms after infection, several developmental defects and miRNA deregulation. Thus, we propose that TMV proteins induce RNA decay pathways (in particular exosome components) to impair antiviral PTGS and this defensive mechanism would constitute an additional counter-defense strategy that lead to disease symptoms. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  1. Accumulation of heavy metals in oil-contaminated peat soils

    Science.gov (United States)

    Vodyanitskii, Yu. N.; Savichev, A. T.; Trofimov, S. Ya.; Shishkonakova, E. A.

    2012-10-01

    X-ray fluorescence and X-ray radiometry represent easy and simple methods to determine concentrations of heavy metals in the ash of peat soils contaminated with oil and can be applied for soil monitoring purposes. Oil spills on peat bogs produce two contamination zones differing in the composition of heavy metals. In the zone of primary contamination, the peat surface is covered by a bitumen crust with V, Ni, Sr, Ba, Ce, and La accumulating there. This zone adjoins the zone of secondary peat contamination, where heavy alkaline-earth metals (Sr, Ba) and lanthanides (Ce and La) are accumulated to a lesser extent. Biological preparations recommended for remediation of oil-contaminated peat soils should be tolerant to high concentrations of heavy metals, particularly, V, Ni, and Ba that are present in the oil contaminated soils in relatively high amounts.

  2. Advancement of the Emerging Field of RNA Nanotechnology

    Science.gov (United States)

    2017-01-01

    The field of RNA nanotechnology has advanced rapidly during the past decade. A variety of programmable RNA nanoparticles with defined shape, size, and stoichiometry have been developed for diverse applications in nanobiotechnology. The rising popularity of RNA nanoparticles is due to a number of factors: (1) removing the concern of RNA degradation in vitro and in vivo by introducing chemical modification into nucleotides without significant alteration of the RNA property in folding and self-assembly; (2) confirming the concept that RNA displays very high thermodynamic stability and is suitable for in vivo trafficking and other applications; (3) obtaining the knowledge to tune the immunogenic properties of synthetic RNA constructs for in vivo applications; (4) increased understanding of the 4D structure and intermolecular interaction of RNA molecules; (5) developing methods to control shape, size, and stoichiometry of RNA nanoparticles; (6) increasing knowledge of regulation and processing functions of RNA in cells; (7) decreasing cost of RNA production by biological and chemical synthesis; and (8) proving the concept that RNA is a safe and specific therapeutic modality for cancer and other diseases with little or no accumulation in vital organs. Other applications of RNA nanotechnology, such as adapting them to construct 2D, 3D, and 4D structures for use in tissue engineering, biosensing, resistive biomemory, and potential computer logic gate modules, have stimulated the interest of the scientific community. This review aims to outline the current state of the art of RNA nanoparticles as programmable smart complexes and offers perspectives on the promising avenues of research in this fast-growing field. PMID:28045501

  3. Monitoring madness

    Energy Technology Data Exchange (ETDEWEB)

    Blankinship, S.

    2006-01-15

    High quality continuous emission monitoring capability can be as essential as high quality emission control equipment. Future mercury monitoring and control requirements add to the justification for better CEMS. The article discusses two prominent mercury measurement methods - the cold vapour atomic absorptive spectrometer (CVAAs) and the atomic absorptive spectrometer (AFS). It stresses the importance of maintaining a CEMS. 1 photo.

  4. Mobility Monitor

    DEFF Research Database (Denmark)

    Schæbel, Anne-Lise; Dybbro, Karina Løvendahl; Andersen, Lisbeth Støvring;

    2015-01-01

    Undersøgelse af digital monitorering af plejehjemsbeboeres vendinger under søvn på Fremtidens Plejehjem, Nørresundby......Undersøgelse af digital monitorering af plejehjemsbeboeres vendinger under søvn på Fremtidens Plejehjem, Nørresundby...

  5. The Cj0588 protein is a Campylobacter jejuni RNA methyltransferase.

    Science.gov (United States)

    Sałamaszyńska-Guz, Agnieszka; Taciak, Bartłomiej; Kwiatek, Agnieszka; Klimuszko, Danuta

    2014-06-06

    TlyA proteins belong to 2'-O-methyltransferases. Methylation is a common posttranscriptional RNA modification. The Campylobacter jejuni Cj0588 protein belongs to the TlyA(I) protein family and is a rRNA methyltransferase. Methylation of ribosomal RNA catalyzed by Cj0588 appears to have an impact on the biology of the cell. Presence of the cj0588 gene in bacteria appears to be important for ribosome stability and virulence properties. Absence of the Cj0588 protein causes accumulation of the 50S ribosomal subunits, reduction in the amount of functional 70S ribosomes and confers increase resistance to capreomycin.

  6. iRNA-seq

    DEFF Research Database (Denmark)

    Madsen, Jesper Grud Skat; Schmidt, Søren Fisker; Larsen, Bjørk Ditlev;

    2015-01-01

    RNA-seq is a sensitive and accurate technique to compare steady-state levels of RNA between different cellular states. However, as it does not provide an account of transcriptional activity per se, other technologies are needed to more precisely determine acute transcriptional responses. Here, we...... have developed an easy, sensitive and accurate novel computational method, IRNA-SEQ: , for genome-wide assessment of transcriptional activity based on analysis of intron coverage from total RNA-seq data. Comparison of the results derived from iRNA-seq analyses with parallel results derived using...... current methods for genome-wide determination of transcriptional activity, i.e. global run-on (GRO)-seq and RNA polymerase II (RNAPII) ChIP-seq, demonstrate that iRNA-seq provides similar results in terms of number of regulated genes and their fold change. However, unlike the current methods that are all...

  7. Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses.

    Science.gov (United States)

    Rabbani, M Ashiq; Maruyama, Kyonoshin; Abe, Hiroshi; Khan, M Ayub; Katsura, Koji; Ito, Yusuke; Yoshiwara, Kyoko; Seki, Motoaki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2003-12-01

    To identify cold-, drought-, high-salinity-, and/or abscisic acid (ABA)-inducible genes in rice (Oryza sativa), we prepared a rice cDNA microarray including about 1700 independent cDNAs derived from cDNA libraries prepared from drought-, cold-, and high-salinity-treated rice plants. We confirmed stress-inducible expression of the candidate genes selected by microarray analysis using RNA gel-blot analysis and finally identified a total of 73 genes as stress inducible including 58 novel unreported genes in rice. Among them, 36, 62, 57, and 43 genes were induced by cold, drought, high salinity, and ABA, respectively. We observed a strong association in the expression of stress-responsive genes and found 15 genes that responded to all four treatments. Venn diagram analysis revealed greater cross talk between signaling pathways for drought, ABA, and high-salinity stresses than between signaling pathways for cold and ABA stresses or cold and high-salinity stresses in rice. The rice genome database search enabled us not only to identify possible known cis-acting elements in the promoter regions of several stress-inducible genes but also to expect the existence of novel cis-acting elements involved in stress-responsive gene expression in rice stress-inducible promoters. Comparative analysis of Arabidopsis and rice showed that among the 73 stress-inducible rice genes, 51 already have been reported in Arabidopsis with similar function or gene name. Transcriptome analysis revealed novel stress-inducible genes, suggesting some differences between Arabidopsis and rice in their response to stress.

  8. Environmental parasitology: Parasites as accumulation bioindicators in the marine environment

    Science.gov (United States)

    Nachev, Milen; Sures, Bernd

    2016-07-01

    Parasites can be used as effective monitoring tools in environmental impact studies as they are able to accumulate certain pollutants (e.g. metals) at levels much higher than those of their ambient environment and of free-living sentinels. Thus, they provide valuable information not only about the chemical conditions of their and their hosts' environment but also deliver insights into the biological availability of allochthonous substances. While a large number of different freshwater parasites (mainly acanthocephalans and cestodes) were investigated in terms of pollutant bioaccumulation, studies based on marine host-parasites systems remain scarce. However, available data show that different marine parasite taxa such as nematodes, cestodes and acanthocephalans exhibit also an excellent metal accumulation capacity. The biological availability of metals and their uptake routes in marine biota and parasites differ from those of freshwater organisms. We assume that a large part of metals and other pollutants are also taken up via the digestive system of the host. Therefore, in addition to environmental conditions the physiology of the host also plays an important role for the accumulation process. Additionally, we highlight some advantages in using parasites as accumulation indicators in marine ecosystems. As parasites occur ubiquitously in marine food webs, the monitoring of metals in their tissues can deliver information about the spatial and trophic distribution of pollutants. Accordingly, parasites as indicators offer an ecological assessment on a broader scale, in contrast to established free-living marine indicators, which are mostly benthic invertebrates and therefore limited in habitat distribution. Globally distributed parasite taxa, which are highly abundant in a large number of host species, are suggested as worldwide applicable sentinels.

  9. PABLM. Accumulated Environment Radiation Dose

    Energy Technology Data Exchange (ETDEWEB)

    Napier, B.A.; Kennedy, W.E.Jr.; Soldat, J.K. [Pacific Northwest Lab., Richland, WA (United States)

    1981-04-01

    PABLM calculates internal radiation doses to man from radionuclides in food products and external radiation doses from radionuclides in the environment. Radiation doses from radionuclides in the environment may be calculated from deposition on the soil or plants during an atmospheric or liquid release, or from exposure to residual radionuclides after the releases have ended. Radioactive decay is considered during the release, after deposition, and during holdup of food after harvest. The radiation dose models consider exposure to radionuclides deposited on the ground or crops from contaminated air or irrigation water, radionuclides in contaminated drinking water, aquatic foods raised in contaminated water, and radionuclides in bodies of water and sediments where people might fish, boat, or swim. For vegetation, the radiation dose model considers both direct deposition and uptake through roots. Doses may be calculated for either a maximum-exposed individual or for a population group. The program is designed to calculate accumulated radiation doses from the chronic ingestion of food products that contain radionuclides and doses from the external exposure to radionuclides in the environment. A first-year committed dose is calculated as well as an integrated dose for a selected number of years.

  10. Sequential biases in accumulating evidence

    Science.gov (United States)

    Huggins, Richard; Dogo, Samson Henry

    2015-01-01

    Whilst it is common in clinical trials to use the results of tests at one phase to decide whether to continue to the next phase and to subsequently design the next phase, we show that this can lead to biased results in evidence synthesis. Two new kinds of bias associated with accumulating evidence, termed ‘sequential decision bias’ and ‘sequential design bias’, are identified. Both kinds of bias are the result of making decisions on the usefulness of a new study, or its design, based on the previous studies. Sequential decision bias is determined by the correlation between the value of the current estimated effect and the probability of conducting an additional study. Sequential design bias arises from using the estimated value instead of the clinically relevant value of an effect in sample size calculations. We considered both the fixed‐effect and the random‐effects models of meta‐analysis and demonstrated analytically and by simulations that in both settings the problems due to sequential biases are apparent. According to our simulations, the sequential biases increase with increased heterogeneity. Minimisation of sequential biases arises as a new and important research area necessary for successful evidence‐based approaches to the development of science. © 2015 The Authors. Research Synthesis Methods Published by John Wiley & Sons Ltd. PMID:26626562

  11. Quantum gravity and inventory accumulation

    CERN Document Server

    Sheffield, Scott

    2011-01-01

    We begin by studying inventory accumulation at a LIFO (last-in-first-out) retailer with two products. In the simplest version, the following occur with equal probability at each time step: first product ordered, first product produced, second product ordered, second product produced. The inventory thus evolves as a simple random walk on Z^2. In more interesting versions, a p fraction of customers orders the "freshest available" product regardless of type. We show that the corresponding random walks scale to Brownian motions with diffusion matrices depending on p. We then turn our attention to the critical Fortuin-Kastelyn random planar map model, which gives, for each q>0, a probability measure on random (discretized) two-dimensional surfaces decorated by loops, related to the q-state Potts model. A longstanding open problem is to show that as the discretization gets finer, the surfaces converge in law to a limiting (loop-decorated) random surface. The limit is expected to be a Liouville quantum gravity surfa...

  12. RNA-modifying enzymes.

    Science.gov (United States)

    Ferré-D'Amaré, Adrian R

    2003-02-01

    A bewildering number of post-transcriptional modifications are introduced into cellular RNAs by enzymes that are often conserved among archaea, bacteria and eukaryotes. The modifications range from those with well-understood functions, such as tRNA aminoacylation, to widespread but more mysterious ones, such as pseudouridylation. Recent structure determinations have included two types of RNA nucleobase modifying enzyme: pseudouridine synthases and tRNA guanine transglycosylases.

  13. Single nucleotide RNA choreography.

    Science.gov (United States)

    Hsiao, Chiaolong; Mohan, Srividya; Hershkovitz, Eli; Tannenbaum, Allen; Williams, Loren Dean

    2006-01-01

    New structural analysis methods, and a tree formalism re-define and expand the RNA motif concept, unifying what previously appeared to be disparate groups of structures. We find RNA tetraloops at high frequencies, in new contexts, with unexpected lengths, and in novel topologies. The results, with broad implications for RNA structure in general, show that even at this most elementary level of organization, RNA tolerates astounding variation in conformation, length, sequence and context. However the variation is not random; it is well-described by four distinct modes, which are 3-2 switches (backbone topology variations), insertions, deletions and strand clips.

  14. Accumulation boundaries: codimension-two accumulation of accumulations in phase diagrams of semiconductor lasers, electric circuits, atmospheric and chemical oscillators.

    Science.gov (United States)

    Bonatto, Cristian; Gallas, Jason Alfredo Carlson

    2008-02-28

    We report high-resolution phase diagrams for several familiar dynamical systems described by sets of ordinary differential equations: semiconductor lasers; electric circuits; Lorenz-84 low-order atmospheric circulation model; and Rössler and chemical oscillators. All these systems contain chaotic phases with highly complicated and interesting accumulation boundaries, curves where networks of stable islands of regular oscillations with ever-increasing periodicities accumulate systematically. The experimental exploration of such codimension-two boundaries characterized by the presence of infinite accumulation of accumulations is feasible with existing technology for some of these systems.

  15. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

    2008-01-01

    Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs....

  16. Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila.

    Science.gov (United States)

    Hur, Junho K; Luo, Yicheng; Moon, Sungjin; Ninova, Maria; Marinov, Georgi K; Chung, Yun D; Aravin, Alexei A

    2016-04-01

    The conserved THO/TREX (transcription/export) complex is critical for pre-mRNA processing and mRNA nuclear export. In metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. Here we show that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small noncoding RNAs that control expression of transposable elements (TEs) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis, resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and colocalize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depend on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, we show that TREX is required for accumulation of nascent piRNA precursors. Our study reveals a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis.

  17. Microbial identification by immunohybridization assay of artificial RNA labels

    Science.gov (United States)

    Kourentzi, Katerina D.; Fox, George E.; Willson, Richard C.

    2002-01-01

    Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

  18. Variance and covariance of accumulated displacement estimates.

    Science.gov (United States)

    Bayer, Matthew; Hall, Timothy J

    2013-04-01

    Tracking large deformations in tissue using ultrasound can enable the reconstruction of nonlinear elastic parameters, but poses a challenge to displacement estimation algorithms. Such large deformations have to be broken up into steps, each of which contributes an estimation error to the final accumulated displacement map. The work reported here measured the error variance for single-step and accumulated displacement estimates using one-dimensional numerical simulations of ultrasound echo signals, subjected to tissue strain and electronic noise. The covariance between accumulation steps was also computed. These simulations show that errors due to electronic noise are negatively correlated between steps, and therefore accumulate slowly, whereas errors due to tissue deformation are positively correlated and accumulate quickly. For reasonably low electronic noise levels, the error variance in the accumulated displacement estimates is remarkably constant as a function of step size, but increases with the length of the tracking kernel.

  19. Revealing accumulation zones of plastic pellets in sandy beaches.

    Science.gov (United States)

    Moreira, Fabiana T; Balthazar-Silva, Danilo; Barbosa, Lucas; Turra, Alexander

    2016-11-01

    Microplastics such as pellets are reported worldwide on sandy beaches, and have possible direct and indirect impacts on the biota and physical characteristics of the habitats where they accumulate. Evaluations of their standing stock at different spatial scales generate data on levels of contamination. This information is needed to identify accumulation zones and the specific beach habitats and communities that are likely to be most affected. Standing stocks of plastic pellets were evaluated in 13 sandy beaches in São Paulo state, Brazil. The sampling strategy incorporated across-shore transects from coastal dunes and backshores, and vertical profiles of the accumulated pellets down to 1 m depth below the sediment surface. Accumulation zones were identified at regional (among beaches) and local (between compartments) scales. At the regional scale pellet density tended to increase at beaches on the central and southwestern coast, near ports and factories that produce and transport the largest amounts of pellets in the country. At the local scale coastal dunes showed larger accumulations of pellets than backshores. For both compartments pellets tended to occur deeper in areas where standing stocks were larger. Most of the pellets were concentrated from the surface down to 0.4 m depth, suggesting that organisms inhabiting this part of the sediment column are more exposed to the risks associated with the presence of pellets. Our findings shed light on the local and regional scales of spatial variability of microplastics and their consequences for assessment and monitoring schemes in coastal compartments. Copyright © 2016. Published by Elsevier Ltd.

  20. ACCUMULATION AND CONSUMPTION IN MICROECONOMIC SYSTEM

    Directory of Open Access Journals (Sweden)

    Serghey A. Amelkin

    2004-12-01

    Full Text Available Two main processes are common for an economic system. They are consumption and accumulation. The first one is described by utility function, either cardinal or ordinal one. The mathematical model for accumulation process can be constructed using wealth function introduced within the frame of irreversible microeconomics. Characteristics of utility and wealth functions are compared and a problem of extreme performance of resources exchange process is solved for a case when both the consumption and accumulation exist.

  1. A cucumber mosaic virus (CMV) RNA 1 transgene mediates suppression of the homologous viral RNA 1 constitutively and prevents CMV entry into the phloem.

    Science.gov (United States)

    Canto, T; Palukaitis, P

    2001-10-01

    Resistance to Cucumber mosaic virus (CMV) in tobacco lines transformed with CMV RNA 1 is characterized by reduced virus accumulation in the inoculated leaf, with specific suppression of accumulation of the homologous viral RNA 1, and by the absence of systemic infection. We show that the suppression of viral RNA 1 occurs in protoplasts from resistant transgenic plants and therefore is not due to a host response activated by the cell-to-cell spread of virus. In contrast, suppression of Tobacco rattle virus vectors carrying CMV RNA 1 sequences did not occur in protoplasts from resistant plants. Furthermore, steady-state levels of transgene mRNA 1 were higher in resistant than in susceptible lines. Thus, the data indicate that sequence homology is not sufficient to induce suppression. Grafting experiments using transgenic resistant or susceptible rootstocks and scions demonstrated that the resistance mechanism exhibited an additional barrier to phloem entry, preventing CMV from moving a long distance in resistant plants. On the other hand, virus from susceptible rootstocks could systemically infect grafted resistant scions via the phloem. Analysis of viral RNA accumulation in the infected scions showed that the mechanism that suppresses the accumulation of viral RNA 1 at the single-cell level was overcome. The data indicate that this transgene-mediated systemic resistance probably is not based on a posttranscriptional gene-silencing mechanism.

  2. Topology of RNA-RNA interaction structures

    DEFF Research Database (Denmark)

    Andersen, Jørgen Ellegaard; Huang, Fenix Wenda; Penner, Robert;

    2012-01-01

    Abstract The topological filtration of interacting RNA complexes is studied, and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that, for two interacting RNAs, called interaction structures, there exist...

  3. Effect of 5' and 3' terminal sequences, overall length, and coding capacity on the accumulation of defective RNAs associated with broad bean mottle bromovirus in planta.

    Science.gov (United States)

    Pogany, J; Romero, J; Bujarski, J J

    1997-02-17

    Broad bean mottle bromovirus (BBMV) was shown to accumulate RNA2-derived defective interfering (DI) RNAs [Romero et al., Virology 194, 576-584 (1993); Pogany et al., Virology 212, 574-586 (1995)]. In this work, we utilize three sets of BBMV RNA2-derived artificial DI RNA constructs to determine factors that affect the accumulation of defective RNAs in planta. One set of deletion constructs was used to localize sequences required for efficient accumulation within the 5' 883 nt and the 3' 387 nt of the DI RNAs. The second set had a gradually increasing size of 3' nested deletions to determine the minimal length required for efficient DI RNA accumulation. The smallest DI RNA still accumulating in plants was found to be 1712 nt long. The third set consisted of frameshift mutants which showed that at least 64.4% of BBMV DI RNA sequences must have the 5' portion of the 2a open reading frame to ensure efficient accumulation. The importance of these factors in the selection of DI RNAs is discussed.

  4. [Probe into monitoring mechanism of Chinese materia medica resources].

    Science.gov (United States)

    Zhang, Xiao-Bo; Li, Da-Ning; Guo, Lan-Ping; Lu, Jian-Wei; Sun, Li-Ying; Huang, Lu-Qi

    2013-10-01

    Focusing on the problems of Chinese materia medica resources,and combining with the national Chinese materia medica resources survey, the paper probes into monitoring mechanism of Chinese materia medica resources. The establishment of the monitoring mechanism needs one organization and management agencies to supervise and guide monitoring work, one network system to gather data information, a group of people to perform monitoring work, a system of technical methods to assure monitoring work scientific and practical, a series of achievements and products to figure out the methods for solving problems, a group of monitoring index system to accumulate basic data, and a plenty of funds to keep normal operation of monitoring work.

  5. Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium.

    Science.gov (United States)

    Nickell, William T; Kleene, Nancy K; Kleene, Steven J

    2007-09-15

    When olfactory receptor neurons respond to odours, a depolarizing Cl(-) efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl(-) against an electrochemical gradient. In isolated olfactory receptor neurons, the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is essential for Cl(-) accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl(-) efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl(-) accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl(-) transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl(-) transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl(-) transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl(-)/HCO(3)(-) exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na(+)-Cl(-) cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl(-)/HCO(3)(-) exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl(-) component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl(-) accumulation in olfactory receptor neurons.

  6. MicroRNA profiling in human neutrophils during bone marrow granulopoiesis and in vivo exudation

    DEFF Research Database (Denmark)

    Larsen, Maria T; Hother, Christoffer; Häger, Mattias

    2013-01-01

    The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, p...

  7. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection

    DEFF Research Database (Denmark)

    Lanford, Robert E; Hildebrandt-Eriksen, Elisabeth S; Petri, Andreas

    2010-01-01

    The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA...

  8. 5S rRNA-assisted DnaK refolding.

    Science.gov (United States)

    Kim, Hyo Kyung; Choi, Seong Il; Seong, Baik L

    2010-01-08

    Although accumulating evidence has revealed that most proteins can fold without the assistance of molecular chaperones, little attention has been paid to other types of chaperoning macromolecules. A variety of proteins interact with diverse RNA molecules in vivo, suggesting a potential role of RNAs for folding of their interacting proteins. Here we show that the in vitro refolding of a representative molecular chaperone, DnaK, an Escherichia coli homolog of Hsp70, could be assisted by its interacting 5S rRNA. The folding enhancement occurred in RNA concentration and its size dependent manner whereas neither the RNA with the reverse sequence of 5S rRNA nor the RNase pretreated 5S rRNA stimulated the folding in vitro. Based on our results, we propose that 5S rRNA could exert the chaperoning activity on DnaK during the folding process. The results suggest an interesting possibility that the folding of RNA-interacting proteins could be assisted by their cognate RNA ligands. Copyright 2009 Elsevier Inc. All rights reserved.

  9. Genome-wide screening for components of small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

    Science.gov (United States)

    Xu, H-J; Chen, T; Ma, X-F; Xue, J; Pan, P-L; Zhang, X-C; Cheng, J-A; Zhang, C-X

    2013-12-01

    The brown planthopper (BPH), Nilaparvata lugens, is a major rice pest in Asia, and accumulated evidence indicates that this species is susceptible to RNA interference (RNAi); however, the mechanism underlying RNAi and parental RNAi has not yet been determined. We comprehensively investigated the repertoire of core genes involved in small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the BPH by comparing its newly assembled transcriptome and genome with those of Drosophila melanogaster, Tribolium castaneum and Caenorhabditis elegans. Our analysis showed that the BPH possesses one drosha and two Dicer (dcr) genes, three dsRNA-binding motif protein genes, two Argonaute (ago) genes, two Eri-1-like genes (eri-1), and a Sid-1-like gene (sid-1). Additionally, we report for first time that parental RNAi might occur in this species, and siRNA pathway and Sid-1 were required for high efficiency of systemic RNAi triggered by exogenous dsRNA. Furthermore, our results also demonstrated that the miRNA pathway was involved in BPH metamorphosis as depletion of the ago1 or dcr1 gene severely impaired ecdysis. The BPH might be a good model system to study the molecular mechanism of systemic RNAi in hemimetabolous insects, and RNAi has potential to be developed to control this pest in agricultural settings. © 2013 Royal Entomological Society.

  10. Monarch Monitoring

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — The US Fish and Wildlife Service has engaged in a multi-partnered, integrated strategy for monitoring conservation of the monarch butterfly (Danaus plexippus...

  11. Monitoring Hadoop

    CERN Document Server

    Singh, Gurmukh

    2015-01-01

    This book is useful for Hadoop administrators who need to learn how to monitor and diagnose their clusters. Also, the book will prove useful for new users of the technology, as the language used is simple and easy to grasp.

  12. Addition of polyadenylate sequences to virus-specific RNA during adenovirus replication.

    Science.gov (United States)

    Philipson, L; Wall, R; Glickman, G; Darnell, J E

    1971-11-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA.

  13. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation......, regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  14. Bayesian Monitoring.

    OpenAIRE

    Kirstein, Roland

    2005-01-01

    This paper presents a modification of the inspection game: The ?Bayesian Monitoring? model rests on the assumption that judges are interested in enforcing compliant behavior and making correct decisions. They may base their judgements on an informative but imperfect signal which can be generated costlessly. In the original inspection game, monitoring is costly and generates a perfectly informative signal. While the inspection game has only one mixed strategy equilibrium, three Perfect Bayesia...

  15. Kaempferol Isolated from Nelumbo nucifera Inhibits Lipid Accumulation and Increases Fatty Acid Oxidation Signaling in Adipocytes.

    Science.gov (United States)

    Lee, Bonggi; Kwon, Misung; Choi, Jae Sue; Jeong, Hyoung Oh; Chung, Hae Young; Kim, Hyeung-Rak

    2015-12-01

    Stamens of Nelumbo nucifera Gaertn have been used as a Chinese medicine due to its antioxidant, hypoglycemic, and antiatherogenic activity. However, the effects of kaempferol, a main component of N. nucifera, on obesity are not fully understood. We examined the effect of kaempferol on adipogenesis and fatty acid oxidation signaling pathways in 3T3-L1 adipocytes. Kaempferol reduced cytoplasmic triglyceride (TG) accumulation in dose and time-dependent manners during adipocyte differentiation. Accumulation of TG was rapidly reversed by retrieving kaempferol treatment. Kaempferol broadly decreased mRNA or protein levels of adipogenic transcription factors and their target genes related to lipid accumulation. Kaempferol also suppressed glucose uptake and glucose transporter GLUT4 mRNA expression in adipocytes. Furthermore, protein docking simulation suggests that Kaempferol can directly bind to and activate peroxisome proliferator-activated receptor (PPAR)-α by forming hydrophobic interactions with VAL324, THR279, and LEU321 residues of PPARα. The binding affinity was higher than a well-known PPARα agonist fenofibrate. Consistently, mRNA expression levels of PPARα target genes were increased. Our study indicates while kaempferol inhibits lipogenic transcription factors and lipid accumulation, it may bind to PPARα and stimulate fatty acid oxidation signaling in adipocytes.

  16. Correlation of Versican Expression, Accumulation, and Degradation during Embryonic Development by Quantitative Immunohistochemistry

    Science.gov (United States)

    Snyder, Jessica M.; Washington, Ida M.; Birkland, Timothy; Chang, Mary Y.; Frevert, Charles W.

    2015-01-01

    Versican, a chondroitin sulfate proteoglycan, is important in embryonic development, and disruption of the versican gene is embryonically lethal in the mouse. Although several studies show that versican is increased in various organs during development, a focused quantitative study on versican expression and distribution during lung and central nervous system development in the mouse has not previously been performed. We tracked changes in versican (Vcan) gene expression and in the accumulation and degradation of versican. Vcan expression and quantitative immunohistochemistry performed from embryonic day (E) 11.5 to E15.5 showed peak Vcan expression at E13.5 in the lungs and brain. Quantitative mRNA analysis and versican immunohistochemistry showed differences in the expression of the versican isoforms in the embryonic lung and head. The expression of Vcan mRNA and accumulation of versican in tissues was complementary. Immunohistochemistry demonstrated co-localization of versican accumulation and degradation, suggesting distinct roles of versican deposition and degradation in embryogenesis. Very little versican mRNA or protein was found in the lungs of 12- to 16-week-old mice but versican accumulation was significantly increased in mice with Pseudomonas aeruginosa lung infection. These data suggest that versican plays an important role in fundamental, overlapping cellular processes in lung development and infection. PMID:26385570

  17. One type of chalcone synthase gene expressed during embryogenesis regulates the flavonoid accumulation in citrus cell cultures.

    Science.gov (United States)

    Moriguchi, T; Kita, M; Tomono, Y; EndoInagaki, T; Omura, M

    1999-06-01

    To elucidate the relationship between the expression of chalcone synthase (CHS) genes and the production of flavonoid in citrus cell cultures, two cDNA clones encoding CHS were isolated (CitCHS1 and CitCHS2) from the citrus. The accumulation of CitCHS2 mRNA was notably induced by embryogenesis but CitCHS1 mRNA was not. There was no detectable accumulation of flavonoid in the undifferentiated calli, but flavonoid accumulated after the morphological changes to embryoids. These results indicate that two CHS genes differentially expressed during citrus somatic embryogenesis and CitCHS2 may regulate the accumulation of flavonoid in citrus cell cultures.

  18. Hepatocellular carcinoma and microRNA: new perspectives on therapeutics and diagnostics.

    Science.gov (United States)

    Yang, Ningning; Ekanem, Nsikak R; Sakyi, Clement A; Ray, Sidhartha D

    2015-01-01

    Hepatocellular carcinoma (HCC) remains one of the most lethal forms of cancer in the world. In this arena, utilities of microRNA (miRNA) as a sophisticated tool, in therapeutics and diagnostics, remains a prime focus among the leading researchers. It is well known that viral hepatitis, chronic alcoholism and metabolic syndrome are the prime causes of HCCs. Nevertheless, HCCs are usually diagnosed at late stages by using current serum biomarkers. Most epidemiological studies have found the survival rate in HCC cases to be relatively low. Therefore, development of effective noninvasive biomarkers for early detention of HCC and new strategies for HCC treatment remains a high priority research area on the shoulders of liver oncologists. Accumulating evidence in cancer diagnostics and therapeutics indicates that miRNAs involve in HCC progression, which may serve as sensitive biomarkers for detecting carcinogenesis and monitoring therapies of HCC. This review focuses on the role of miRNAs in the diagnostics and therapeutics of HCC.

  19. Molecular Beacon-Based MicroRNA Imaging During Neurogenesis.

    Science.gov (United States)

    Lee, Jonghwan; Kim, Soonhag

    2016-01-01

    The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h.

  20. Testglass changer for direct optical monitoring

    Science.gov (United States)

    Zoeller, A.; Hagedorn, H.; Weinrich, W.; Wirth, E.

    2011-09-01

    For the production of high performance multilayer systems with tight specifications and large numbers of layers optical monitoring is essential. Substantial progress was achieved by the introduction of direct monitoring on the rotating substrate holder. Pre production analysis by computer simulation of coating processes helps to optimise monitoring strategies and reduces the effort for expensive and time consuming test runs significantly. However not in any case we can find error compensating monitoring strategies. Also we have to deal with error accumulation effects especially with multi layer systems with large number of layers. Changing the monitor glass after the layer stack is deposited partly is a useful method to discontinue accumulation or to simplify the monitoring strategy. A testglass changer which helps to suppress error accumulation was developed and automized. The testglasses are located on the rotating substrate holder which may be a calotte or a plane substrate holder. It combines the advantages of direct monitoring with the flexibility to change testglasses in a fully automatic process. The basic principle will be described. Results of multilayer systems demonstrate the benefits of the newly developed testglass changer.

  1. Assessing integrity of insect RNA

    Science.gov (United States)

    Assessing total RNA integrity is important for the success of downstream RNA applications. The 2100 Bioanalyzer system with the RNA Integrity Number (RIN) provides a quantitative measure of RNA degradation. Although RINs may not be ascertained for RNA from all organisms, namely those with unusual or...

  2. 40 CFR 262.34 - Accumulation time.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Accumulation time. 262.34 Section 262...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.34 Accumulation time... removed from the drip pad and the sump or collection system and the date and time of removal; and/or...

  3. Charge accumulation in lossy dielectrics: a review

    DEFF Research Database (Denmark)

    Rasmussen, Jørgen Knøster; McAllister, Iain Wilson; Crichton, George C

    1999-01-01

    such that the material parameters which influence charge accumulation are clearly identified; viz. the conductivity, permittivity and dimensions of the insulating media. The two former parameters, together with the applied voltage, govern both the magnitude and polarity of the accumulated charge....

  4. Annual Greenland accumulation rates (2009-2012) from airborne snow radar

    Science.gov (United States)

    Koenig, Lora S.; Ivanoff, Alvaro; Alexander, Patrick M.; MacGregor, Joseph A.; Fettweis, Xavier; Panzer, Ben; Paden, John D.; Forster, Richard R.; Das, Indrani; McConnell, Joesph R.; Tedesco, Marco; Leuschen, Carl; Gogineni, Prasad

    2016-08-01

    Contemporary climate warming over the Arctic is accelerating mass loss from the Greenland Ice Sheet through increasing surface melt, emphasizing the need to closely monitor its surface mass balance in order to improve sea-level rise predictions. Snow accumulation is the largest component of the ice sheet's surface mass balance, but in situ observations thereof are inherently sparse and models are difficult to evaluate at large scales. Here, we quantify recent Greenland accumulation rates using ultra-wideband (2-6.5 GHz) airborne snow radar data collected as part of NASA's Operation IceBridge between 2009 and 2012. We use a semiautomated method to trace the observed radiostratigraphy and then derive annual net accumulation rates for 2009-2012. The uncertainty in these radar-derived accumulation rates is on average 14 %. A comparison of the radar-derived accumulation rates and contemporaneous ice cores shows that snow radar captures both the annual and long-term mean accumulation rate accurately. A comparison with outputs from a regional climate model (MAR) shows that this model matches radar-derived accumulation rates in the ice sheet interior but produces higher values over southeastern Greenland. Our results demonstrate that snow radar can efficiently and accurately map patterns of snow accumulation across an ice sheet and that it is valuable for evaluating the accuracy of surface mass balance models.

  5. Ab initio RNA folding.

    Science.gov (United States)

    Cragnolini, Tristan; Derreumaux, Philippe; Pasquali, Samuela

    2015-06-17

    RNA molecules are essential cellular machines performing a wide variety of functions for which a specific three-dimensional structure is required. Over the last several years, the experimental determination of RNA structures through x-ray crystallography and NMR seems to have reached a plateau in the number of structures resolved each year, but as more and more RNA sequences are being discovered, the need for structure prediction tools to complement experimental data is strong. Theoretical approaches to RNA folding have been developed since the late nineties, when the first algorithms for secondary structure prediction appeared. Over the last 10 years a number of prediction methods for 3D structures have been developed, first based on bioinformatics and data-mining, and more recently based on a coarse-grained physical representation of the systems. In this review we are going to present the challenges of RNA structure prediction and the main ideas behind bioinformatic approaches and physics-based approaches. We will focus on the description of the more recent physics-based phenomenological models and on how they are built to include the specificity of the interactions of RNA bases, whose role is critical in folding. Through examples from different models, we will point out the strengths of physics-based approaches, which are able not only to predict equilibrium structures, but also to investigate dynamical and thermodynamical behavior, and the open challenges to include more key interactions ruling RNA folding.

  6. Plant RNA binding proteins for control of RNA virus infection

    OpenAIRE

    Huh, Sung Un; Paek, Kyung-Hee

    2013-01-01

    Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific b...

  7. CRISPR guide RNA design for research applications.

    Science.gov (United States)

    Mohr, Stephanie E; Hu, Yanhui; Ewen-Campen, Benjamin; Housden, Benjamin E; Viswanatha, Raghuvir; Perrimon, Norbert

    2016-09-01

    The rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on-target and effective guide RNAs (gRNAs). Here, we review the state of the art in CRISPR gRNA design for research applications of the CRISPR-Cas9 system, including knockout, activation, and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high-quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off-targets and effectiveness metrics.

  8. Are NORMs Accumulated in Filters of Drinking Water Facilities?

    Science.gov (United States)

    Choung, S.; Shin, W.; Park, M.; Han, J. H.; Ryu, J. S.; Han, W. S.; Chang, B. U.

    2016-12-01

    Groundwater is used as raw water to produce mineral drinking water through filtering processes in bottled water facilities. Although natural occurring radioactive materials (NORMs) exist in groundwater, accumulation of NORMs were rarely studied due to their low concentrations in groundwater. The goal of this study is to evaluate potential accumulation of NORMs in filters used at the drinking water facilities. Raw water and treated water after each filtering step, bottled water, and used filters were collected from a total of 13 bottled water facilities to analyze major dissolved ions and NORMs. Additionally, surface radioactive dose rate were measured at individual filter housings. The measured concentrations of NORMs in raw and treated water were quite low. However, the surface radioactivity dose rates dramatically increased around filter housing located at very first step regardless of filter type (i.e., activated carbon or membrane filter) in 4 out of 6 facilities. Some used filters showed approximately 20 times greater contents of Pb-210 than the Korean regulation level of 1 Bq g-1. Also, the concentrations of uranium and thorium were detected up to 75 µg g-1filter and 2 µg g-1filter, respectively, in 4 water facilities. These results implies potential accumulation of NORMs in filters used at bottled water facilities. Therefore, the filters need to be monitored during manufacturing processes of bottled water, and may be properly managed after use.

  9. Accumulation of Transcripts Abundance after Barley Inoculation with Cochliobolus sativus

    Directory of Open Access Journals (Sweden)

    Mohammad Imad Eddin Arabi

    2015-03-01

    Full Text Available Spot blotch caused by the hemibiotrophic pathogen Cochliobolus sativus has been the major yield-reducing factor for barley production during the last decade. Monitoring transcriptional reorganization triggered in response to this fungus is an essential first step for the functional analysis of genes involved in the process. To characterize the defense responses initiated by barley resistant and susceptible cultivars, a survey of transcript abundance at early time points of C. sativus inoculation was conducted. A notable number of transcripts exhibiting significant differential accumulations in the resistant and susceptible cultivars were detected compared to the non-inoculated controls. At the p-value of 0.0001, transcripts were divided into three general categories; defense, regulatory and unknown function, and the resistant cultivar had the greatest number of common transcripts at different time points. Quantities of differentially accumulated gene transcripts in both cultivars were identified at 24 h post infection, the approximate time when the pathogen changes trophic lifestyles. The unique and common accumulated transcripts might be of considerable interest for enhancing effective resistance to C. sativus.

  10. microRNA

    OpenAIRE

    Xin, Xiong; Ning, Zhou

    2007-01-01

    MicroRNAs (miRNAs) are short, 20-22 nucleotide RNA molecules that function as negative regulators of gene expression in eukaryotic organisms. RNA mediated gene silencing pathways have essential roles in development, cell differentiation, proliferation, and cell death. It is becoming clear that microRNAs can play a very important role in regulation of gene expression. Understanding the basic mechanism of miRNA biogenesis is one of the central aims of molecular biologists in the future. MicroRN...

  11. RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

    Science.gov (United States)

    Hunter, Lydia J R; Brockington, Samuel F; Murphy, Alex M; Pate, Adrienne E; Gruden, Kristina; MacFarlane, Stuart A; Palukaitis, Peter; Carr, John P

    2016-03-16

    Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

  12. Triglyceride accumulation inhibitory effects of new chromone glycosides from Drynaria fortunei.

    Science.gov (United States)

    Han, Lifeng; Zheng, Fang; Zhang, Yi; Liu, Erwei; Li, Wei; Xia, Minghui; Wang, Tao; Gao, Xiumei

    2015-01-01

    Two new chromone glycosides, drynachromosides C (1) and D (2), along with five known chromones (3-7), were isolated from the rhizomes of Drynaria fortunei. The structures of the two new compounds were elucidated on the basis of physico-chemical property and spectroscopic data. Triglyceride (TG) accumulation inhibitory effects of the obtained chromones on 3T3-L1 cells were investigated. The results showed that 1, 2 and 5 exhibited inhibitory activity on TG accumulation. Effects of compounds 1 and 2 on mRNA expression of PPARγ, C/EBPα and aP2 in 3T3-L1 cells were also investigated.

  13. Accumulation of cadmium and uranium in arable soils in Switzerland.

    Science.gov (United States)

    Bigalke, Moritz; Ulrich, Andrea; Rehmus, Agnes; Keller, Armin

    2017-02-01

    Mineral phosphorus (P) fertilizers contain contaminants that are potentially hazardous to humans and the environment. Frequent mineral P fertilizer applications can cause heavy metals to accumulate and reach undesirable concentrations in agricultural soils. There is particular concern about Cadmium (Cd) and Uranium (U) accumulation because these metals are toxic and can endanger soil fertility, leach into groundwater, and be taken up by crops. We determined total Cd and U concentrations in more than 400 topsoil and subsoil samples obtained from 216 agricultural sites across Switzerland. We also investigated temporal changes in Cd and U concentrations since 1985 in soil at six selected Swiss national soil monitoring network sites. The mean U concentrations were 16% higher in arable topsoil than in grassland topsoil. The Cd concentrations in arable and grassland soils did not differ, which we attribute to soil management practices and Cd sources other than mineral P fertilizers masking Cd inputs from mineral P fertilizers. The mean Cd and U concentrations were 58% and 9% higher, respectively, in arable topsoil than in arable subsoil, indicating that significant Cd and U inputs to arable soils occurred in the past. Geochemical mass balances confirmed this, indicating an accumulation of 52% for Cd and 6% for U. Only minor temporal changes were found in the Cd concentrations in topsoil from the six soil-monitoring sites, but U concentrations in topsoil from three sites had significantly increased since 1985. Sewage sludge and atmospheric deposition were previously important sources of Cd to agricultural soils, but today mineral P fertilizers are the dominant sources of Cd and U. Future Cd and U inputs to agricultural soils may be reduced by using optimized management practices, establishing U threshold values for mineral P fertilizers and soils, effectively enforcing threshold values, and developing and using clean recycled P fertilizers. Copyright © 2016 Elsevier Ltd

  14. [Synthesis in E. coli cells of short RNA encoded in plasmids].

    Science.gov (United States)

    Dontsova, O A; Bogdanova, S L; Kopylov, A M

    1989-05-01

    The synthesis of 5S rRNA and 4.5S RNA in E. coli HB 101 cells harbouring plasmids pKK 5-1 and pKK 247-2 was studied. The plasmids were derived from pBK 322 and contained genes coding for 5S rRNA and 4.5S RNA with regulatory elements of an rRNA transcription operon rrn B. When the cells were grown on enriched or minimal media (2 and 0.3 duplications per hour), the synthesis of both 5S rRNA and 4.5S RNA was proportional to the gene dosage and was greater in the plasmid than in the host strain. Such RNA accumulation did not change the cell growth parameters and was thus not toxic for the cells. At high growth rates, the RNA synthesis in the cells became excessive, and the processing system was upset with the accumulation of RNA precursors. The fact confirms the hypothesis, according to which the whole rRNA operon is essential for its own feedback regulation.

  15. Study on Monitoring Model for Nitrogen Accumulation in Peanut (Arachis Hypogasa L.) Leaves Based on Hyper- Spectral Remote Sensing%基于高光谱遥感的花生叶片氮积累量监测模型的研究

    Institute of Scientific and Technical Information of China (English)

    张晓艳; 王丽丽; 刘锋; 封文杰; 刘淑云; 朱建华

    2012-01-01

    作物氮素状况是评价长势、提高产量和改善品质的重要指标,因此叶片氮积累量的实时无损估测对作物生产的氮素管理具有重要意义.本研究选用大花生品种丰花1号为试验材料,在大田生产条件下,分析了花生叶片氮积累量与冠层高光谱参数的定量关系.结果表明,叶片氮素含量随生育进程逐渐下降,不同处理之间差异较小;叶片氮素积累量随生育时期推进呈现先升后降的单峰曲线变化趋势,在结荚期达到高峰.花生冠层光谱反射率在740 ~1100nm波段内随叶片氮积累量的增加而增加,叶片氮积累量的敏感波段主要存在于近红外平台和可见光区,其中,“红边”区域表现最为显著.通过微分等技术构造多种植被指数,对高光谱参数和叶片氮积累量进行相关回归分析,红边振幅(Dr)、氮素反射指数(NRI)、归一化植被指数(NDVI)各波段组合平均值及比值植被指数( RVI)与叶片氮积累量关系最密切,方程拟合决定系数分别为0.9194、0.8984、0.8918、0.8899、0.8794、0.8797.经另外一组独立数据的检验表明,对叶片氮积累量的预测以红边位置(REP)和Dr两个参数表现最优,预测的根均方差(RMSE)分别为1.78和1.10,相对误差为5.29%和3.59%.NDVI[ Average(1230,1240,1250,1260),640]和土壤调整植被指数(SAVI)两个光谱参数预测的RMSE分别为1.15和1.19,预测相对误差为5.42%和7.41%.比较而言,Dr为自变量建立的模型,可以更好地评估不同条件下叶片氮素积累状况.%Nitrogen condition of crops was an important indicator for evaluating growth vigor, increasing yield and improving quality, so it was significant of real - time and lossless estimation of nitrogen accumulation in leaves for nitrogen management during crop production. In this study, the peanut variety Fenghua 1 with large seed was used as experimental material to study the relationship between nitrogen accumulation in

  16. 47 CFR 36.505 - Accumulated amortization-Tangible-Account 3400 (Class B Telephone Companies); Accumulated...

    Science.gov (United States)

    2010-10-01

    ... (Class B Telephone Companies); Accumulated amortization-Capital Leases-Account 3410 (Class A Telephone... and Deferrals § 36.505 Accumulated amortization—Tangible—Account 3400 (Class B Telephone Companies); Accumulated amortization—Capital Leases—Account 3410 (Class A Telephone Companies). (a) Amounts in...

  17. [Status of lead exposure and its impact on health of workers in an accumulator factory].

    Science.gov (United States)

    Liang, Jiabin; Zhang, Jian; Guo, Xiaojing; Mai, Jianping; Wang, Zhi; Liu, Yimin

    2014-02-01

    To identify the occupational hazard factors in an accumulator factory, to analyze the status of internal and external lead exposure and evaluate the impact of lead exposure on the health of workers in the accumulator industry, and to provide a theoretical basis for improved lead exposure criteria and technical support for the control of lead contamination in the accumulator industry. An on-site investigation was carried out to monitor and evaluate the lead fume and dust in the workplaces of an accumulator factory, and occupational health examination was performed in all workers. The occupational hazard safeguards in the accumulator factory were unadvanced. The contamination of lead fume and dust was serious. The abnormal rate of blood lead was up to 79.80%, and many workers developed anemia and mild peripheral nerve disease. Lead contamination is serious in the accumulator factory, leading to poor health of workers. It is essential to take effective control measures, improve the working environment, provide occupational health education, increase workers' self-protection awareness, and periodically conduct occupational hazard monitoring and health surveillance. The government must reinforce occupational health supervision of such enterprises.

  18. Generation of siRNA Nanosheets for Efficient RNA Interference

    Science.gov (United States)

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-04-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances.

  19. Seasonal Arsenic Accumulation in Stream Sediments at a Groundwater Discharge Zone

    DEFF Research Database (Denmark)

    MacKay, Allison A.; Gan, Ping; Yu, Ran

    2014-01-01

    Seasonal changes in arsenic and iron accumulation rates were examined in the sediments of a brook that receives groundwater discharges of arsenic and reduced iron. Clean glass bead columns were deployed in sediments for known periods over the annual hydrologic cycle to monitor changes in arsenic ...

  20. Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.

    Science.gov (United States)

    Kang, Eunju; Wang, Xinjian; Tippner-Hedges, Rebecca; Ma, Hong; Folmes, Clifford D L; Gutierrez, Nuria Marti; Lee, Yeonmi; Van Dyken, Crystal; Ahmed, Riffat; Li, Ying; Koski, Amy; Hayama, Tomonari; Luo, Shiyu; Harding, Cary O; Amato, Paula; Jensen, Jeffrey; Battaglia, David; Lee, David; Wu, Diana; Terzic, Andre; Wolf, Don P; Huang, Taosheng; Mitalipov, Shoukhrat

    2016-05-05

    The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

  1. Incorporation of radiometric tracers in peat and implications for estimating accumulation rates

    Energy Technology Data Exchange (ETDEWEB)

    Hansson, Sophia V., E-mail: sophia.hansson@emg.umu.se [Department of Ecology and Environmental Science, Umeå University, SE-901 87 Umeå (Sweden); Kaste, James M. [Geology Department, The College of William and Mary, Williamsburg, VA 23187 (United States); Olid, Carolina; Bindler, Richard [Department of Ecology and Environmental Science, Umeå University, SE-901 87 Umeå (Sweden)

    2014-09-15

    Accurate dating of peat accumulation is essential for quantitatively reconstructing past changes in atmospheric metal deposition and carbon burial. By analyzing fallout radionuclides {sup 210}Pb, {sup 137}Cs, {sup 241}Am, and {sup 7}Be, and total Pb and Hg in 5 cores from two Swedish peatlands we addressed the consequence of estimating accumulation rates due to downwashing of atmospherically supplied elements within peat. The detection of {sup 7}Be down to 18–20 cm for some cores, and the broad vertical distribution of {sup 241}Am without a well-defined peak, suggest some downward transport by percolating rainwater and smearing of atmospherically deposited elements in the uppermost peat layers. Application of the CRS age–depth model leads to unrealistic peat mass accumulation rates (400–600 g m{sup −2} yr{sup −1}), and inaccurate estimates of past Pb and Hg deposition rates and trends, based on comparisons to deposition monitoring data (forest moss biomonitoring and wet deposition). After applying a newly proposed IP-CRS model that assumes a potential downward transport of {sup 210}Pb through the uppermost peat layers, recent peat accumulation rates (200–300 g m{sup −2} yr{sup −1}) comparable to published values were obtained. Furthermore, the rates and temporal trends in Pb and Hg accumulation correspond more closely to monitoring data, although some off-set is still evident. We suggest that downwashing can be successfully traced using {sup 7}Be, and if this information is incorporated into age–depth models, better calibration of peat records with monitoring data and better quantitative estimates of peat accumulation and past deposition are possible, although more work is needed to characterize how downwashing may vary between seasons or years. - Highlights: • {sup 210}Pb, {sup 137}Cs, {sup 241}Am and {sup 7}Be, and tot-Pb and tot Hg were measured in 5 peat cores. • Two age–depth models were applied resulting in different accumulation rates

  2. Aflatoxin Accumulation in a Maize Diallel Cross

    Directory of Open Access Journals (Sweden)

    W. Paul Williams

    2015-06-01

    Full Text Available Aflatoxins, produced by the fungus Aspergillus flavus, occur naturally in maize. Contamination of maize grain with aflatoxin is a major food and feed safety problem and greatly reduces the value of the grain. Plant resistance is generally considered a highly desirable approach to reduction or elimination of aflatoxin in maize grain. In this investigation, a diallel cross was produced by crossing 10 inbred lines with varying degrees of resistance to aflatoxin accumulation in all possible combinations. Three lines that previously developed and released as sources of resistance to aflatoxin accumulation were included as parents. The 10 parental inbred lines and the 45 single crosses making up the diallel cross were evaluated for aflatoxin accumulation in field tests conducted in 2013 and 2014. Plants were inoculated with an A. flavus spore suspension seven days after silk emergence. Ears were harvested approximately 60 days later and concentration of aflatoxin in the grain determined. Parental inbred lines Mp717, Mp313E, and Mp719 exhibited low levels (3–12 ng/g of aflatoxin accumulation. In the diallel analysis, both general and specific combining ability were significant sources of variation in the inheritance of resistance to aflatoxin accumulation. General combining ability effects for reduced aflatoxin accumulation were greatest for Mp494, Mp719, and Mp717. These lines should be especially useful in breeding for resistance to aflatoxin accumulation. Breeding strategies, such as reciprocal recurrent selection, would be appropriate.

  3. Shapes of interacting RNA complexes

    DEFF Research Database (Denmark)

    Fu, Benjamin Mingming; Reidys, Christian

    2014-01-01

    Shapes of interacting RNA complexes are studied using a filtration via their topological genus. A shape of an RNA complex is obtained by (iteratively) collapsing stacks and eliminating hairpin loops.This shape-projection preserves the topological core of the RNA complex and for fixed topological...... genus there are only finitely many such shapes. Our main result is a new bijection that relates the shapes of RNA complexes with shapes of RNA structures. This allows to compute the shape polynomial of RNA complexes via the shape polynomial of RNA structures. We furthermore present a linear time uniform...... sampling algorithm for shapes of RNA complexes of fixed topological genus....

  4. rRNA and Poly-β-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast and Famine Cycles

    Science.gov (United States)

    Frigon, Dominic; Muyzer, Gerard; van Loosdrecht, Mark; Raskin, Lutgarde

    2006-01-01

    Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-β-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological

  5. Selenoprotein P Inhibits Radiation-Induced Late Reactive Oxygen Species Accumulation and Normal Cell Injury

    Energy Technology Data Exchange (ETDEWEB)

    Eckers, Jaimee C.; Kalen, Amanda L.; Xiao, Wusheng; Sarsour, Ehab H.; Goswami, Prabhat C., E-mail: prabhat-goswami@uiowa.edu

    2013-11-01

    Purpose: Radiation is a common mode of cancer therapy whose outcome is often limited because of normal tissue toxicity. We have shown previously that the accumulation of radiation-induced late reactive oxygen species (ROS) precedes cell death, suggesting that metabolic oxidative stress could regulate cellular radiation response. The purpose of this study was to investigate whether selenoprotein P (SEPP1), a major supplier of selenium to tissues and an antioxidant, regulates late ROS accumulation and toxicity in irradiated normal human fibroblasts (NHFs). Methods and Materials: Flow cytometry analysis of cell viability, cell cycle phase distribution, and dihydroethidium oxidation, along with clonogenic assays, were used to measure oxidative stress and toxicity. Human antioxidant mechanisms array and quantitative real-time polymerase chain reaction assays were used to measure gene expression during late ROS accumulation in irradiated NHFs. Sodium selenite addition and SEPP1 overexpression were used to determine the causality of SEPP1 regulating late ROS accumulation and toxicity in irradiated NHFs. Results: Irradiated NHFs showed late ROS accumulation (4.5-fold increase from control; P<.05) that occurs after activation of the cell cycle checkpoint pathways and precedes cell death. The mRNA levels of CuZn- and Mn-superoxide dismutase, catalase, peroxiredoxin 3, and thioredoxin reductase 1 increased approximately 2- to 3-fold, whereas mRNA levels of cold shock domain containing E1 and SEPP1 increased more than 6-fold (P<.05). The addition of sodium selenite before the radiation treatment suppressed toxicity (45%; P<.05). SEPP1 overexpression suppressed radiation-induced late ROS accumulation (35%; P<.05) and protected NHFs from radiation-induced toxicity (58%; P<.05). Conclusion: SEPP1 mitigates radiation-induced late ROS accumulation and normal cell injury.

  6. Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro.

    Science.gov (United States)

    Sharwood, Robert E; Hotto, Amber M; Bollenbach, Thomas J; Stern, David B

    2011-02-01

    Post-transcriptional regulation in the chloroplast is exerted by nucleus-encoded ribonucleases and RNA-binding proteins. One of these ribonucleases is RNR1, a 3'-to-5' exoribonuclease of the RNase II family. We have previously shown that Arabidopsis rnr1-null mutants exhibit specific abnormalities in the expression of the rRNA operon, including the accumulation of precursor 23S, 16S, and 4.5S species and a concomitant decrease in the mature species. 5S rRNA transcripts, however, accumulate to a very low level in both precursor and mature forms, suggesting that they are unstable in the rnr1 background. Here we demonstrate that rnr1 plants overaccumulate an antisense RNA, AS5, that is complementary to the 5S rRNA, its intergenic spacer, and the downstream trnR gene, which encodes tRNA(Arg), raising the possibility that AS5 destabilizes 5S rRNA or its precursor and/or blocks rRNA maturation. To investigate this, we used an in vitro system that supports 5S rRNA and trnR processing. We show that AS5 inhibits 5S rRNA maturation from a 5S-trnR precursor, and shorter versions of AS5 demonstrate that inhibition requires intergenic sequences. To test whether the sense and antisense RNAs form double-stranded regions in vitro, treatment with the single-strand-specific mung bean nuclease was used. These results suggest that 5S-AS5 duplexes interfere with a sense-strand secondary structure near the endonucleolytic cleavage site downstream from the 5S rRNA coding region. We hypothesize that these duplexes are degraded by a dsRNA-specific ribonuclease in vivo, contributing to the 5S rRNA deficiency observed in rnr1.

  7. Manganese accumulation in the brain: MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    Uchino, A.; Nomiyama, K.; Takase, Y.; Nakazono, T.; Nojiri, J.; Kudo, S. [Saga Medical School, Department of Radiology, Saga (Japan); Noguchi, T. [Kyushu University, Department of Clinical Radiology, Graduate School of Medicine, Fukuoka (Japan)

    2007-09-15

    Manganese (Mn) accumulation in the brain is detected as symmetrical high signal intensity in the globus pallidi on T1-weighted MR images without an abnormal signal on T2-weighted images. In this review, we present several cases of Mn accumulation in the brain due to acquired or congenital diseases of the abdomen including hepatic cirrhosis with a portosystemic shunt, congenital biliary atresia, primary biliary cirrhosis, congenital intrahepatic portosystemic shunt without liver dysfunction, Rendu-Osler-Weber syndrome with a diffuse intrahepatic portosystemic shunt, and patent ductus venosus. Other causes of Mn accumulation in the brain are Mn overload from total parenteral nutrition and welding-related Mn intoxication. (orig.)

  8. Recent accumulation rate at Dome A, Antarctica

    Institute of Scientific and Technical Information of China (English)

    HOU ShuGui; LI YuanSheng; XIAO CunDe; REN JiaWen

    2007-01-01

    Based on the horizon of β activity and the density profiles, recent accumulation rate at Dome A, Antarctica is calculated to be 0.023 m water equivalent per year. This value is comparative to the accumulation rates deduced from the other inland sites of Antarctica. Clear-sky precipitation (or diamond dust) dominates the total precipitation at Dome A region. We speculate Dome A as a potential site to discover the oldest ice in Antarctica due to its tremendous ice thickness (>3000 m), extremely low accumulation rate, and low ice velocity.

  9. Microbial accumulation of uranium, radium, and cesium

    Energy Technology Data Exchange (ETDEWEB)

    Strandberg, G.W.; Shumate, S.E. II; Parrott, J.R. Jr.; North, S.E.

    1981-05-01

    Diverse microbial species varied considerably in their ability to accumulate uranium, cesium, and radium. Mechanistic differences in uranium uptake by Saccharomyces cerevisiae and Pseudomonas aeruginosa were indicated. S. serevisiae exhibited a slow (hours) surface accumulation of uranium which was subject to environmental factors, while P. aeruginosa accumulated uranium rapidly (minutes) as dense intracellular deposits and did not appear to be affected by environmental parameters. Metabolism was not required for uranium uptake by either organism. Cesium and radium were concentrated to a considerably lesser extent than uranium by the several species tested.

  10. Accumulation of Norfloxacin by Bacteroides fragilis

    OpenAIRE

    Ricci, Vito; Piddock, Laura J. V.

    2000-01-01

    The accumulation of norfloxacin by Bacteroides fragilis NCTC 9343 was determined by the modified fluorescence method. The time required to achieve a steady-state concentration (SSC) after allowing B. fragilis to accumulate norfloxacin in an aerobic or an anaerobic environment was ∼2 min; the SSC achieved in air was 90.28 ± 9.32 ng of norfloxacin/mg (dry weight) of cells, and that achieved anaerobically was 98.45 ± 3.7 ng of norfloxacin/mg (dry weight) of cells. Initial rates of accumulation w...

  11. Effects of ulvoid (Ulva spp.) accumulation on the structure and function of eelgrass (Zostera marina L.) bed

    Energy Technology Data Exchange (ETDEWEB)

    Sugimoto, Kenji [Hiroshima Environment and Health Association, 9-11, Hirosekitamachi Naka-ku, Hiroshima 730-8631 (Japan)], E-mail: kenji.sugimoto@kanhokyo.or.jp; Hiraoka, Kiyonori [Hiroshima Environment and Health Association, 9-11, Hirosekitamachi Naka-ku, Hiroshima 730-8631 (Japan); Ohta, Seiji [Hiroshima Defense Facilities Administration Bureau, 6-30, Kamihattyobori, Naka-ku, Hiroshima 730-0012 (Japan); Niimura, Yoko [National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5 Maruishi Ohno, Saeki, Hiroshima 739-0452 (Japan); Terawaki, Toshinobu [Fisheries Research Agency, Queen' s Tower-B 15F, Minato-Mirai, West, Yokohama 220-6115 (Japan); Okada, Mitsumasa [Hiroshima University, 1-4-1, Kagamiyama, Higashihiroshima 739-8527 (Japan)

    2007-10-15

    The objective of this study is to clarify the effect of ulvoid (Ulva spp.) accumulation on the structure and function of an eelgrass bed by the coast of Iwakuni, Seto Inland Sea, Japan. We monitored eelgrass shoot density and volume of ulvoid accumulation in the study site and evaluated effects of the accumulated ulvoid canopy on the percent survival, seedling density, growth rates, photosynthetic photon flux density (PPFD) and carbon contents of eelgrass. Eelgrass shoot density decreased by the accumulation of ulvoid. Also, seedling density decreased by the increase in the ulvoid volumes. Shoot density, seedling density and leaf elongation were negatively correlated with ulvoid volume. Carbon contents in eelgrass decreased by the accumulation of ulvoid (canopy height: 25 cm). These results suggest that accumulation of ulvoid bloom has significant negative impacts on the structure and function of eelgrass bed, i.e. decreases in vegetative shoot density, seedling density, shoot height and growth rate.

  12. Effects of ulvoid (Ulva spp.) accumulation on the structure and function of eelgrass (Zostera marina L.) bed.

    Science.gov (United States)

    Sugimoto, Kenji; Hiraoka, Kiyonori; Ohta, Seiji; Niimura, Yoko; Terawaki, Toshinobu; Okada, Mitsumasa

    2007-10-01

    The objective of this study is to clarify the effect of ulvoid (Ulva spp.) accumulation on the structure and function of an eelgrass bed by the coast of Iwakuni, Seto Inland Sea, Japan. We monitored eelgrass shoot density and volume of ulvoid accumulation in the study site and evaluated effects of the accumulated ulvoid canopy on the percent survival, seedling density, growth rates, photosynthetic photon flux density (PPFD) and carbon contents of eelgrass. Eelgrass shoot density decreased by the accumulation of ulvoid. Also, seedling density decreased by the increase in the ulvoid volumes. Shoot density, seedling density and leaf elongation were negatively correlated with ulvoid volume. Carbon contents in eelgrass decreased by the accumulation of ulvoid (canopy height: 25cm). These results suggest that accumulation of ulvoid bloom has significant negative impacts on the structure and function of eelgrass bed, i.e. decreases in vegetative shoot density, seedling density, shoot height and growth rate.

  13. Inhibition of RNA recruitment and replication of an RNA virus by acridine derivatives with known anti-prion activities.

    Directory of Open Access Journals (Sweden)

    Zsuzsanna Sasvari

    Full Text Available BACKGROUND: Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV, a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ and quinacrine (QC, which are active against prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii reduction of minus-strand synthesis by the tombusvirus replicase; and (iii inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication. CONCLUSION/SIGNIFICANCE: Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.

  14. Biological analysis of chronic lymphocytic leukemia: integration of mRNA and microRNA expression profiles.

    Science.gov (United States)

    Dong, L; Bi, K H; Huang, N; Chen, C Y

    2016-01-08

    Chronic lymphocytic leukemia (CLL) is a disease that involves progressive accumulation of nonfunctioning lymphocytes and has a low cure rate. There is an urgent requirement to determine the molecular mechanism underlying this disease in order to improve the early diagnosis and treatment of CLL. In this study, genes differentially expressed between CLL samples and age-matched controls were identified using microRNA (miRNA) and mRNA expression profiles. Differentially expressed (DE) miRNA targets were predicted by combining five algorithms. Common genes were obtained on overlapping the DE mRNA and DE miRNA targets. Then, network and module analyses were performed. A total of 239 miRNA targets were predicted and 357 DE mRNAs were obtained. On intersecting miRNA targets and DE mRNAs, 33 common genes were obtained. The protein-protein interaction network and module analysis identified several crucial genes and modules that might be associated with the development of CLL. These DE mRNAs were significantly enriched in the hematopoietic cell lineage (P = 2.58E-4), mitogen-activated protein kinase signaling pathway (P = 0.0025), and leukocyte transendothelial migration pathway (P = 0.0026). Thus, we conducted biological analysis on integration of DE mRNAs and DE miRNAs in CLL, determined gene expression patterns, and screened out several important genes that might be related to CLL.

  15. Smarter radiation monitors for safeguards and security

    Science.gov (United States)

    Fehlau, P. E.; Pratt, J. C.; Markin, J. T.; Scurry, T., Jr.

    Versatile microprocessor systems permit more efficient, and more useful methods for monitoring nuclear materials. One such method is simple stepwise monitoring, which has variable alarm levels to expedite monitoring where extended monitoring periods are required. Another method, sequential probability ratio logic, tests data as it accumulates against two hypothesis - background, or background plus a transient diversion signal - and terminates monitoring as soon as a decision can be made that meets false alarm and detection confidence requirements. A third method, quntitative monitoring for personnel, calculates count ratios of high to low energy gamma ray regions to predict whether the material detected is a small quantity of bare material or a larger quantity of shielded material. Microprocessor system subprograms can assist in detector calibration and trouble shooting. Examples of subprograms are a variance analysis technique to set hias levels in plastic scintillators and a state of health routine for detecting malfunctions in digital circuit components.

  16. RNA-Dependent RNA Polymerase Activity in Influenza Virions

    Science.gov (United States)

    Penhoet, Edward; Miller, Henry; Doyle, Michael; Blatti, Stanley

    1971-01-01

    An RNA-dependent RNA polymerase activity has been detected in purified preparations of influenza virus. In contrast to the replicase activity induced in influenza-infected cells, the virion-associated enzyme has an absolute requirement for Mn++. Most of the RNA synthesized in vitro is complementary to virion RNA. PMID:5288388

  17. Studying RNA-protein interactions in vivo by RNA immunoprecipitation

    DEFF Research Database (Denmark)

    Selth, Luke A; Close, Pierre; Svejstrup, Jesper Q

    2011-01-01

    The crucial roles played by RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of transcription, have become increasingly evident. Moreover, other factors that do not directly interact with RNA molecules can nevertheless function proximally to RNA polymerases an...

  18. Hepatic Copper Accumulation: A Novel Feature in Transient Infantile Liver Failure Due to TRMU Mutations?

    Science.gov (United States)

    Grover, Z; Lewindon, P; Clousten, A; Shaag, A; Elpeleg, O; Coman, D

    2015-01-01

    Defects in the mitochondrial respiratory chain can induce a heterogeneous range of clinical and biochemical manifestations. Hepatic involvement includes acute fulminant hepatic failure, microvesicular steatosis, neonatal non-alloimmune haemochromatosis and cirrhosis. Recently pathogenic mutations in tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) gene (OMIM 610230) have been demonstrated to cause transient infantile liver failure (OMIM 613070). The human TRMU gene encodes a mitochondrial protein, 5-methylaminomethyl-2-thiouridylate methyltransferase, whose molecular function is that of mitochondrial tRNA modification.We report an infant who presented with acute liver failure, in whom we observed hepatic copper intoxication and cirrhosis on liver biopsy. We postulate that the hepatic copper intoxication observed in our patient is most likely a secondary event associated with cholangiopathy. Periportal copper accumulation has been implicated in causing secondary mitochondrial dysfunction; the impact of copper accumulation in patients with TRMU mutations is unclear and warrants long-term clinical follow-up.

  19. Subnuclear compartmentalization of transiently expressed polyadenylated pri-microRNAs: processing at transcription sites or accumulation in SC35 foci.

    Science.gov (United States)

    Pawlicki, Jan M; Steitz, Joan A

    2009-02-01

    MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate expression of their target messenger RNAs. We recently demonstrated that primary miRNA transcripts (pri-miRNAs) retained at transcription sites are processed with enhanced efficiency, suggesting that pri-miRNA processing is coupled to transcription in mammalian cells. We also observed that transiently expressed pri-miRNAs accumulate in nuclear foci with splicing factor SC35 and Microprocessor components, Drosha and DGCR8. Here, we show that pri-miRNAs containing a self-cleaving hepatitis delta ribozyme accumulate in the nucleoplasm after release from their transcription sites, but are not efficiently processed. Pri-miRNAs with ribozyme-generated 3' ends do not localize to SC35-containing foci, whereas cleaved and polyadenylated pri-miRNA transcripts with or without the pre-miRNA hairpin do. Pri-miRNA/SC35 foci contain a number of proteins normally associated with SC35 domains, including ASF/SF2, PABII, and the prolyl isomerase, Pin1. In contrast, RNA polymerase II and PM/Scl-100 do not strongly colocalize with pri-miRNAs in SC35-containing foci. These data argue that pri-miRNA/SC35-containing foci are not major sites of pri-miRNA processing and that pri-miRNA processing is coupled to transcription. We discuss the implications of our findings relative to recent insights into miRNA biogenesis, mRNA metabolism, and the nuclear organization of gene expression.

  20. Towards innovative roadside monitoring

    Science.gov (United States)

    Ojha, G.; Appel, E.; Magiera, T.

    2012-04-01

    Soil contamination along roadsides is an important factor of anthropogenic point source pollution. Climatic and traffic-specific factors influence the amount and characteristics of pollution emitted and deposited in the roadside soil. In our present study we focus on monitoring typical traffic pollutants (heavy metals HM, platinum group elements, polycyclic hydrocarbons PAH), and investigate the use of magnetic parameters, especially magnetic susceptibility (MS) as proxy. Monitoring plots were installed along roadside in areas with different climatic conditions and different traffic-specific activities (traffic density and speed, vehicle types, abrasion of tires, brake linings, petrol/diesel compounds and road maintenance). For monitoring we removed 10-15 cm of top soil at 1 m distance from the roadside edge and placed 30 plastic boxes there filled with clean quartz sand, to be sampled after regular intervals within two years. Preliminary data from the first year of monitoring are presented. Magnetic results revealed that a coarse grained magnetite-like phase is responsible for the enhancement of magnetic concentration. The mass-specific MS and concentration of pollutants (HM, PAH) all show a significant increase with time, however, there are obviously also seasonal and site-dependent effects which lead to more stable values over several months or even some decrease in the upper few cm due to migration into depth. Source identification indicates that the accumulated PAHs are primarily emissions from traffic. In order to be able to discriminate in between different kinds of transport and deposition (surface run off from the road and neighbouring soil material, splash water, air transport), we additionally established pillars at the roadside with clean quartz sampling boxes at different heights (surface, 0.5 m, 2 m). As a first surprising result we observed that the increase in the boxes at surface is not necessarily higher than at 0.5 m height. The results from our

  1. Phosphorylation of the viral coat protein regulates RNA virus infection

    Directory of Open Access Journals (Sweden)

    Hoover HS

    2016-11-01

    Full Text Available Haley S Hoover, C Cheng Kao Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA Abstract: Coat proteins (CPs are the most abundant protein produced during a viral infection. CPs have been shown to regulate the infection processes of RNA viruses, including RNA replication and gene expression. The numerous activities of the CP in infection are likely to require regulation, possibly through posttranslational modifications. Protein posttranslational modifications are involved in signal transduction, expanding and regulating protein function, and responding to changes in the environment. Accumulating evidence suggests that phosphorylation of viral CPs is involved in the regulation of the viral infection process from enabling virion disassembly to regulation of viral protein synthesis and replication. CP phosphorylation also affects viral trafficking and virion assembly. This review focuses on the regulatory roles that phosphorylation of CPs has in the life cycle of viruses with RNA genomes. Keywords: viral capsid protein, posttranslational modification, phosphorylation, protein–RNA interaction

  2. Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes.

    Science.gov (United States)

    Da Veiga, Cyrielle; Mezher, Joelle; Dumas, Philippe; Ennifar, Eric

    2016-01-01

    The success rate of nucleic acids/ligands co-crystallization can be significantly improved by performing preliminary biophysical analyses. Among suitable biophysical approaches, isothermal titration calorimetry (ITC) is certainly a method of choice. ITC can be used in a wide range of experimental conditions to monitor in real time the formation of the RNA- or DNA-ligand complex, with the advantage of providing in addition the complete binding profile of the interaction. Following the ITC experiment, the complex is ready to be concentrated for crystallization trials. This chapter describes a detailed experimental protocol for using ITC as a tool for monitoring RNA/small molecule binding, followed by co-crystallization.

  3. Factors influencing the cardiac MIBG accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Takatsu, Hisato; Fujiwara, Hisayoshi [Gifu Univ. (Japan). School of Medicine

    1997-02-01

    Following factors possibly influencing the cardiac MIBG accumulation were examined mainly in mice. 1. The specific activity of the MIBG (meta-iodo-benzyl guanidine) on the neuronal and non-neuronal fractions. 2. Motor restriction stress on MIBG accumulation and washout. 3. Loading and restriction of sodium chloride on the accumulation and effect of suppression of renin-angiotensin system. 4. Examinations in Dahl rats. 125I- or 131I-MIBG was intravenously administered to mice at 74 kBq. At 30 min or 4 hr after administration, mice were sacrificed and their left ventricles were dissected out for measurement of radioactivity in a liquid scintillation counter. Salt-sensitive and -resistant Dahl rats were given with 37 MBq of 123I-MIBG and cardiac radioactivity was measured externally for calculation of washout. Factors examined were found highly correlated with the accumulation of MIBG and measurement of its washout was considered useful for evaluating sympathetic activity. (K.H.)

  4. Rock bed heat accumulators. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Riaz, M.

    1977-12-01

    The principal objectives of the research program on rock bed heat accumulators (or RBHA) are: (1) to investigate the technical and economic feasibility of storing large amounts of thermal energy (in the tens of MWt range) at high temperature (up to 500/sup 0/C) over extended periods of time (up to 6 months) using native earth or rock materials; (2) to conduct studies to establish the performance characteristics of large rock bed heat accumulators at various power and temperature levels compatible with thermal conversion systems; and (3) to assess the materials and environmental problems associated with the operation of such large heat accumulators. Results of the study indicate that rock bed heat accumulators for seasonal storage are both technically and economically feasible, and hence could be exploited in various applications in which storage plays an essential role such as solar power and total energy systems, district and cogeneration heating systems.

  5. Accumulation of nickel in transgenic tobacco

    Science.gov (United States)

    Sidik, Nik Marzuki; Othman, Noor Farhan

    2013-11-01

    The accumulation of heavy metal Ni in the roots and leaves of four T1 transgenic lines of tobacco (T(1)20E, T(1)24C, T(1)18B1 and T(1)20B) expressing eiMT1 from E.indica was assessed. The aim of the study was to investigate the level of Ni accumulation in the leaves and roots of each transgenic lines and to evaluate the eligibility of the plants to be classified as a phytoremediation agent. All of the transgenic lines showed different ability in accumulating different metals and has translocation factor (TF) less than 1 (TFmetal treatment. Among the 4 transgenic lines, transgenic line T(1)24C showed the highest accumulation of Ni (251.9 ± 0.014 mg/kg) and the lowest TF value (TFT(1)24C=0.0875) at 60 ppm Ni.

  6. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

    Science.gov (United States)

    Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

  7. The RNA infrastructure: an introduction to ncRNA networks.

    Science.gov (United States)

    Collins, Lesley J

    2011-01-01

    The RNA infrastructure connects RNA-based functions. With transcription-to-translation processing forming the core of the network, we can visualise how RNA-based regulation, cleavage and modification are the backbone of cellular function. The key to interpreting the RNA-infrastructure is in understanding how core RNAs (tRNA, mRNA and rRNA) and other ncRNAs operate in a spatial-temporal manner, moving around the nucleus, cytoplasm and organelles during processing, or in response to environmental cues. This chapter summarises the concept of the RNA-infrastructure, and highlights examples of RNA-based networking within prokaryotes and eukaryotes. It describes how transcription-to-translation processes are tightly connected, and explores some similarities and differences between prokaryotic and eukaryotic RNA networking.

  8. NEURODEGENERATION WITH IRON ACCUMULATION TYPE1

    Directory of Open Access Journals (Sweden)

    Shrikhande D Y

    2010-03-01

    Full Text Available Neurodegeneration with iron accumulation type 1 is a rare degenerative disorder presenting with dementia and progressive extrapyramidal dysfunction. A 10 yrs old girl reported with complaints of difficulty in speech and involuntary movements. MRI Brain showed ‘eye of tiger appearance’ which is suggestive of neurodegeneration with iron accumulation type 1. Treatment is symptomatic and chelating agents have no effect. The disease is progressivelyfatal

  9. Evaluation of carrier-mediated siRNA delivery

    DEFF Research Database (Denmark)

    Colombo, Stefano; Nielsen, Hanne Mørck; Foged, Camilla

    2013-01-01

    RNA delivered by use of carriers remains an analytical challenge. The purpose of the present study was to optimize and validate an analytical protocol based on stem-loop reverse transcription quantitative polymerase chain reaction (RT qPCR) to quantitatively monitor the carrier-mediated intracellular si......RNA delivery. An in vitro cell culture model system expressing enhanced green fluorescent protein (EGFP) was used to develop the assay, which was based on the intracellular quantification of a full-length double-stranded Dicer substrate siRNA by stem-loop RT qPCR. The result is a well-documented protocol...

  10. Clusters of basic amino acids contribute to RNA binding and nucleolar localization of ribosomal protein L22.

    Directory of Open Access Journals (Sweden)

    Jennifer L Houmani

    Full Text Available The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80-93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80-93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA.

  11. Topology of RNA-RNA interaction structures

    CERN Document Server

    Andersen, Jørgen E; Penner, Robert C; Reidys, Christian M

    2011-01-01

    The topological filtration of interacting RNA complexes is studied and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that for two interacting RNAs, called interaction structures, there exist for fixed genus only finitely many irreducible shadows. This implies that for fixed genus there are only finitely many classes of interaction structures. In particular the simplest case of genus zero already provides the formalism for certain types of structures that occur in nature and are not covered by other filtrations. This case of genus zero interaction structures is already of practical interest, is studied here in detail and found to be expressed by a multiple context-free grammar extending the usual one for RNA secondary structures. We show that in $O(n^6)$ time and $O(n^4)$ space complexity, this grammar for genus zero interaction structures provides not only minimum free energy solutions but also the complete partit...

  12. Sucrose induces vesicle accumulation and autophagy.

    Science.gov (United States)

    Higuchi, Takahiro; Nishikawa, Jun; Inoue, Hiroko

    2015-04-01

    It has been shown that the treatment of mammalian cells with sucrose leads to vacuole accumulation associated with lysosomes and upregulation of lysosomal enzyme expression and activity. Autophagy is an evolutionarily conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes, thus it is probable that sucrose affects the autophagic activity. The role of sucrose in autophagy is unknown; however, another disaccharide, trehalose has been shown to induce autophagy. In the current study, we used mouse embryonic fibroblasts to investigate whether sucrose induces autophagy and whether vesicle formation is associated with autophagy. The results showed that sucrose induces autophagy while being accumulated within the endosomes/lysosomes. These vesicles were swollen and packed within the cytoplasm. Furthermore, trehalose and the trisaccharide raffinose, which are not hydrolyzed in mammalian cells, increased the rate of vesicles accumulation and LC3-II level (a protein marker of autophagy). However, fructose and maltose did not show the same effects. The correlation between the two processes, vesicle accumulation and autophagy induction, was confirmed by treatment of cells with sucrose plus invertase, or maltose plus acarbose-the α-glucosidase inhibitor-and by sucrose deprivation. Results also showed that vesicle accumulation was not affected by autophagy inhibition. Therefore, the data suggest that sucrose-induced autophagy through accumulation of sucrose-containing vesicles is caused by the absence of hydrolysis enzymes.

  13. Accumulation of radionuclides by lichen symbionts

    Energy Technology Data Exchange (ETDEWEB)

    Nifontova, M.G.; Kulikov, N.V. (AN SSSR, Sverdlovsk. Inst. Ehkologii Rastenij i Zhivotnykh)

    1983-01-01

    The aim of investigation is the quantitative estimation of ability and role of separate symbionts in the accumulation of radionuclides. As investigation volumes, durably cultivated green lichen alga Trebouxia erici and lichen fungi extracted from Cladonia rangiferina, Parmelia caperata and Acarospora fuscata are used. The accumulation of radioactive isotopes with fungi and seaweeds is estimated according to accumulation coefficients (AC) which are the ratio of radiation concentration in plants and agarized medium. Radionuclide content (/sup 90/Sr and /sup 137/Cs) is determined radiometrically. A special series of experiments is done to investigate radionuclide accumulation dependences with lichen seaweed and fungi on light conditions. It is shown that both symbionts of lichen-seaweed and fungus take part in the accumulation of radionuclide from outer medium (atmospheric fall-out and soil). However fungus component constituting the base of structural organization of thallus provides the greater part of radionuclides accumulated by the plant. Along with this the violation of viability of seaweed symbionts particularly in the case of light deficiency brings about the reduction of /sup 137/Cs sorption by seaweeds and tells on the total content of radiocesium in plant thallus.

  14. Control of mRNA stability during development of Dictyostelium discoideum.

    Science.gov (United States)

    Mangiarotti, G

    1989-01-01

    A large group of mRNA species (which are mainly pre-spore specific) accumulate only after the formation of multicellular aggregates. They are transcribed at a constant rate from the beginning of development and their accumulation is controlled by a 10-20-fold increase in their stability. This mRNA stabilization is dependent upon multicellularity. When aggregates are dispersed, the mRNAs are destabilized; if cells are allowed to reaggregate, the destabilization is reversed. Destabilization is not due to a selective exclusion of mRNA from polyribosomes, but is a primary control event. It does not require synthesis of new RNA or protein, but it may require an interaction between ribosome and the 5'-end of mRNA molecules.

  15. Small noncoding RNA modulates japanese encephalitis virus replication and translation in trans

    Directory of Open Access Journals (Sweden)

    Fan Yi-Hsin

    2011-11-01

    Full Text Available Abstract Background Sequence and structural elements in the 3'-untranslated region (UTR of Japanese encephalitis virus (JEV are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA which is collinear with the highly conserved regions of the 3'-UTR in JEV-infected cells. However, function of the sfRNA in JEV life cycle remains unknown. Results Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. Trans-acting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation. Conclusions Our results indicate that sfRNA modulates viral translation and replication in trans.

  16. Chaperoning 5S RNA assembly

    National Research Council Canada - National Science Library

    Madru, Clément; Lebaron, Simon; Blaud, Magali; Delbos, Lila; Pipoli, Juliana; Pasmant, Eric; Réty, Stéphane; Leulliot, Nicolas

    2015-01-01

    ...—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP...

  17. The Functions of RNA-Dependent RNA Polymerases in Arabidopsis

    Science.gov (United States)

    Willmann, Matthew R.; Endres, Matthew W.; Cook, Rebecca T.; Gregory, Brian D.

    2011-01-01

    One recently identified mechanism that regulates mRNA abundance is RNA silencing, and pioneering work in Arabidopsis thaliana and other genetic model organisms helped define this process. RNA silencing pathways are triggered by either self-complementary fold-back structures or the production of double-stranded RNA (dsRNA) that gives rise to small RNAs (smRNAs) known as microRNAs (miRNAs) or small-interfering RNAs (siRNAs). These smRNAs direct sequence-specific regulation of various gene transcripts, repetitive sequences, viruses, and mobile elements via RNA cleavage, translational inhibition, or transcriptional silencing through DNA methylation and heterochromatin formation. Early genetic screens in Arabidopsis were instrumental in uncovering numerous proteins required for these important regulatory pathways. Among the factors identified by these studies were RNA-dependent RNA polymerases (RDRs), which are proteins that synthesize siRNA-producing dsRNA molecules using a single-stranded RNA (ssRNA) molecule as a template. Recently, a growing body of evidence has implicated RDR-dependent RNA silencing in many different aspects of plant biology ranging from reproductive development to pathogen resistance. Here, we focus on the specific functions of the six Arabidopsis RDRs in RNA silencing, their ssRNA substrates and resulting RDR-dependent smRNAs, and the numerous biological functions of these proteins in plant development and stress responses. PMID:22303271

  18. Energy Monitoring

    DEFF Research Database (Denmark)

    Hansen, Claus T.; Madsen, Dines; Christiensen, Thomas

    Energy measurement has become an important aspect of our daily lives since we have learned that energy consumption, is one of the main source of global warming. Measuring instruments varies from a simple watt-meter to more sophisticated microprocessor control devices. The negative effects...... that fossil fuels induce on our environment has forced us to research renewable energy such as sunlight, wind etc. This new environmental awareness has also helped us to realize the importance of monitoring and controlling our energy use. The main purpose in this research is to introduce a more sophisticated...... but affordable way to monitor energy consumption of individuals or groups of home appliances. By knowing their consumption the utilization can be regulated for more efficient use. A prototype system has been constructed to demonstrate our idea....

  19. Energy Monitoring

    DEFF Research Database (Denmark)

    Hansen, Claus T.; Madsen, Dines; Christiensen, Thomas

    Energy measurement has become an important aspect of our daily lives since we have learned that energy consumption, is one of the main source of global warming. Measuring instruments varies from a simple watt-meter to more sophisticated microprocessor control devices. The negative effects...... that fossil fuels induce on our environment has forced us to research renewable energy such as sunlight, wind etc. This new environmental awareness has also helped us to realize the importance of monitoring and controlling our energy use. The main purpose in this research is to introduce a more sophisticated...... but affordable way to monitor energy consumption of individuals or groups of home appliances. By knowing their consumption the utilization can be regulated for more efficient use. A prototype system has been constructed to demonstrate our idea....

  20. Material monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Kotter, W.; Zirker, L.; Hancock, J.A.

    1995-11-01

    Waste Reduction Operations Complex (WROC) facilities are located at the Idaho National Engineering Laboratory (INEL). The overall goal for the Pollution Prevention/Waste Minimization Unit is to identify and establish the correct amount of waste generated so that it can be reduced. Quarterly, the INEL Pollution Prevention (P2) Unit compares the projected amount of waste generated per process with the actual amount generated. Examples of waste streams that would be addresses for our facility would include be are not limited to: Maintenance, Upgrades, Office and Scrap Metal. There are three potential sources of this variance: inaccurate identification of those who generate the waste; inaccurate identification of the process that generates the waste; and inaccurate measurement of the actual amount generated. The Materials Monitoring Program was proposed to identify the sources of variance and reduce the variance to an acceptable level. Prior to the implementation of the Material Monitoring Program, all information that was gathered and recorded was done so through an informal estimation of waste generated by various personnel concerned with each processes. Due to the inaccuracy of the prior information gathering system, the Material Monitoring Program was established. The heart of this program consists of two main parts. In the first part potential waste generators provide information on projected waste generation process. In the second part, Maintenance, Office, Scrap Metal and Facility Upgrade wastes from given processes is disposed within the appropriate bin dedicated to that process. The Material Monitoring Program allows for the more accurate gathering of information on the various waste types that are being generated quarterly.

  1. Panicovirus accumulation is governed by two membrane-associated proteins with a newly identified conserved motif that contributes to pathogenicity

    Directory of Open Access Journals (Sweden)

    Turina Massimo

    2006-03-01

    Full Text Available Abstract Panicum mosaic virus (PMV has a positive-sense, single-stranded RNA genome that serves as the mRNA for two 5'-proximal genes, p48 and p112. The p112 open reading frame (ORF has a GDD-motif, a feature of virus RNA-dependent RNA polymerases. Replication assays in protoplasts showed that p48 and p112 are sufficient for replication of PMV and its satellite virus (SPMV. Differential centrifugation of extracts from PMV-infected plants showed that the p48 and p112 proteins are membrane-associated. The same fractions exhibited RNA polymerase activity in vitro on viral RNA templates, suggesting that p48 and p112 represent the viral replication proteins. Moreover, we identified a domain spanning amino acids 306 to 405 on the p48 and p112 PMV ORFs that is common to the Tombusviridae. Alanine scanning mutagenesis of the conserved domain (CD revealed that several substitutions were lethal or severely debilitated PMV accumulation. Other substitutions did not affect RNA accumulation, yet they caused variable phenotypes suggestive of plant-dependent effects on systemic invasion and symptom induction. The mutants that were most debilitating to PMV replication were hydrophobic amino acids that we hypothesize are important for membrane localization and functional replicase activity.

  2. Simultaneous RNA-DNA FISH.

    Science.gov (United States)

    Lai, Lan-Tian; Meng, Zhenyu; Shao, Fangwei; Zhang, Li-Feng

    2016-01-01

    A highly useful tool for studying lncRNAs is simultaneous RNA-DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA-DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA.

  3. Stochastic Kinetics of Nascent RNA

    Science.gov (United States)

    Xu, Heng; Skinner, Samuel O.; Sokac, Anna Marie; Golding, Ido

    2016-09-01

    The stochastic kinetics of transcription is typically inferred from the distribution of RNA numbers in individual cells. However, cellular RNA reflects additional processes downstream of transcription, hampering this analysis. In contrast, nascent (actively transcribed) RNA closely reflects the kinetics of transcription. We present a theoretical model for the stochastic kinetics of nascent RNA, which we solve to obtain the probability distribution of nascent RNA per gene. The model allows us to evaluate the kinetic parameters of transcription from single-cell measurements of nascent RNA. The model also predicts surprising discontinuities in the distribution of nascent RNA, a feature which we verify experimentally.

  4. SIRT1 regulates accumulation of oxidized LDL in HUVEC via the autophagy-lysosomal pathway.

    Science.gov (United States)

    Zhang, Yanlin; Sun, Juanjuan; Yu, Xiaoyan; Shi, Luyao; Du, Wenxiu; Hu, Lifang; Liu, Chunfeng; Cao, Yongjun

    2016-01-01

    Autophagy is involved in the degradation of oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs). Sirtuin1 (SIRT1), a new anti-atherosclerotic factor, can induce autophagy in cardiac myocytes. In the present study, we observed the effect of SIRT1 on the accumulation of ox-LDL in HUVECs, and elucidated whether its effect is relative with the autophagy-lysosomal pathway. The results showed that treatment with either SIRT1 siRNA or SIRT1 inhibitor nicotinamide (NAM) increased Dil-labelled-ox-LDL (Dil-ox-LDL) accumulation in HUVECs, and the SIRT1 inducer resveratrol (RSV) decreased it. Knockdown of autophagy-related protein 5 or inhibit the lysosomal degradation by chloroquine (CQ) decreased the effect of RSV. In HUVECs with ox-LDL, expression of LC3II and LC3 puncta was decreased by treatment with SIRT1 siRNA or NAM, but increased by RSV treatment; sequestosome 1 p62 expression showed the opposite effects. Moreover, Dil-ox-LDL combined with SIRT1 siRNA or NAM showed a much smaller degree of overlap of Lamp1 or Cathepsin D with Dil-ox-LDL than in cells with Dil-ox-LDL alone, and RSV treatment resulted in a greater degree of overlap. These results suggest that SIRT1 can decrease the accumulation of ox-LDL in HUVECs, and this effect is related to the autophagy-lysosomal pathway.

  5. Monitoring assembly of ribonucleoprotein complexes by isothermal titration calorimetry

    OpenAIRE

    Recht, Michael I; Ryder, Sean P.; Williamson, James R.

    2008-01-01

    Isothermal titration calorimetry (ITC) is a useful technique to study RNA-protein interactions, as it provides the only method by which the thermodynamic parameters of free energy, enthalpy, and entropy can be directly determined. This chapter presents a general procedure for studying RNA-protein interactions using ITC, and gives specific examples for monitoring the binding of Caenorhabditis elegans GLD-1 STAR domain to TGE RNA and the binding of Aquifex aeolicus S6:S18 ribosomal protein hete...

  6. Local administration of siRNA through Microneedle: Optimization, Bio-distribution, Tumor Suppression and Toxicity

    Science.gov (United States)

    Tang, Tao; Deng, Yan; Chen, Jiao; Zhao, Yi; Yue, Ruifeng; Choy, Kwong Wai; Wang, Chi Chiu; Du, Quan; Xu, Yan; Han, Linxiao; Chung, Tony Kwok Hung

    2016-07-01

    Although RNA interference may become a novel therapeutic approach for cancer treatment, target-site accumulation of siRNA to achieve therapeutic dosage will be a major problem. Microneedle represents a better way to deliver siRNAs and we have evaluated for the first time the capability of a silicon microneedle array for delivery of Gapdh siRNA to the skin in vivo and the results showed that the microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. For the further study in this field, we evaluated the efficacy of the injectable microneedle device for local delivery of siRNA to the mouse xenograft. The results presented here indicate that local administration of siRNA through injectable microneedle could effectively deliver siRNA into the tumor region, and inhibit tumor progression without major adverse effects.

  7. Yeast nuclear RNA processing

    Institute of Scientific and Technical Information of China (English)

    Jade; Bernstein; Eric; A; Toth

    2012-01-01

    Nuclear RNA processing requires dynamic and intricately regulated machinery composed of multiple enzymes and their cofactors.In this review,we summarize recent experiments using Saccharomyces cerevisiae as a model system that have yielded important insights regarding the conversion of pre-RNAs to functional RNAs,and the elimination of aberrant RNAs and unneeded intermediates from the nuclear RNA pool.Much progress has been made recently in describing the 3D structure of many elements of the nuclear degradation machinery and its cofactors.Similarly,the regulatory mechanisms that govern RNA processing are gradually coming into focus.Such advances invariably generate many new questions,which we highlight in this review.

  8. Oxidative stress affects FET proteins localization and alternative pre-mRNA processing in cellular models of ALS.

    Science.gov (United States)

    Svetoni, Francesca; Caporossi, Daniela; Paronetto, Maria Paola

    2014-10-01

    FUS/TLS, EWS and TAF15 are members of the FET family of DNA and RNA binding proteins, involved in multiple steps of DNA and RNA processing and implicated in the regulation of gene expression and cell-signaling. All members of the FET family contribute to human pathologies, as they are involved in sarcoma translocations and neurodegenerative diseases. Mutations in FUS/TLS, in EWSR1 and in TAF15 genescause Amyotrophic Lateral Sclerosis (ALS), a fatal human neurodegenerative disease that affects primarily motor neurons and is characterized by the progressive loss of motor neurons and degradation of the neuromuscular junctions.ALS-associated FET mutations cause FET protein relocalization into cytoplasmic aggregates, thus impairing their normal function. Protein aggregation has been suggested as a co-opting factor during the disease pathogenesis. Cytoplasmic mislocalization of FET proteins contributes to the formation of cytoplasmic aggregates that may alter RNA processing and initiate motor neuron degeneration. Interestingly, oxidative stress, which is implicated in the pathogenesis of ALS, triggers the accumulation of mutant FUS in cytoplasmic stress granules where it binds and sequester wild-type FUS.In order to evaluate the role of FET proteins in ALS and their involvement in the response to oxidative stress, we have developed cellular models of ALS expressing ALS-related FET mutants in neuroblastoma cell lines. Upon treatment with sodium arsenite, cells were analysed by immunofluorescence to monitor the localization of wild-type and mutated FET proteins. Furthermore, we have characterized signal transduction pathways and cell survival upon oxidative stress in our cellular models of ALS. Interestingly, we found that EWS mutant proteins display a different localization from FUS mutants and neither wild-type nor mutated EWS protein translocate into stress granules upon oxidative stress treatment. Collectively, our data provide a new link between the oxidative stress

  9. SPATIAL AND TEMPORAL ACCUMULATION OF MESSENGER-RNAS ENCODING 2 COMMON LIGNIN PEROXIDASES IN PHANEROCHAETE-CHRYSOSPORIUM

    NARCIS (Netherlands)

    MOUKHA, SM; WOSTEN, HAB; MYLIUS, EJ; ASTHER, M; WESSELS, JGH

    Accumulation of peroxidases and their mRNAs was localized in colonies of Phanerochaete chrysosporium sandwiched between perforated polycarbonate membranes. Northern (RNA) blot analyses of colonial rings and in situ hybridizations with specific probes for manganese(II)-dependent peroxidase (MnP-1)

  10. An oleaginous bacterium that intrinsically accumulates long-chain free Fatty acids in its cytoplasm.

    Science.gov (United States)

    Katayama, Taiki; Kanno, Manabu; Morita, Naoki; Hori, Tomoyuki; Narihiro, Takashi; Mitani, Yasuo; Kamagata, Yoichi

    2014-02-01

    Medium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family Erysipelotrichaceae in the phylum Firmicutes but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited β-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.

  11. Sensing of RNA viruses

    DEFF Research Database (Denmark)

    Jensen, Søren; Thomsen, Allan Randrup

    2012-01-01

    pathogen-associated molecular patterns have emerged in great detail. This review presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecular structures these receptors sense, and the involved downstream signaling pathways.......Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral...

  12. Three Acyltransferases and Nitrogen-responsive Regulator Are Implicated in Nitrogen Starvation-induced Triacylglycerol Accumulation in Chlamydomonas*

    Science.gov (United States)

    Boyle, Nanette R.; Page, Mark Dudley; Liu, Bensheng; Blaby, Ian K.; Casero, David; Kropat, Janette; Cokus, Shawn J.; Hong-Hermesdorf, Anne; Shaw, Johnathan; Karpowicz, Steven J.; Gallaher, Sean D.; Johnson, Shannon; Benning, Christoph; Pellegrini, Matteo; Grossman, Arthur; Merchant, Sabeeha S.

    2012-01-01

    Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions. PMID:22403401

  13. Reduction of polygalacturonase activity in tomato fruit by antisense RNA.

    Science.gov (United States)

    Sheehy, R E; Kramer, M; Hiatt, W R

    1988-12-01

    Polygalacturonase [PG; poly(1,4-alpha-D-galacturonide) glycanhydrolase; EC 3.2.1.15] is expressed in tomato only during the ripening stage of fruit development. PG becomes abundant during ripening and has a major role in cell wall degradation and fruit softening. Tomato plants were transformed to produce antisense RNA from a gene construct containing the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in reverse orientation. The construct was integrated into the tomato genome by Agrobacterium-mediated transformation. The constitutive synthesis of PG antisense RNA in transgenic plants resulted in a substantial reduction in the levels of PG mRNA and enzymatic activity in ripening fruit. The steady-state levels of PG antisense RNA in green fruit of transgenic plants were lower than the levels of PG mRNA normally attained during ripening. However, analysis of transcription in isolated nuclei demonstrated that the antisense RNA construct was transcribed at a higher rate than the tomato PG gene(s). Analysis of fruit from transgenic plants demonstrated a reduction in PG mRNA and enzymatic activity of 70-90%. The reduction in PG activity did not prevent the accumulation of the red pigment lycopene.

  14. Regulation of small RNA stability: methylation and beyond

    Institute of Scientific and Technical Information of China (English)

    Lijuan Ji; Xuemei Chen

    2012-01-01

    As central components of RNA silencing,small RNAs play diverse and important roles in many biological processes in eukaryotes.Aberrant reduction or elevation in the levels of small RNAs is associated with many developmental and physiological defects.The in vivo levels of small RNAs are precisely regulated through modulating the rates of their biogenesis and turnover.2'-O-methylation on the 3' terminal ribose is a major mechanism that increases the stability of small RNAs.The small RNA methyltransferase HUA ENHANCER1 (HEN1) and its homologs methylate microRNAs and small interfering RNAs (siRNAs) in plants,Piwi-interacting RNAs (piRNAs) in animals,and siRNAs in Drosophila.3' nucleotide addition,especially uridylation,and 3'-5' exonucleolytic degradation are major mechanisms that turnover small RNAs.Other mechanisms impacting small RNA stability include complementary RNAs,cis-elements in small RNA sequences and RNA-binding proteins.Investigations are ongoing to further understand how small RNA stability impacts their accumulation in vivo in order to improve the utilization of RNA silencing in biotechnology and therapeutic applications.

  15. Enhancing endosomal escape for nanoparticle mediated siRNA delivery

    Science.gov (United States)

    Ma, Da

    2014-05-01

    Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

  16. [Synthesis of messenger-like RNA in plasma cels producing different clases of immunoglobulins].

    Science.gov (United States)

    Nikitin, A V; Babichev, V A

    1976-01-01

    Electrophoretic analysis of the nuclear poly A RNA cells of the MOPC 21, MOPC 406 and MOPC 41 plasmocytomas demonstrated a similarity in their distribution by the mol wt. Kinetics of the label accumulation in the nuclear poly A RNA of different plasma cells was unitypical. Individual peculiarities of the distribution of the cytoplasmic poly A RNA were expressed for each individual line of the myeloma cells. The majority of the molecules of the heterogenous RNA in the plasma cells were subjected to posttranscription polyadenylation.

  17. Reversal of multidrug resistance by small interfering RNA (siRNA) in doxorubicin-resistant MCF-7 breast cancer cells.

    Science.gov (United States)

    Dönmez, Yaprak; Gündüz, Ufuk

    2011-03-01

    Resistance to anticancer drugs is a serious obstacle to cancer chemotherapy. A common form of multidrug resistance (MDR) is caused by the overexpression of transmembrane transporter proteins P-glycoprotein (P-gp) and multidrug resistance-associated protein-1 (MRP1), encoded by MDR1 and MRP1 genes, respectively. These proteins lead to reduced intracellular drug concentration and decreased cytotoxicity by means of their ability to pump the drugs out of the cells. Breast cancer tumor resistance is mainly associated with overexpression of P-gp/MDR1. Although some chemical MDR modulators aim to overcome MDR by interfering functioning of P-gp, their toxicities limit their usage in clinics. Consequently, RNA interference mediated sequence specific inhibition of the expression of P-gp/MDR1 mRNA may be an efficient tool to reverse MDR phenotype and increase the success of chemotherapy. Aim of this study was resensitizing doxorubicin-resistant breast cancer cells to anticancer agent doxorubicin by selective downregulation of P-gp/MDR1 mRNA. The effect of the selected MDR1 siRNA, and MRP1 expression after MDR1 silencing was determined by qPCR analysis. Intracellular drug accumulation and localization was investigated by confocal laser scanning microscopy after treatment with MDR1 siRNA. XTT cell proliferation assay was performed to determine the effect of MDR1 silencing on doxorubicin sensitivity. The results demonstrated that approximately 90% gene silencing occurred by the selected siRNA targeting MDR1 mRNA. However, the level of MRP1 mRNA did not change after MDR1 downregulation. Silencing of P-gp encoding MDR1 gene resulted in almost complete restoration of the intracellular doxorubicin accumulation and relocalization of the drug in the nuclei. Introduction of siRNA resulted in about 70% resensitization to doxorubicin. Selected siRNA duplex was shown to effectively inhibit MDR1 gene expression, restore doxorubicin accumulation and localization, and enhance

  18. Cotranslational Coat Protein-Mediated Inhibition of Potyviral RNA Translation

    Science.gov (United States)

    Besong-Ndika, Jane; Ivanov, Konstantin I.; Hafrèn, Anders; Michon, Thierry

    2015-01-01

    ABSTRACT Potato virus A (PVA) is a single-stranded positive-sense RNA virus and a member of the family Potyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating in trans and CP translated from viral RNA in cis are required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection. IMPORTANCE The main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the

  19. Patterns and regulation of ribosomal RNA transcription in Borrelia burgdorferi

    Directory of Open Access Journals (Sweden)

    Schwartz Ira

    2011-01-01

    Full Text Available Abstract Background Borrelia burgdorferi contains one 16S and two tandem sets of 23S-5S ribosomal (r RNA genes whose patterns of transcription and regulation are unknown but are likely to be critical for survival and persistence in its hosts. Results RT-PCR of B. burgdorferi N40 and B31 revealed three rRNA region transcripts: 16S rRNA-alanine transfer RNA (tRNAAla; tRNAIle; and both sets of 23S-5S rRNA. At 34°C, there were no differences in growth rate or in accumulation of total protein, DNA and RNA in B31 cultured in Barbour-Stoenner-Kelly (BSK-H whether rabbit serum was present or not. At 23°C, B31 grew more slowly in serum-containing BSK-H than at 34°C. DNA per cell was higher in cells in exponential as compared to stationary phase at either temperature; protein per cell was similar at both temperatures in both phases. Similar amounts of rRNA were produced in exponential phase at both temperatures, and rRNA was down-regulated in stationary phase at either temperature. Interestingly, a relBbu deletion mutant unable to generate (pppGpp did not down-regulate rRNA at transition to stationary phase in serum-containing BSK-H at 34°C, similar to the relaxed phenotype of E. coli relA mutants. Conclusions We conclude that rRNA transcription in B. burgdorferi is complex and regulated both by growth phase and by the stringent response but not by temperature-modulated growth rate.

  20. Technology monitoring; Technologie-Monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Eicher, H.; Rigassi, R. [Eicher und Pauli AG, Liestal (Switzerland); Ott, W. [Econcept AG, Zuerich (Switzerland)

    2003-07-01

    This study made for the Swiss Federal Office of Energy (SFOE) examines ways of systematically monitoring energy technology development and the cost of such technologies in order to pave the way to a basis for judging the economic development of new energy technologies. Initial results of a survey of the past development of these technologies are presented and estimates are made of future developments in the areas of motor-based combined heat and power systems, fuel-cell heating units for single-family homes and apartment buildings, air/water heat pumps for new housing projects and high-performance thermal insulation. The methodology used for the monitoring and analysis of the various technologies is described. Tables and diagrams illustrate the present situation and development potential of various fields of technology.

  1. [Ribosomal RNA Evolution

    Science.gov (United States)

    1997-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith

  2. RNA STRAND: The RNA Secondary Structure and Statistical Analysis Database

    Directory of Open Access Journals (Sweden)

    Andronescu Mirela

    2008-08-01

    Full Text Available Abstract Background The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures. Results In this paper we describe RNA STRAND – the RNA secondary STRucture and statistical ANalysis Database, a curated database containing known secondary structures of any type and organism. Our new database provides a wide collection of known RNA secondary structures drawn from public databases, searchable and downloadable in a common format. Comprehensive statistical information on the secondary structures in our database is provided using the RNA Secondary Structure Analyser, a new tool we have developed to analyse RNA secondary structures. The information thus obtained is valuable for understanding to which extent and with which probability certain structural motifs can appear. We outline several ways in which the data provided in RNA STRAND can facilitate research on RNA structure, including the improvement of RNA energy models and evaluation of secondary structure prediction programs. In order to keep up-to-date with new RNA secondary structure experiments, we offer the necessary tools to add solved RNA secondary structures to our database and invite researchers to contribute to RNA STRAND. Conclusion RNA STRAND is a carefully assembled database of trusted RNA secondary structures, with easy on-line tools for searching, analyzing and downloading user selected entries, and is publicly available at http://www.rnasoft.ca/strand.

  3. Microbial detection with low molecular weight RNA

    Science.gov (United States)

    Kourentzi, K. D.; Fox, G. E.; Willson, R. C.

    2001-01-01

    The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

  4. Accumulation of swimming bacteria near an interface

    Science.gov (United States)

    Tang, Jay; Li, Guanglai

    2012-11-01

    Microbes inhabit planet earth over billions of years and have adapted to diverse physical environment of water, soil, and particularly at or near interfaces. We focused our attention on the locomotion of Caulobacter crescentus, a singly flagellated bacterium, at the interface of water/solid or water/air. We measured the distribution of a forward swimming strain of C. crescentus near a surface using a three-dimensional tracking technique based on dark field microscopy and found that the swimming bacteria accumulate heavily within a micrometer from the surface. We attribute this accumulation to frequent collisions of the swimming cells with the surface, causing them to align parallel to the surface as they continually move forward. The extent of accumulation at the steady state is accounted for by balancing alignment caused by these collisions with rotational Brownian motion of the micrometer-sized bacteria. We performed a simulation based on this model, which reproduced the measured results. Additional simulations demonstrate the dependence of accumulation on swimming speed and cell size, showing that longer and faster cells accumulate more near a surface than shorter and slower ones do. The overarching goal of our study is to describe interfacial microbial behavior through detailed analysis of their motion. We acknowledge support by NSF PHY 1058375.

  5. Branched RNA: A New Architecture for RNA Interference

    Directory of Open Access Journals (Sweden)

    Anna Aviñó

    2011-01-01

    Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

  6. Verification of chemical evolution of RNA under hydrothermal environments on the primitive Earth

    Directory of Open Access Journals (Sweden)

    Kawamura Kunio

    2015-01-01

    Full Text Available The RNA World hypothesis proposes that primitive forms of life used polymers resembling RNA both as catalysts and as carriers of genetic information. It has also been suggested that the origin of life occurred in hydrothermal conditions, but this implies that the ester bonds of nucleic acids are sufficiently stable to survive in aqueous conditions at elevated temperatures. Here, we summarize the results of experimental tests of RNA in simulated hydrothermal conditions in which stability is monitored at elevated temperatures and pressures. This perspective provides insight into the evolutionary pathway from small nucleotides to functional RNA molecules and the feasibility of RNA-based life.

  7. Incorporation of radiometric tracers in peat and implications for estimating accumulation rates.

    Science.gov (United States)

    Hansson, Sophia V; Kaste, James M; Olid, Carolina; Bindler, Richard

    2014-09-15

    Accurate dating of peat accumulation is essential for quantitatively reconstructing past changes in atmospheric metal deposition and carbon burial. By analyzing fallout radionuclides (210)Pb, (137)Cs, (241)Am, and (7)Be, and total Pb and Hg in 5 cores from two Swedish peatlands we addressed the consequence of estimating accumulation rates due to downwashing of atmospherically supplied elements within peat. The detection of (7)Be down to 18-20 cm for some cores, and the broad vertical distribution of (241)Am without a well-defined peak, suggest some downward transport by percolating rainwater and smearing of atmospherically deposited elements in the uppermost peat layers. Application of the CRS age-depth model leads to unrealistic peat mass accumulation rates (400-600 g m(-2) yr(-1)), and inaccurate estimates of past Pb and Hg deposition rates and trends, based on comparisons to deposition monitoring data (forest moss biomonitoring and wet deposition). After applying a newly proposed IP-CRS model that assumes a potential downward transport of (210)Pb through the uppermost peat layers, recent peat accumulation rates (200-300 g m(-2) yr(-1)) comparable to published values were obtained. Furthermore, the rates and temporal trends in Pb and Hg accumulation correspond more closely to monitoring data, although some off-set is still evident. We suggest that downwashing can be successfully traced using (7)Be, and if this information is incorporated into age-depth models, better calibration of peat records with monitoring data and better quantitative estimates of peat accumulation and past deposition are possible, although more work is needed to characterize how downwashing may vary between seasons or years.

  8. Monitoring microcirculation.

    Science.gov (United States)

    Ocak, Işık; Kara, Atila; Ince, Can

    2016-12-01

    The clinical relevance of microcirculation and its bedside observation started gaining importance in the 1990s since the introduction of hand-held video microscopes. From then, this technology has been continuously developed, and its clinical relevance has been established in more than 400 studies. In this paper, we review the different types of video microscopes, their application techniques, the microcirculation of different organ systems, the analysis methods, and the software and scoring systems. The main focus of this review will be on the state-of-art technique, CytoCam-incident dark-field imaging, and the most recent technological and technical updates concerning microcirculation monitoring.

  9. [Accumulation of polycyclic arenes in Baltic Sea algae].

    Science.gov (United States)

    Veldre, I A; Itra, A R; Paal'me, L P; Kukk, Kh A

    1985-01-01

    The paper presents data on the level of benzo(a)pyrene (BP) and some other polycyclic arenes in alga and phanerogam specimens from different gulfs of the Baltic Sea. Algae were shown to absorb BP from sea water. The mean concentration of BP in sea water was under 0.004 microgram/1, while in algae it ranged 0.1-21.2 micrograms/kg dry weight. Algae accumulate BP to a higher degree than phanerogams. The highest concentrations of BP were found in algae Enteromorpha while the lowest ones in Furcellaria. In annual green algae, BP level was higher in autumn, i. e. at the end of vegetation period, than in spring. Brown algae Fucus vesiculosus is recommended for monitoring polycyclic arene pollution in the area from Vormsi Island to Käsmu and green algae Cladophora or Enteromorpha in the eastern part of the Finnish Gulf.

  10. Shaping tRNA

    Science.gov (United States)

    Priano, Christine

    2013-01-01

    This model-building activity provides a quick, visual, hands-on tool that allows students to examine more carefully the cloverleaf structure of a typical tRNA molecule. When used as a supplement to lessons that involve gene expression, this exercise reinforces several concepts in molecular genetics, including nucleotide base-pairing rules, the…

  11. The RNA interference revolution

    Directory of Open Access Journals (Sweden)

    G. Lenz

    2005-12-01

    Full Text Available The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

  12. RNA and RNP as Building Blocks for Nanotechnology and Synthetic Biology.

    Science.gov (United States)

    Ohno, Hirohisa; Saito, Hirohide

    2016-01-01

    Recent technologies that aimed to elucidate cellular function have revealed essential roles for RNA molecules in living systems. Our knowledge concerning functional and structural information of naturally occurring RNA and RNA-protein (RNP) complexes is increasing rapidly. RNA and RNP interaction motifs are structural units that function as building blocks to constitute variety of complex structures. RNA-central synthetic biology and nanotechnology are constructive approaches that employ the accumulated information and build synthetic RNA (RNP)-based circuits and nanostructures. Here, we describe how to design and construct synthetic RNA (RNP)-based devices and structures at the nanometer-scale for biological and future therapeutic applications. RNA/RNP nanostructures can also be utilized as the molecular scaffold to control the localization or interactions of target molecule(s). Moreover, RNA motifs recognized by RNA-binding proteins can be applied to make protein-responsive translational "switches" that can turn gene expression "on" or "off" depending on the intracellular environment. This "synthetic RNA and RNP world" will expand tools for nanotechnology and synthetic biology. In addition, these reconstructive approaches would lead to a greater understanding of building principle in naturally occurring RNA/RNP molecules and systems. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Cardiac event monitors

    Science.gov (United States)

    ... ECG) - ambulatory; Continuous electrocardiograms (EKGs); Holter monitors; Transtelephonic event monitors ... attached. You can carry or wear a cardiac event monitor up to 30 days. You carry the ...

  14. Cohabitation history, marriage, and wealth accumulation.

    Science.gov (United States)

    Vespa, Jonathan; Painter, Matthew A

    2011-08-01

    This study extends research on the relationship between wealth accumulation and union experiences, such as marriage and cohabitation. Using data from the National Longitudinal Survey of Youth 1979, we explore the wealth trajectories of married individuals in light of their premarital cohabitation histories. Over time, marriage positively correlates with wealth accumulation. Most married persons with a premarital cohabitation history have wealth trajectories that are indistinguishable from those without cohabitation experience, with one exception: individuals who marry their one and only cohabiting partner experience a wealth premium that is twice as large as that for married individuals who never cohabited prior to marrying. Results remain robust over time despite cohabiters' selection out of marriage, yet vary by race/ethnicity. We conclude that relationship history may shape long-term wealth accumulation, and contrary to existing literature, individuals who marry their only cohabiting partners experience a beneficial marital outcome. It is therefore important to understand the diversity of cohabitation experiences among the married.

  15. A transgenic zebrafish model for monitoring xbp1 splicing and endoplasmic reticulum stress in vivo.

    Science.gov (United States)

    Li, Junling; Chen, Zhiliang; Gao, Lian-Yong; Colorni, Angelo; Ucko, Michal; Fang, Shengyun; Du, Shao Jun

    2015-08-01

    Accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) triggers ER stress that initiates unfolded protein response (UPR). XBP1 is a transcription factor that mediates one of the key signaling pathways of UPR to cope with ER stress through regulating gene expression. Activation of XBP1 involves an unconventional mRNA splicing catalyzed by IRE1 endonuclease that removes an internal 26 nucleotides from xbp1 mRNA transcripts in the cytoplasm. Researchers have taken advantage of this unique activation mechanism to monitor XBP1 activation, thereby UPR, in cell culture and transgenic models. Here we report a Tg(ef1α:xbp1δ-gfp) transgenic zebrafish line to monitor XBP1 activation using GFP as a reporter especially in zebrafish oocytes and developing embryos. The Tg(ef1α:xbp1δ-gfp) transgene was constructed using part of the zebrafish xbp1 cDNA containing the splicing element. ER stress induced splicing results in the cDNA encoding a GFP-tagged partial XBP1 without the transactivation activation domain (XBP1Δ-GFP). The results showed that xbp1 transcripts mainly exist as the spliced active isoform in unfertilized oocytes and zebrafish embryos prior to zygotic gene activation at 3 hours post fertilization. A strong GFP expression was observed in unfertilized oocytes, eyes, brain and skeletal muscle in addition to a weak expression in the hatching gland. Incubation of transgenic zebrafish embryos with (dithiothreitol) DTT significantly induced XBP1Δ-GFP expression. Collectively, these studies unveil the presence of maternal xbp1 splicing in zebrafish oocytes, fertilized eggs and early stage embryos. The Tg(ef1α:xbp1δ-gfp) transgenic zebrafish provides a useful model for in vivo monitoring xbp1 splicing during development and under ER stress conditions.

  16. Molecular Beacons: Powerful Tools for Imaging RNA in Living Cells

    Science.gov (United States)

    Monroy-Contreras, Ricardo; Vaca, Luis

    2011-01-01

    Recent advances in RNA functional studies highlights the pivotal role of these molecules in cell physiology. Diverse methods have been implemented to measure the expression levels of various RNA species, using either purified RNA or fixed cells. Despite the fact that fixed cells offer the possibility to observe the spatial distribution of RNA, assays with capability to real-time monitoring RNA transport into living cells are needed to further understand the role of RNA dynamics in cellular functions. Molecular beacons (MBs) are stem-loop hairpin-structured oligonucleotides equipped with a fluorescence quencher at one end and a fluorescent dye (also called reporter or fluorophore) at the opposite end. This structure permits that MB in the absence of their target complementary sequence do not fluoresce. Upon binding to targets, MBs emit fluorescence, due to the spatial separation of the quencher and the reporter. Molecular beacons are promising probes for the development of RNA imaging techniques; nevertheless much work remains to be done in order to obtain a robust technology for imaging various RNA molecules together in real time and in living cells. The present work concentrates on the different requirements needed to use successfully MB for cellular studies, summarizing recent advances in this area. PMID:21876785

  17. Molecular Beacons: Powerful Tools for Imaging RNA in Living Cells

    Directory of Open Access Journals (Sweden)

    Ricardo Monroy-Contreras

    2011-01-01

    Full Text Available Recent advances in RNA functional studies highlights the pivotal role of these molecules in cell physiology. Diverse methods have been implemented to measure the expression levels of various RNA species, using either purified RNA or fixed cells. Despite the fact that fixed cells offer the possibility to observe the spatial distribution of RNA, assays with capability to real-time monitoring RNA transport into living cells are needed to further understand the role of RNA dynamics in cellular functions. Molecular beacons (MBs are stem-loop hairpin-structured oligonucleotides equipped with a fluorescence quencher at one end and a fluorescent dye (also called reporter or fluorophore at the opposite end. This structure permits that MB in the absence of their target complementary sequence do not fluoresce. Upon binding to targets, MBs emit fluorescence, due to the spatial separation of the quencher and the reporter. Molecular beacons are promising probes for the development of RNA imaging techniques; nevertheless much work remains to be done in order to obtain a robust technology for imaging various RNA molecules together in real time and in living cells. The present work concentrates on the different requirements needed to use successfully MB for cellular studies, summarizing recent advances in this area.

  18. Landscape Evolution and Carbon Accumulation: Uniformitarianism Revisited

    Science.gov (United States)

    Rosenbloom, N. A.; Harden, J. W.; Neff, J. C.; Schimel, D. S.

    2003-12-01

    What is the role of hillslope transport in long-term carbon accumulation in soils? How do parent material, climate, and landform interact to produce the landscapes we observe today and to what extent can we use present day conditions to infer the dominant processes of the past? We use the CREEP [Rosenbloom, N.A. et al., 2001] process-response model to ask these questions, exploring the time-evolution of landscape form, soil distribution, and carbon accumulation in an undisturbed prairie site in western Iowa [Harden, J.W. et al., 2002]. The CREEP model simulates differential transport of soil particles, blanket deposition of atmospheric 10Be with eolian dust, and passive advection of soil carbon and 10Be, enabling the preferential enrichment and burial of rapidly moving soil constituents. By comparing landscape-wide average accumulations of 10Be to borehole observations at three hillslope positions, we conclude that the distribution of clay-adsorbed 10Be cannot be explained by co-transport with clay particles alone. Rather, 10Be appears to behave as a more complex tracer than originally assumed, requiring an explicit, independent parameterization of wet deposition and transport. By comparison, model carbon accumulation strongly reflects patterns of clay redistribution indicating that in situ carbon turnover is faster than redistribution. Observed vertical distributions of soil properties, including 10Be, could only be explained by assuming variations in deposition and erosion rates, specifically periods of accumulation, followed by periods of transport. This effect might not be apparent if only landform shape, geometry, and soil depth were considered and vertical distributions of soil properties were not explicitly simulated. The current landscape reflects a history of strong shifts in erosion and accumulation rates that cannot be simulated using a uniform parameterization of long-term landscape-evolution processes.

  19. Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

    Energy Technology Data Exchange (ETDEWEB)

    Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S., E-mail: Connie.s.schmaljohn.civ@mail.mil

    2015-12-15

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Rift Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.

  20. The ticking tail: daily oscillations in mRNA poly(A) tail length drive circadian cycles in protein synthesis.

    Science.gov (United States)

    Gotic, Ivana; Schibler, Ueli

    2012-12-15

    In this issue of Genes & Development, Kojima and colleagues (pp. 2724-2736) examined the impact of mRNA poly(A) tail length on circadian gene expression. Their study demonstrates how dynamic changes in transcript poly(A) tail length can lead to rhythmic protein expression, irrespective of whether mRNA accumulation is circadian or constitutive.

  1. Accumulation of nanocarriers in the ovary

    DEFF Research Database (Denmark)

    Schädlich, Andreas; Hoffmann, Stefan; Mueller, Thomas;

    2012-01-01

    Several nanocarrier systems are frequently used in modern pharmaceutical therapies. Within this study a potential toxicity risk of all nanoscaled drug delivery systems was found. An accumulation of several structurally different nanocarriers but not of soluble polymers was detected in rodent...... vivo multispectral fluorescence imaging and confocal laser scanning microscopy. The findings of this study emphasise the role of early and comprehensive in vivo studies in pharmaceutical research. Nanocarrier accumulation in the ovaries may also comprise an important toxicity issue in humans...... but the results might as well open a new field of targeted ovarian therapies....

  2. Stationarity Testing of Accumulated Ethernet Traffic

    Directory of Open Access Journals (Sweden)

    Zhiping Lu

    2013-01-01

    Full Text Available We investigate the stationarity property of the accumulated Ethernet traffic series. We applied several widely used stationarity and unit root tests, such as Dickey-Fuller test and its augmented version, Phillips-Perron test, as well as the Kwiatkowski-Phillips-Schmidt-Shin test and some of its generalizations, to the assessment of the stationarity of the traffic traces at the different time scales. The quantitative results in this research provide evidence that when the time scale increases, the accumulated traffic series are more stationary.

  3. Accumulation of Norfloxacin by Bacteroides fragilis

    Science.gov (United States)

    Ricci, Vito; Piddock, Laura J. V.

    2000-01-01

    The accumulation of norfloxacin by Bacteroides fragilis NCTC 9343 was determined by the modified fluorescence method. The time required to achieve a steady-state concentration (SSC) after allowing B. fragilis to accumulate norfloxacin in an aerobic or an anaerobic environment was ∼2 min; the SSC achieved in air was 90.28 ± 9.32 ng of norfloxacin/mg (dry weight) of cells, and that achieved anaerobically was 98.45 ± 3.7 ng of norfloxacin/mg (dry weight) of cells. Initial rates of accumulation were determined with a range of external concentrations, as up to 8 μg/ml the concentration of norfloxacin accumulated increased proportionally to the external concentration, 12.13 ng/mg (dry weight) of cells per μg of exogenous norfloxacin per ml. At concentrations above 10 μg/ml no increase in the rate of norfloxacin accumulation was observed. From the kinetic data, a Lineweaver-Burk plot calculated a Km of 5.03 μg/ml and a Vmax of 25.1 ng of norfloxacin/s. With an increase in temperature of between 0 and 30°C, the concentration of norfloxacin accumulated also increased proportionally at 4.722 ng of norfloxacin/mg (dry weight) of cells/°C. At low concentrations of glucose (norfloxacin accumulated was decreased. With the addition of 100 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) the mean SSC of norfloxacin was increased to 116 ± 7.01 ng of norfloxacin/mg (dry weight) of cells; glucose had no significant effect in the presence of CCCP. Magnesium chloride (20 mM) decreased the SSC of norfloxacin to 40.5 ± 3.76 ng of norfloxacin per mg (dry weight) of cells. These data suggest that the mechanism of accumulation of norfloxacin by B. fragilis is similar to that of aerobic bacteria and that the fluoresence procedure is suitable for use with an anaerobic bacterium. PMID:10952580

  4. FTO, RNA epigenetics and epilepsy

    OpenAIRE

    2012-01-01

    Several recent landmark papers describing N6-methyladenosine (m6A) RNA modifications have provided valuable new insights as to the importance of m6A in the RNA transcriptome and in furthering the understanding of RNA epigenetics. One endogenous enzyme responsible for demethylating RNA m6A, FTO, is highly expressed in the CNS and is likely involved in mRNA metabolism, splicing or other nuclear RNA processing events. microRNAs (miRNAs), a family of small, non-coding transcripts that bind to tar...

  5. Messenger RNA (mRNA) nanoparticle tumour vaccination

    Science.gov (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  6. RNA tectonics (tectoRNA) for RNA nanostructure design and its application in synthetic biology.

    Science.gov (United States)

    Ishikawa, Junya; Furuta, Hiroyuki; Ikawa, Yoshiya

    2013-01-01

    RNA molecules are versatile biomaterials that act not only as DNA-like genetic materials but also have diverse functions in regulation of cellular biosystems. RNA is capable of regulating gene expression by sequence-specific hybridization. This feature allows the design of RNA-based artificial gene regulators (riboregulators). RNA can also build complex two-dimensional (2D) and 3D nanostructures, which afford protein-like functions and make RNA an attractive material for nanobiotechnology. RNA tectonics is a methodology in RNA nanobiotechnology for the design and construction of RNA nanostructures/nanoobjects through controlled self-assembly of modular RNA units (tectoRNAs). RNA nanostructures designed according to the concept of RNA tectonics are also attractive as tools in synthetic biology, but in vivo RNA tectonics is still in the early stages. This review presents a summary of the achievements of RNA tectonics and its related researches in vitro, and also introduces recent developments that facilitated the use of RNA nanostructures in bacterial cells.

  7. Is oxidative stress related to cadmium accumulation in the Mollusc Crassostrea angulata?

    Energy Technology Data Exchange (ETDEWEB)

    Macías-Mayorga, Dayanara, E-mail: dayanara.macias@uleam.edu.ec [Instituto de Ciencias Marinas de Andalucía (CSIC), Campus Río San Pedro, S/N, 11510 Puerto Real, Cádiz (Spain); Departamento Central De Investigación (DCI), Universidad Laica Eloy Alfaro de Manabí, Vía San Mateo, Manta (Ecuador); Laiz, Irene [Departamento de Física Aplicada, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Campus Río San Pedro, S/N, 11510 Puerto Real, Cádiz (Spain); Moreno-Garrido, Ignacio; Blasco, Julián [Instituto de Ciencias Marinas de Andalucía (CSIC), Campus Río San Pedro, S/N, 11510 Puerto Real, Cádiz (Spain)

    2015-04-15

    Highlights: • The cadmium accumulation in C. angulata tended toward a stationary state. • Metallothionein-like protein (MTLP) is clearly induced by Cd accumulation. • The MTLP detoxification mechanism is affected at high Cd concentrations. • Cadmium toxicity causes GSH levels to decrease and inhibits antioxidant enzymes. - Abstract: The kinetics of cadmium (Cd) accumulation in the gills and digestive gland of Crassotrea angulata at three concentrations of cadmium (0.088 μM, 0.44 μM and 2.22 μM) was monitored for 28 days. The relationship between accumulation and toxicity was studied using metallothionein-like protein (MTLP) concentration and reduced glutathione levels (GSH) as biochemical endpoints. The activity of enzymes which form part of the antioxidant defense system, in particular glutathione reductase (GR), total glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), as enzymatic endpoints, was also assessed. A first order kinetic model demonstrated that the accumulation process does not take place linearly, as the Cd concentration in gills and digestive gland tended toward a stationary state. Metallothionein-like protein is clearly induced by Cd accumulation; however, at high Cd concentrations the detoxification mechanism of this protein is affected. High Cd concentrations (2.22 μM) lead to a decrease in GSH levels, and also inhibit antioxidant enzyme activities, demonstrating the adverse effect of this metal on the antioxidant balance system.

  8. Annual Greenland accumulation rates (2009–2012 from airborne Snow Radar

    Directory of Open Access Journals (Sweden)

    L. S. Koenig

    2015-12-01

    Full Text Available Contemporary climate warming over the Arctic is accelerating mass loss from the Greenland Ice Sheet (GrIS through increasing surface melt, emphasizing the need to closely monitor surface mass balance (SMB in order to improve sea-level rise predictions. Here, we quantify accumulation rates, the largest component of GrIS SMB, at a higher spatial resolution than currently available, using Snow Radar stratigraphy. We use a semi-automated method to derive annual-net accumulation rates from airborne Snow Radar data collected by NASA's Operation IceBridge from 2009 to 2012. An initial comparison of the accumulation rates from the Snow Radar and the outputs of a regional climate model (MAR shows that, in general, the radar-derived accumulation matches closely with MAR in the interior of the ice sheet but MAR estimates are high over the southeast GrIS. Comparing the radar-derived accumulation with contemporaneous ice cores reveals that the radar captures the annual and long-term mean. The radar-derived accumulation rates resolve large-scale patterns across the GrIS with uncertainties of up to 11 %, attributed mostly to uncertainty in the snow/firn density profile.

  9. Annual Greenland accumulation rates (2009-2012) from airborne Snow Radar

    Science.gov (United States)

    Koenig, L. S.; Ivanoff, A.; Alexander, P. M.; MacGregor, J. A.; Fettweis, X.; Panzer, B.; Paden, J. D.; Forster, R. R.; Das, I.; McConnell, J.; Tedesco, M.; Leuschen, C.; Gogineni, P.

    2015-12-01

    Contemporary climate warming over the Arctic is accelerating mass loss from the Greenland Ice Sheet (GrIS) through increasing surface melt, emphasizing the need to closely monitor surface mass balance (SMB) in order to improve sea-level rise predictions. Here, we quantify accumulation rates, the largest component of GrIS SMB, at a higher spatial resolution than currently available, using Snow Radar stratigraphy. We use a semi-automated method to derive annual-net accumulation rates from airborne Snow Radar data collected by NASA's Operation IceBridge from 2009 to 2012. An initial comparison of the accumulation rates from the Snow Radar and the outputs of a regional climate model (MAR) shows that, in general, the radar-derived accumulation matches closely with MAR in the interior of the ice sheet but MAR estimates are high over the southeast GrIS. Comparing the radar-derived accumulation with contemporaneous ice cores reveals that the radar captures the annual and long-term mean. The radar-derived accumulation rates resolve large-scale patterns across the GrIS with uncertainties of up to 11 %, attributed mostly to uncertainty in the snow/firn density profile.

  10. Breakage of resistance to Cucumber mosaic virus by co-infection with Zucchini yellow mosaic virus: enhancement of CMV accumulation independent of symptom expression.

    Science.gov (United States)

    Wang, Y; Lee, K C; Gaba, V; Wong, S M; Palukaitis, P; Gal-On, A

    2004-02-01

    Resistance to the cucumovirus Cucumber mosaic virus (CMV) in cucumber cv. Delila was manifested as a very low level of accumulation of viral RNA and capsid protein, and an absence of CMV-induced symptoms. In addition, resistance was observed at the single cell level, with a reduction in accumulation of CMV RNAs, compared to accumulation in cells of the susceptible cucumber cv. Bet Alpha. Resistance to CMV in cv. Delila was broken by co-infection with the potyvirus Zucchini yellow mosaic virus (ZYMV). Resistance breakage in cv. Delila plants was manifested by an increase in the accumulation of (+) and (-) CMV RNA as well as CMV capsid protein, with no increase in the level of accumulation of ZYMV. Resistance breakage in the resistant cultivar by ZYMV also occurred at the single cell level. Thus, synergistic interactions known to occur between a potyvirus and a cucumovirus led to resistance breakage during a double infection. However, resistance breakage was not accompanied by an increase in disease symptoms beyond those induced by ZYMV itself. On co-inoculation with an asymptomatic variant of ZYMV-AG an enhancement of CMV infection occurred without disease manifestation. Consequently, intensification of viral RNA and capsid protein accumulation can occur without a corresponding increase in disease development, suggesting that different host genes regulate viral accumulation and disease development in the CMV-resistant cucumber plants.

  11. Freiburg RNA Tools: a web server integrating IntaRNA, ExpaRNA and LocARNA

    OpenAIRE

    Smith, Cameron; Heyne, Steffen; Richter, Andreas S.; Will, Sebastian; Backofen, Rolf

    2010-01-01

    The Freiburg RNA tools web server integrates three tools for the advanced analysis of RNA in a common web-based user interface. The tools IntaRNA, ExpaRNA and LocARNA support the prediction of RNA–RNA interaction, exact RNA matching and alignment of RNA, respectively. The Freiburg RNA tools web server and the software packages of the stand-alone tools are freely accessible at http://rna.informatik.uni-freiburg.de.

  12. Strategies underlying RNA silencing suppression by negative strand RNA viruses

    NARCIS (Netherlands)

    Hemmes, J.C.

    2007-01-01

    The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double strand

  13. Strategies underlying RNA silencing suppression by negative strand RNA viruses

    NARCIS (Netherlands)

    Hemmes, J.C.

    2007-01-01

    The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double strand

  14. LigandRNA: computational predictor of RNA-ligand interactions.

    Science.gov (United States)

    Philips, Anna; Milanowska, Kaja; Lach, Grzegorz; Bujnicki, Janusz M

    2013-12-01

    RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA-small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA-ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a "meta-predictor" leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.

  15. Quantitative Model of microRNA-mRNA interaction

    Science.gov (United States)

    Noorbakhsh, Javad; Lang, Alex; Mehta, Pankaj

    2012-02-01

    MicroRNAs are short RNA sequences that regulate gene expression and protein translation by binding to mRNA. Experimental data reveals the existence of a threshold linear output of protein based on the expression level of microRNA. To understand this behavior, we propose a mathematical model of the chemical kinetics of the interaction between mRNA and microRNA. Using this model we have been able to quantify the threshold linear behavior. Furthermore, we have studied the effect of internal noise, showing the existence of an intermediary regime where the expression level of mRNA and microRNA has the same order of magnitude. In this crossover regime the mRNA translation becomes sensitive to small changes in the level of microRNA, resulting in large fluctuations in protein levels. Our work shows that chemical kinetics parameters can be quantified by studying protein fluctuations. In the future, studying protein levels and their fluctuations can provide a powerful tool to study the competing endogenous RNA hypothesis (ceRNA), in which mRNA crosstalk occurs due to competition over a limited pool of microRNAs.

  16. Monitoring Leverage

    DEFF Research Database (Denmark)

    Geanakoplos, John; Heje Pedersen, Lasse

    2014-01-01

    We argue that leverage is a central element of economic cycles and discuss how leverage can be properly monitored. While traditionally the interest rate has been regarded as the single key feature of a loan, we contend that the size of the loan, i.e., the leverage, is in fact a more important...... measure of systemic risk. Indeed, systemic crises tend to erupt when highly leveraged economic agents are forced to deleverage, sending the economy into recession. We emphasize the importance of measuring both the average leverage on old loans (which captures the economy's vulnerability) and the leverage...... offered on new loans (which captures current credit conditions) since the economy enters a crisis when leverage on new loans is low and leverage on old loans is high. While leverage plays an important role in several economic models, the data on leverage is model-free and simply needs to be collected...

  17. Treaty Monitoring

    DEFF Research Database (Denmark)

    Canty, M.; Lingenfelder, I.; Nielsen, Allan Aasbjerg;

    2009-01-01

    This volume provides the reader with an overview of the state-of-the-art Earth Observation (EO) related research that deals with national and international security. An interdisciplinary approach was adopted in this book in order to provide the reader with a broad understanding on the uses...... of remote sensing technologies. The book therefore comprises management aspects (issues and priorities of security research, crisis response), applied methodologies and process chains (treaty monitoring, estimation of population densities and characteristics, border permeability models, damage assessment......, as well as project managers and decision makers working in the field of security having an interest in technical solutions. The integrative use of many figures and sample images are ideal in enabling the non-technical reader to grasp quickly the modern technologies that are being researched in the area...

  18. Intracranial pressure monitoring

    Science.gov (United States)

    ICP monitoring; CSF pressure monitoring ... There are 3 ways to monitor pressure in the skull (intracranial pressure). INTRAVENTRICULAR CATHETER The intraventricular catheter is the most accurate monitoring method. To insert an intraventricular catheter, a ...

  19. Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

    Directory of Open Access Journals (Sweden)

    Mario Gustavo Mayer

    2012-06-01

    Full Text Available The addition of a capped mini-exon [spliced leader (SL] through trans-splicing is essential for the maturation of RNA polymerase (pol II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1 in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin, we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

  20. A Novel Mechanism Underlying the Innate Immune Response Induction upon Viral-Dependent Replication of Host Cell mRNA: A Mistake of +sRNA Viruses' Replicases

    Science.gov (United States)

    Delgui, Laura R.; Colombo, María I.

    2017-01-01

    Viruses are lifeless particles designed for setting virus-host interactome assuring a new generation of virions for dissemination. This interactome generates a pressure on host organisms evolving mechanisms to neutralize viral infection, which places the pressure back onto virus, a process known as virus-host cell co-evolution. Positive-single stranded RNA (+sRNA) viruses are an important group of viral agents illustrating this interesting phenomenon. During replication, their genomic +sRNA is employed as template for translation of viral proteins; among them the RNA-dependent RNA polymerase (RdRp) is responsible of viral genome replication originating double-strand RNA molecules (dsRNA) as intermediates, which accumulate representing a potent threat for cellular dsRNA receptors to initiate an antiviral response. A common feature shared by these viruses is their ability to rearrange cellular membranes to serve as platforms for genome replication and assembly of new virions, supporting replication efficiency increase by concentrating critical factors and protecting the viral genome from host anti-viral systems. This review summarizes current knowledge regarding cellular dsRNA receptors and describes prototype viruses developing replication niches inside rearranged membranes. However, for several viral agents it's been observed both, a complex rearrangement of cellular membranes and a strong innate immune antiviral response induction. So, we have included recent data explaining the mechanism by, even though viruses have evolved elegant hideouts, host cells are still able to develop dsRNA receptors-dependent antiviral response. PMID:28164038

  1. Plastic Accumulation in the Mediterranean Sea

    Science.gov (United States)

    Cózar, Andrés; Sanz-Martín, Marina; Martí, Elisa; González-Gordillo, J. Ignacio; Ubeda, Bárbara; Gálvez, José Á.; Irigoien, Xabier; Duarte, Carlos M.

    2015-01-01

    Concentrations of floating plastic were measured throughout the Mediterranean Sea to assess whether this basin can be regarded as a great accumulation region of plastic debris. We found that the average density of plastic (1 item per 4 m2), as well as its frequency of occurrence (100% of the sites sampled), are comparable to the accumulation zones described for the five subtropical ocean gyres. Plastic debris in the Mediterranean surface waters was dominated by millimeter-sized fragments, but showed a higher proportion of large plastic objects than that present in oceanic gyres, reflecting the closer connection with pollution sources. The accumulation of floating plastic in the Mediterranean Sea (between 1,000 and 3,000 tons) is likely related to the high human pressure together with the hydrodynamics of this semi-enclosed basin, with outflow mainly occurring through a deep water layer. Given the biological richness and concentration of economic activities in the Mediterranean Sea, the affects of plastic pollution on marine and human life are expected to be particularly frequent in this plastic accumulation region. PMID:25831129

  2. 40 CFR 94.220 - Service accumulation.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Service accumulation. 94.220 Section 94.220 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED... than recommended lubrication and filter changes or maintenance otherwise allowed by this part, may...

  3. Plastic accumulation in the Mediterranean sea.

    Directory of Open Access Journals (Sweden)

    Andrés Cózar

    Full Text Available Concentrations of floating plastic were measured throughout the Mediterranean Sea to assess whether this basin can be regarded as a great accumulation region of plastic debris. We found that the average density of plastic (1 item per 4 m2, as well as its frequency of occurrence (100% of the sites sampled, are comparable to the accumulation zones described for the five subtropical ocean gyres. Plastic debris in the Mediterranean surface waters was dominated by millimeter-sized fragments, but showed a higher proportion of large plastic objects than that present in oceanic gyres, reflecting the closer connection with pollution sources. The accumulation of floating plastic in the Mediterranean Sea (between 1,000 and 3,000 tons is likely related to the high human pressure together with the hydrodynamics of this semi-enclosed basin, with outflow mainly occurring through a deep water layer. Given the biological richness and concentration of economic activities in the Mediterranean Sea, the affects of plastic pollution on marine and human life are expected to be particularly frequent in this plastic accumulation region.

  4. Plastic accumulation in the Mediterranean sea.

    Science.gov (United States)

    Cózar, Andrés; Sanz-Martín, Marina; Martí, Elisa; González-Gordillo, J Ignacio; Ubeda, Bárbara; Gálvez, José Á; Irigoien, Xabier; Duarte, Carlos M

    2015-01-01

    Concentrations of floating plastic were measured throughout the Mediterranean Sea to assess whether this basin can be regarded as a great accumulation region of plastic debris. We found that the average density of plastic (1 item per 4 m2), as well as its frequency of occurrence (100% of the sites sampled), are comparable to the accumulation zones described for the five subtropical ocean gyres. Plastic debris in the Mediterranean surface waters was dominated by millimeter-sized fragments, but showed a higher proportion of large plastic objects than that present in oceanic gyres, reflecting the closer connection with pollution sources. The accumulation of floating plastic in the Mediterranean Sea (between 1,000 and 3,000 tons) is likely related to the high human pressure together with the hydrodynamics of this semi-enclosed basin, with outflow mainly occurring through a deep water layer. Given the biological richness and concentration of economic activities in the Mediterranean Sea, the affects of plastic pollution on marine and human life are expected to be particularly frequent in this plastic accumulation region.

  5. Systems of Accumulation and the Evolving MEC

    NARCIS (Netherlands)

    S. Ashman (Sam); B. Fine (Ben); S.A. Newman (Susan)

    2013-01-01

    textabstractThe limitations of the Developmental State Paradigm were discussed in the introductory chapter to this volume. This chapter offers an alternative approach to the DSP through use of the notion of systems of (capital) accumulation and its specific application to South Africa’s evolving pol

  6. Plastic Accumulation in the Mediterranean Sea

    KAUST Repository

    Cózar, Andrés

    2015-04-01

    Concentrations of floating plastic were measured throughout the Mediterranean Sea to assess whether this basin can be regarded as a great accumulation region of plastic debris. We found that the average density of plastic (1 item per 4 m2), as well as its frequency of occurrence (100% of the sites sampled), are comparable to the accumulation zones described for the five subtropical ocean gyres. Plastic debris in the Mediterranean surface waters was dominated by millimeter-sized fragments, but showed a higher proportion of large plastic objects than that present in oceanic gyres, reflecting the closer connection with pollution sources. The accumulation of floating plastic in the Mediterranean Sea (between 1,000 and 3,000 tons) is likely related to the high human pressure together with the hydrodynamics of this semi-enclosed basin, with outflow mainly occurring through a deep water layer. Given the biological richness and concentration of economic activities in the Mediterranean Sea, the affects of plastic pollution on marine and human life are expected to be particularly frequent in this plastic accumulation region.

  7. Mitochondrial accumulation of APP and Abeta

    DEFF Research Database (Denmark)

    Pavlov, Pavel F; Petersen, Anna Camilla Hansson; Glaser, Elzbieta

    2009-01-01

    Accumulating evidence suggest that alterations in energy metabolism are among the earliest events that occur in the Alzheimer disease (AD) affected brain. Energy consumption is drastically decreased in the AD-affected regions of cerebral cortex and hippocampus pointing towards compromised mitocho...

  8. Intracellular accumulation of norfloxacin in Mycobacterium smegmatis.

    Science.gov (United States)

    Corti, S; Chevalier, J; Cremieux, A

    1995-11-01

    To evaluate the intracellular accumulation of norfloxacin in mycobacteria, two methods were used with Mycobacterium smegmatis. A radiometric method (K. V. Cundy, C. E. Fasching, K. E. Willard, and L. R. Peterson, J. Antimicrob. Chemother. 28:491-497, 1991) was used without great modification, but the fluorometric method (P. G. S. Mortimer and L. J. V. Piddock, J. Antimicrob. Chemother. 28:639-653, 1991) was changed considerably. Indeed, adsorption of the quinolone to the bacterial surface was characterized by measuring the level of accumulation of 0 degree C. Taking into account the adsorption, the pH of the washing buffer was increased from 7.0 to 9.0 to improve the desorption of norfloxacin from the cell surface. Both the fluorometric method, with the technical improvement, and the radiometric method could be used to estimate the intracellular accumulation of norfloxacin, which resulted from the difference between the whole uptake measured at 37 degrees C and the adsorption measured at 0 degrees C. A total of 35 ng of norfloxacin per mg of cells (dry weight) penetrated into the M. smegmatis cell, and the steady state was achieved in 5 min. Use of inhibitors of the proton motive force revealed that transport of norfloxacin was energy independent. Thus, the same mechanisms of quinolone accumulation that occur in eubacteria seem to occur in mycobacteria, at least in M. smegmatis.

  9. Accumulating Project Management Knowledge Using Process Theory

    NARCIS (Netherlands)

    Niederman, Fred; March, Salvatore T.; Mueller, Benjamin

    2016-01-01

    Process theory has become an important mechanism for the accumulation of knowledge in a number of disciplines. In contrast with variance theory, which focuses on co-variation of dependent and independent variables, process theory focuses on sequences of activities, their duration and the intervals

  10. siRNA and miRNA%干扰小RNA与微RNA

    Institute of Scientific and Technical Information of China (English)

    何晨; 陈薇; 谭军; 聂能

    2005-01-01

    干扰小RNA(small interfering RNA;siRNA)和微RNA(microRNA;miRNA)是两种序列特异性地转录后基因表达的调节因子,它们的相关性密切,既具有相似性,又具有差异性.该文主要介绍这两种小RNA分子的产生、作用机制,以及两者之间的联系与区别.

  11. Validation of RNA interferences as technique to study CNS anti-obesity drug targets

    NARCIS (Netherlands)

    Gestel, M.A. van

    2015-01-01

    RNA interference offers the opportunity to unravel the roles of genes in a specific area of the brain. Genes implicated in energy balance are perfect targets for the application of RNA interference as their phenotypes can be easily monitored and the neural circuits involved in energy homeostasis are

  12. Comparative transcriptome analyses of deltamethrin-resistant and -susceptible Anopheles gambiae mosquitoes from Kenya by RNA-Seq.

    Directory of Open Access Journals (Sweden)

    Mariangela Bonizzoni

    Full Text Available Malaria causes more than 300 million clinical cases and 665,000 deaths each year, and the majority of the mortality and morbidity occurs in sub-Saharan Africa. Due to the lack of effective vaccines and wide-spread resistance to antimalarial drugs, mosquito control is the primary method of malaria prevention and control. Currently, malaria vector control relies on the use of insecticides, primarily pyrethroids. The extensive use of insecticides has imposed strong selection pressures for resistance in the mosquito populations. Consequently, resistance to pyrethroids in Anopheles gambiae, the main malaria vector in sub-Saharan Africa, has become a major obstacle for malaria control. A key element of resistance management is the identification of resistance mechanisms and subsequent development of reliable resistance monitoring tools. Field-derived An. gambiae from Western Kenya were phenotyped as deltamethrin-resistant or -susceptible by the standard WHO tube test, and their expression profile compared by RNA-seq. Based on the current annotation of the An. gambiae genome, a total of 1,093 transcripts were detected as significantly differentially accumulated between deltamethrin-resistant and -susceptible mosquitoes. These transcripts are distributed over the entire genome, with a large number mapping in QTLs previously linked to pyrethorid resistance, and correspond to heat-shock proteins, metabolic and transport functions, signal transduction activities, cytoskeleton and others. The detected differences in transcript accumulation levels between resistant and susceptible mosquitoes reflect transcripts directly or indirectly correlated with pyrethroid resistance. RNA-seq data also were used to perform a de-novo Cufflinks assembly of the An. gambiae genome.

  13. Accumulation of Radiocesium in Eleutherococcus sciadophylloides

    Energy Technology Data Exchange (ETDEWEB)

    Sugiura, Y.; Takenaka, C.; Kanasashi, T. [Graduate School of Bioagricultural Sciences, Nagoya University, 464-8601, Nagoya City, Aichi Prefecture (Japan); Deguchi, S. [School of Agricultural Sciences, Nagoya University, Nagoya City, Aichi Prefecture, 464-8601 (Japan); Matsuda, Y. [Graduate School of Bioresources, Mie University, Tsu City, Mie Prefecture, 514-0102 (Japan); Ozawa, H. [Fukushima Prefectural Forestry Research Centre, Koriyama City Fukushima Prefecture, 963-0112 (Japan)

    2014-07-01

    1. Introduction: After Fukushima Daiichi Nuclear Power Plant accident, radiocesium ({sup 137}Cs) had deposited on forests in Fukushima Prefecture. In order to comprehend radiocesium circulation in forest ecosystem, it is important to understand about properties of {sup 137}Cs accumulation of each plant species. In addition, {sup 137}Cs accumulator plants would be candidates of phyto-remediation, which is a remediation method using plants to remove pollutants from environment. We aimed to find {sup 137}Cs accumulator plants and to clarify the accumulate mechanisms. 2. Materials and Methods: We collected soil and plant samples at 22 points in Fukushima Prefecture more than once a year from May 2011 to October 2013. Surface (0-5 cm) soils were collected at the same site as the plant sampling. The soil samples were air-dried for 2-3 weeks and then passed through a 2 mm sieve. Foliar samples were washed with tap water to remove soil particles and rinsed with deionized water for {sup 137}Cs and other elements analysis. The samples were dried at 80 deg. C for 48 hr and ground with a mill mixer. {sup 137}Cs activities in soil and plant samples were determined by means of high-purity Ge detector (HPGe). The elements concentrations of the plant samples were determined by inductively coupled plasma mass spectrometry (ICP-MS) after wet digestion with HNO{sub 3}. 3. Results and Discussion: As a whole trend, evergreen tree species such as Camellia japonica and Cryptomeria japonica contained {sup 137}Cs at high concentration due to the deposited {sup 137}Cs on old leaves and foliar absorption. The activities in leaves of deciduous tree species were lower than those in evergreen trees. However, we confirmed that a deciduous tree species, Eleutherococcus sciadophylloides, collected in 2012 and 2013 accumulated {sup 137}Cs, whereas that collected in 2011 did not accumulate {sup 137}Cs. The {sup 137}Cs concentration of E. sciadophylloides in 2012 and 2013 were higher than those of

  14. RNA interference in Lepidoptera

    DEFF Research Database (Denmark)

    Terenius, Ole; Papanicolaou, Alexie; Garbutt, Jennie S.

    2011-01-01

    Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive...... is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success...... in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our...

  15. Chemical evolution of life-like system under hydrothermal environments: prebiotic formation, degradation, and functions regarding protein-like molecules and RNA

    Science.gov (United States)

    Kawamura, Kunio

    The accumulation of biopolymers without enzymes is an essential step for the chemical evolu-tion towards a primitive life-like system. Previously, we discussed the relationship between the RNA world hypothesis and the hydrothermal origin of life hypothesis on the basis of the em-pirical data of RNA behaviors under the hydrothermal environments examined using real-time monitoring technique for hydrothermal reactions within the millisecond to second time scale. On the other hand, we have also examined the stabilities and behaviors of amino acids, pep-tides, and proteins under the hydrothermal environments. These observations have shown the possibility that oligopeptides could have been accumulated under near submarine hydrother-mal vent environments on primitive Earth within the relatively short time scale. However, the formation of oligopeptides under the simulated hydrothermal conditions is not so effective in the absence of catalysts and condensation agents. Thus, the investigation of the roles of min-eral catalysis and condensation reagents are very important since these materials could have enhanced efficiently the formation of peptides and stabilize primitive protein-like molecules. Recently, we investigated the roles of condensation reagents for the elongation of oligopeptides in the presence of minerals. In addition, we have designed a mineral-mediated hydrothermal flow reactor system (MHFR), which enables monitoring hydrothermal reactions in the presence of solid particles. By using MHFR, we attempted to examine naturally occurring minerals, such as apatite and quartz, for the elongation of oligopeptides at temperatures over 200 o C within 10 -30 sec. According to these data, the chemical evolution of protein-like molecules on primitive Earth will be discussed.

  16. The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways.

    Science.gov (United States)

    Csorba, Tibor; Bovi, Aurelie; Dalmay, Tamás; Burgyán, József

    2007-11-01

    One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3' overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised.

  17. Accumulation of carbon in northern mire ecosystems

    Energy Technology Data Exchange (ETDEWEB)

    Tolonen, K.; Turunen, J.; Alm, J. [Joensuu Univ. (Finland). Dept. of Biology; Korhola, A. [Helsinki Univ. (Finland). Lab. of Physical Geography; Jungner, H. [Helsinki Univ. (Finland). Dating Lab.; Vasander, H. [Helsinki Univ. (Finland). Dept. of Forest Ecology

    1996-12-31

    The basic feature in the functional ecology of any mire ecosystem is retardation of the effective decay of organic material resulting in a conspicuous accumulation of plant debris as peat overtime. The carbon accumulation process is slow, and climatic change may have an impact on the carbon cycle of peatlands, therefore, it has been of interest to study the rate of carbon accumulation by geological methods from dated peat strata. The approach is hampered by several facts. First, the mires vary enormously as to their vegetation and hydrology and hence their production and decay properties. It follows that a great number of study sites are needed. Second, the peat in mires expands both vertically and laterally, and this requires a spatial reconstruction of carbon accumulation within a mire basin. Third, simple geological methods cannot account for the actual rate of carbon accumulation in peat, and finally, an additional carbon sink in the mire ecosystems can be the mineral subsoil beneath peat. The proposed warming will perhaps shift northwards the existing climatic mire regimes and, thus, the northern aapa fens will change to Sphagnum bogs that are more effective in sequestering carbon, but distinctly less effective in their CH{sub 4} and N{sub 2}O emanation. The role of mire fires in more remote northern areas may then become another important factor. The answer to the important question of future total sequestration of carbon to peatlands depends on the precipitation and its seasonal distribution pattern. Most climatic scenarios predict a decrease in the evaporation surplus during the summer at northern regions. Presumably, the consequent lowering of the water table would improve growth of forest on mires and simultaneously decrease the methane fluxes from peat. The combined net effect could be a clear restraining of the radiative forcing

  18. Monitoring assembly of ribonucleoprotein complexes by isothermal titration calorimetry

    Science.gov (United States)

    Recht, Michael I.; Ryder, Sean P.; Williamson, James R.

    2010-01-01

    Isothermal titration calorimetry (ITC) is a useful technique to study RNA-protein interactions, as it provides the only method by which the thermodynamic parameters of free energy, enthalpy, and entropy can be directly determined. This chapter presents a general procedure for studying RNA-protein interactions using ITC, and gives specific examples for monitoring the binding of Caenorhabditis elegans GLD-1 STAR domain to TGE RNA and the binding of Aquifex aeolicus S6:S18 ribosomal protein heterodimer to an S15-rRNA complex. PMID:18982287

  19. Generation of RNA in abiotic conditions.

    Science.gov (United States)

    di Mauro, Ernesto

    Generation of RNA in abiotic conditions. Ernesto Di Mauro Dipartimento di Genetica Bi-ologia Molecolare, Universit` "Sapienza" Roma, Italy. a At least four conditions must be satisfied for the spontaneous generation of (pre)-genetic poly-mers: 1) availability of precursors that are activated enough to spontaneously polymerize. Preliminary studies showed that (a) nucleic bases and acyclonucleosides can be synthesized from formamide H2NCOH by simply heating with prebiotically available mineral catalysts [last reviewed in (1)], and that b) nucleic bases can be phosphorylated in every possible posi-tion [2'; 3'; 5'; cyclic 2',3'; cyclic 3',5' (2)]. The higher stability of the cyclic forms allows their accumulation. 2) A polymerization mechanism. A reaction showing the formation of RNA polymers starting from prebiotically plausible precursors (3',5' cyclic GMP and 3', 5'cyclic AMP) was recently reported (3). Polymerization in these conditions is thermodynamically up-hill and an equilibrium is attained that limits the maximum length of the polymer produced to about 40 nucleotides for polyG and 100 nucleotides for polyA. 3) Ligation of the synthesized oligomers. If this type of reaction could occur according to a terminal-joining mechanism and could generate canonical 3',5' phosphodiester bonds, exponential growth would be obtained of the generated oligomers. This type of reaction has been reported (4) , limited to homogeneous polyA sequences and leading to the production of polyA dimers and tetramers. What is still missing are: 4) mechanisms that provide the proof of principle for the generation of sequence complexity. We will show evidence for two mechanisms providing this proof of principle for simple complementary sequences. Namely: abiotic sequence complementary-driven terminal ligation and sequence-complementary terminal growth. In conclusion: all the steps leading to the generation of RNA in abiotic conditions are satisfied. (1) R Saladino, C Crestini, F

  20. RNA-protein complexes identified by crosslinking of polysomes.

    Science.gov (United States)

    Sköld, S E

    1981-01-01

    The bifunctional cleavable reagent diepoxybutane was used to investigate the crosslinking of proteins to the 16S and 23S RNA in Escherichia coli ribosomes. The crosslinking patterns from polysomes, accumulated in the absence and presence of oxytetracycline, as well as reassociated 70S ribosomes were compared. The 30S proteins: S3, S4, S5, S7, S8, S9, S12, S13, S14 and S18 were recovered crosslinked to the 16S RNA and the 50S: proteins L1, L2, L4, L13, L14-L21, L15, L16, L17, L18-L23, L19-22-24, L27 and L28 were recovered crosslinked to the 23S RNA, in all three associated states. Proteins crosslinked to the RNA of the heterologous subunit and therefore considered to be at or near the ribosomal subunit interface were, for all three states, proteins S1, S4, S6, S9, S12, S13, S14 and S18 from the small subunit and proteins L16, L17, L20 and L27 from the large subunit. Finally, the recovery of intrasubunit crosslinks was measured for the isolated subunits. Additional crosslinked complexes were observed between 16S RNA and S1, S2 as well as S6 from the 30S subunit; and between 23S RNA and L10, L11, L7/12 from the 50S subunit.

  1. Poly(A) RNA a new component of Cajal bodies.

    Science.gov (United States)

    Kołowerzo, Agnieszka; Smoliński, Dariusz Jan; Bednarska, Elzbieta

    2009-07-01

    In European larch microsporocytes, spherical structures 0.5 to 6 microm in diameter are present in which poly(A) RNA accumulates. There were one to several bodies per cell and they were often present in the vicinity of the nucleolus. No nascent transcripts were observed within them. Splicing factors of the SR family, including protein SC35, which participates in bringing the 3' and 5' sites closer in the splicing reaction, were also not observed. The absence of the above-mentioned elements within bodies containing poly(A) RNA disqualifies them as sites of synthesis and preliminary stages of primary transcript maturation. However, they contained abundant elements of the splicing machinery commonly occurring in Cajal bodies, i.e., Sm proteins or small nuclear RNA (snRNA). The molecular composition as well as the characteristic ultrastructure of bodies containing poly(A) RNA proves that these were Cajal bodies. This is the first report of such poly(A) RNA localization.

  2. CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use

    Directory of Open Access Journals (Sweden)

    Mercereau-Puijalon Odile

    2005-03-01

    Full Text Available Abstract Background Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. Results To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS or Staphylococcus aureus strain Cowan (SAC. Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS. Conclusion

  3. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Science.gov (United States)

    Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

  4. Elucidation of pathways of ribosomal RNA degradation: an essential role for RNase E

    Science.gov (United States)

    Sulthana, Shaheen; Basturea, Georgeta N.; Deutscher, Murray P.

    2016-01-01

    Although normally stable in growing cells, ribosomal RNAs are degraded under conditions of stress, such as starvation, and in response to misassembled or otherwise defective ribosomes in a process termed RNA quality control. Previously, our laboratory found that large fragments of 16S and 23S rRNA accumulate in strains lacking the processive exoribonucleases RNase II, RNase R, and PNPase, implicating these enzymes in the later steps of rRNA breakdown. Here, we define the pathways of rRNA degradation in the quality control process and during starvation, and show that the essential endoribonuclease, RNase E, is required to make the initial cleavages in both degradative processes. We also present evidence that explains why the exoribonuclease, RNase PH, is required to initiate the degradation of rRNA during starvation. The data presented here provide the first detailed description of rRNA degradation in bacterial cells. PMID:27298395

  5. Screening of As-accumulating plants using a foliar application and a native accumulation of As.

    Science.gov (United States)

    Zhang, Z; Sugawara, K; Hatayama, M; Huang, Y; Inoue, Chihiro

    2014-01-01

    The discovery of novel accumulating plants is useful for efficient phytoremediation due to the demands of various conditions of impacted sites such as land use, soil properties, concentration of pollutants, and climate. In the present study, we investigated foliar application or a field with highly bioavailable arsenic (As) to screen As-accumulating plants. Plants grown in the downstream of a hot springs area were analyzed for native As accumulation and As foliar application, and the rhizosphere soils were collected. The water-soluble As in the rhizosphere soils had a high average, 144 microg/kg, whereas total As was similar to normal soil in Japan. Among 34 herbaceous plants and 17 woody plants, Chelidonium majus var. asiaticum accumulated a relatively high As level, 8.07 mg/kg DW (93.6% of As added), that was not revealed by native accumulation. In a further pot experiment, C. majus accumulated a moderately high As level (314 mg/kg DW) in the roots but not in the shoot (30.1 mg/kg DW), and exhibited a low transfer factor (TF = 0.096). Thus, a foliar application would be a simple and high-throughput method to screen plants that accumulate and tolerate As. C. majus would be useful as a tool for phytostabilization of As.

  6. Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation.

    Directory of Open Access Journals (Sweden)

    Johannes H Urban

    2008-03-01

    Full Text Available Small noncoding RNAs (sRNA can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3' end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms.

  7. RNase MRP cleaves pre-tRNASer-Met in the tRNA maturation pathway.

    Science.gov (United States)

    Saito, Yuichiro; Takeda, Jun; Adachi, Kousuke; Nobe, Yuko; Kobayashi, Junya; Hirota, Kouji; Oliveira, Douglas V; Taoka, Masato; Isobe, Toshiaki

    2014-01-01

    Ribonuclease mitochondrial RNA processing (RNase MRP) is a multifunctional ribonucleoprotein (RNP) complex that is involved in the maturation of various types of RNA including ribosomal RNA. RNase MRP consists of a potential catalytic RNA and several protein components, all of which are required for cell viability. We show here that the temperature-sensitive mutant of rmp1, the gene for a unique protein component of RNase MRP, accumulates the dimeric tRNA precursor, pre-tRNA(Ser-Met). To examine whether RNase MRP mediates tRNA maturation, we purified the RNase MRP holoenzyme from the fission yeast Schizosaccharomyces pombe and found that the enzyme directly and selectively cleaves pre-tRNA(Ser-Met), suggesting that RNase MRP participates in the maturation of specific tRNA in vivo. In addition, mass spectrometry-based ribonucleoproteomic analysis demonstrated that this RNase MRP consists of one RNA molecule and 11 protein components, including a previously unknown component Rpl701. Notably, limited nucleolysis of RNase MRP generated an active catalytic core consisting of partial mrp1 RNA fragments, which constitute "Domain 1" in the secondary structure of RNase MRP, and 8 proteins. Thus, the present study provides new insight into the structure and function of RNase MRP.

  8. Extensive microheterogeneity of serine tRNA genes from Drosophila melanogaster.

    Science.gov (United States)

    Cribbs, D L; Leung, J; Newton, C H; Hayashi, S; Miller, R C; Tener, G M

    1987-10-05

    The nucleotide sequences of nine genes corresponding to tRNA(Ser)4 or tRNA(Ser)7 of Drosophila melanogaster were determined. Eight of the genes compose the major tRNA(Ser)4,7 cluster at 12DE on the X chromosome, while the other is from 23E on the left arm of chromosome 2. Among the eight X-linked genes, five different, interrelated, classes of sequence were found. Four of the eight genes correspond to tRNA(Ser)4 and tRNA(Ser)7 (which are 96% homologous), two appear to result from single crossovers between tRNA(Ser)4 and tRNA(Ser)7 genes, one is an apparent double crossover product, and the last differs from a tRNA(Ser)4 gene by a single C to T transition at position 50. The single autosomal gene corresponds to tRNA(Ser)7. Comparison of a pair of genes corresponding to tRNA(Ser)4 from D. melanogaster and Drosophila simulans showed that, while gene flanking sequences may diverge considerably by accumulation of point changes, gene sequences are maintained intact. Our data indicate that recombination occurs between non-allelic tRNA(Ser) genes, and suggest that at least some recombinational events may be intergenic conversions.

  9. RNA-SSPT: RNA Secondary Structure Prediction Tools.

    Science.gov (United States)

    Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

    2013-01-01

    The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes.

  10. iRNA-PseU: Identifying RNA pseudouridine sites

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2016-01-01

    Full Text Available As the most abundant RNA modification, pseudouridine plays important roles in many biological processes. Occurring at the uridine site and catalyzed by pseudouridine synthase, the modification has been observed in nearly all kinds of RNA, including transfer RNA, messenger RNA, small nuclear or nucleolar RNA, and ribosomal RNA. Accordingly, its importance to basic research and drug development is self-evident. Despite some experimental technologies have been developed to detect the pseudouridine sites, they are both time-consuming and expensive. Facing the explosive growth of RNA sequences in the postgenomic age, we are challenged to address the problem by computational approaches: For an uncharacterized RNA sequence, can we predict which of its uridine sites can be modified as pseudouridine and which ones cannot? Here a predictor called “iRNA-PseU” was proposed by incorporating the chemical properties of nucleotides and their occurrence frequency density distributions into the general form of pseudo nucleotide composition (PseKNC. It has been demonstrated via the rigorous jackknife test, independent dataset test, and practical genome-wide analysis that the proposed predictor remarkably outperforms its counterpart. For the convenience of most experimental scientists, the web-server for iRNA-PseU was established at http://lin.uestc.edu.cn/server/iRNA-PseU, by which users can easily get their desired results without the need to go through the mathematical details.

  11. Internal RNA Replication Elements are Prevalent in Tombusviridae</