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Sample records for island-encoded srna isrm

  1. The ISRM suggested methods for rock characterization, testing and monitoring 2007-2014

    CERN Document Server

    2015-01-01

    This book is a collection of ISRM suggested methods for testing or measuring properties of rocks and rock masses both in the laboratory and in situ, as well as for monitoring the performance of rock engineering structures. The first collection (Yellow Book) has been published in 1981. In order to provide access to all the Suggested Methods in one volume, the ISRM Blue Book was published in 2007 (by the ISRM via the Turkish National Group) and contains the complete set of Suggested Methods from 1974 to 2006 inclusive. The papers in this most recent volume have been published during the last seven years in international journals, mainly in Rock Mechanics and Rock Engineering. They offer guidance for rock characterization procedures and laboratory and field testing and monitoring in rock engineering. These methods provide a definitive procedure for the identification, measurement and evaluation of one or more qualities, characteristics, or properties of rocks or rock systems that produces a test result.

  2. The United States initiative for international radioactive source management (ISRM)

    International Nuclear Information System (INIS)

    Naraine, N.; Karhnak, J.

    1999-01-01

    The United States takes seriously the potential problems from uncontrolled radioactive sources. To address these problems, the United States Department of State is leading the development of an initiative for International Radioactive Source Management (ISRM). The Department of State, through a number of Federal and state agencies, regulatory bodies and private industry, will endeavor to provide coordinated support to the international community, particularly through IAEA, to assist in the development and implementation of risk-based clearance levels to support import/export of radioactive contaminated metals and the tracking, management, identification, remediation, and disposition of 'lost sources' entering nation states and targeted industries. The United States believes that the international control of radioactive sources is critical in avoiding wide-spread contamination of the world metal supply. Thus the initiative has four objectives: (1) Protect sources from becoming lost (Tracking management); (2) Identify primary locations where sources have been lost (Stop future losses); (3) Locate lost sources (monitor and retrieve); and (4) Educate and train (deploy knowledge and technology). A number of efforts already underway in the United States support the overall initiative. The EPA has provided a grant to the Conference of Radiation Program Control Directors (CRCPD) to develop a nation-wide program for the disposition of orphaned radioactive sources. This program now has internet visibility and a toll-free telephone number to call for assistance in the disposal of sources. The Nuclear Regulatory Commission (NRC), the Department of Energy (DOE), and other government agencies as well as private companies are assisting CRCPD in this program. The NRC has begun a program to improve control of radioactive sources in the United States, and also intends to promulgate a regulation defining conditions for the release of materials from licensed facilities. The DOE is

  3. A Model for an Information Security Risk Management (ISRM) Framework for Saudi Arabian Organisations

    Science.gov (United States)

    Alshareef, Naser

    2016-01-01

    Countries in the Gulf represent thriving, globally important commercial centres. They have embraced technology and modern management methods, often originating in the western countries. In adapting to quite different cultures these do not always operate as successfully. The adoption and practices of the Information Security Risk Management (ISRM)…

  4. EVALUATION OF AMENDMENTS FOR MENDING THE INSITU REDOX MANIPULATION (ISRM) BARRIER

    Energy Technology Data Exchange (ETDEWEB)

    PETERSEN, S.W.

    2006-02-07

    In May of 2004, the U.S. Department of Energy (DOE) Richland and Fluor Hanford requested technical assistance from DOE Headquarters EM-23 to provide a team of technical experts to evaluate likely chemical/biological amendments for mending the In Situ Redox Manipulation (ISRM) Barrier in the 100-D Area of the Hanford Site. This request was a follow-on to an earlier request for assistance regarding the cause of chromium (Cr) breakthrough and recommendations for mending the barrier (March 2004 workshop). This report provides written documentation of the team's findings and recommendations. In 1995, a plume of dissolved hexavalent chromium [Cr(VI)] was discovered along the Columbia River shoreline and in the 100-D Area. Between 1999 and 2003, a reactive barrier using the ISRM technology, was installed at a distance of 680 meters along the river to reduce the Cr(VI) in the groundwater. The ISRM technology creates a treatment zone within the aquifer by injection of sodium dithionite, a strong reducing agent that scavenges dissolved oxygen (DO) from the aquifer and reduces ferric iron [Fe(III)], related metals, and oxy-ions. Bench-scale and field-scale treatability tests were conducted to demonstrate proof-of principle and to estimate barrier longevity, calculated to be in excess of twenty years. However, several years after initial and secondary treatment, groundwater in approximately 17 wells has been found to contain elevated Cr concentrations. The March 2004 technical assistance team (TAT) identified potential causes of Cr breakthrough as likely related to physical and chemical heterogeneity within the aquifer (including loss of reductive capacity within preferential flow paths) and the presence of other oxidants (DO and nitrate) significantly affecting the reductive capacity of the treated aquifer. These aquifer characteristics may limit the ability of alternative amendments to extend the reducing capacity of the barrier. A 2001 Bechtel Hanford report and

  5. Application of ISRM testing methods to fracture toughness testing of graphite

    International Nuclear Information System (INIS)

    Hashida, T.; Fukasawa, T.; Takahashi, H.; Ishiyama, S.; Oku, T.

    1987-01-01

    Fracture toughness measurements of nuclear grade graphites, IG11 and PGX, were made by means of AE technique. Tests were conducted on edge-notched round bend bar, edge-notched short bar and round compact tension specimens. These round-shaped specimens used in this study have been proposed for standard fracture toughness tests of rock as a draft of testing standard of International Society for Rock Mechanics (ISRM). Taking the observed nonlinear deformation behavior into account, J-integral approach was utilized to determine the fracture toughness of the graphites. It is shown that the critical J integral determined by AE technique, J iAE , is independent of specimen geometry. Based on this experimental results, the fracture toughness K IC of the graphites was determined from the J iAE values. K IC value of IG11 was 1.04 MPa√m, and 0.77 MPa√m for PGX respectively. Furthermore, the specimen size effect of the fracture toughness determined by the J-integral/AE method is discussed. (author)

  6. Proton therapy Monte Carlo SRNA-VOX code

    Directory of Open Access Journals (Sweden)

    Ilić Radovan D.

    2012-01-01

    Full Text Available The most powerful feature of the Monte Carlo method is the possibility of simulating all individual particle interactions in three dimensions and performing numerical experiments with a preset error. These facts were the motivation behind the development of a general-purpose Monte Carlo SRNA program for proton transport simulation in technical systems described by standard geometrical forms (plane, sphere, cone, cylinder, cube. Some of the possible applications of the SRNA program are: (a a general code for proton transport modeling, (b design of accelerator-driven systems, (c simulation of proton scattering and degrading shapes and composition, (d research on proton detectors; and (e radiation protection at accelerator installations. This wide range of possible applications of the program demands the development of various versions of SRNA-VOX codes for proton transport modeling in voxelized geometries and has, finally, resulted in the ISTAR package for the calculation of deposited energy distribution in patients on the basis of CT data in radiotherapy. All of the said codes are capable of using 3-D proton sources with an arbitrary energy spectrum in an interval of 100 keV to 250 MeV.

  7. Proton absorbed dose distribution in human eye simulated by SRNA-2KG code

    International Nuclear Information System (INIS)

    Ilic, R. D.; Pavlovic, R.

    2004-01-01

    The model of Monte Carlo SRNA code is described together with some numerical experiments to show feasibility of this code to be used in proton therapy, especially for tree dimensional proton absorption dose calculation in human eye. (author) [sr

  8. A broad range quorum sensing inhibitor working through sRNA inhibition

    DEFF Research Database (Denmark)

    Jakobsen, Tim H.; Warming, Anders N.; Vejborg, Rebecca M.

    2017-01-01

    that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, we found that ajoene......-spectrum compounds transcending the Gram negative-positive borderline....

  9. Using small RNA (sRNA) deep sequencing to understand global virus distribution in plants

    Science.gov (United States)

    Small RNAs (sRNAs), a class of regulatory RNAs, have been used to serve as the specificity determinants of suppressing gene expression in plants and animals. Next generation sequencing (NGS) uncovered the sRNA landscape in most organisms including their associated microbes. In the current study, w...

  10. Distribution of absorbed dose in human eye simulated by SRNA-2KG computer code

    International Nuclear Information System (INIS)

    Ilic, R.; Pesic, M.; Pavlovic, R.; Mostacci, D.

    2003-01-01

    Rapidly increasing performances of personal computers and development of codes for proton transport based on Monte Carlo methods will allow, very soon, the introduction of the computer planning proton therapy as a normal activity in regular hospital procedures. A description of SRNA code used for such applications and results of calculated distributions of proton-absorbed dose in human eye are given in this paper. (author)

  11. Balancing gene expression without library construction via a reusable sRNA pool.

    Science.gov (United States)

    Ghodasara, Amar; Voigt, Christopher A

    2017-07-27

    Balancing protein expression is critical when optimizing genetic systems. Typically, this requires library construction to vary the genetic parts controlling each gene, which can be expensive and time-consuming. Here, we develop sRNAs corresponding to 15nt 'target' sequences that can be inserted upstream of a gene. The targeted gene can be repressed from 1.6- to 87-fold by controlling sRNA expression using promoters of different strength. A pool is built where six sRNAs are placed under the control of 16 promoters that span a ∼103-fold range of strengths, yielding ∼107 combinations. This pool can simultaneously optimize up to six genes in a system. This requires building only a single system-specific construct by placing a target sequence upstream of each gene and transforming it with the pre-built sRNA pool. The resulting library is screened and the top clone is sequenced to determine the promoter controlling each sRNA, from which the fold-repression of the genes can be inferred. The system is then rebuilt by rationally selecting parts that implement the optimal expression of each gene. We demonstrate the versatility of this approach by using the same pool to optimize a metabolic pathway (β-carotene) and genetic circuit (XNOR logic gate). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Srna-Monte Carlo codes for proton transport simulation in combined and voxelized geometries

    CERN Document Server

    Ilic, R D; Stankovic, S J

    2002-01-01

    This paper describes new Monte Carlo codes for proton transport simulations in complex geometrical forms and in materials of different composition. The SRNA codes were developed for three dimensional (3D) dose distribution calculation in proton therapy and dosimetry. The model of these codes is based on the theory of proton multiple scattering and a simple model of compound nucleus decay. The developed package consists of two codes: SRNA-2KG and SRNA-VOX. The first code simulates proton transport in combined geometry that can be described by planes and second order surfaces. The second one uses the voxelized geometry of material zones and is specifically adopted for the application of patient computer tomography data. Transition probabilities for both codes are given by the SRNADAT program. In this paper, we will present the models and algorithms of our programs, as well as the results of the numerical experiments we have carried out applying them, along with the results of proton transport simulation obtaine...

  13. GLASSgo – Automated and Reliable Detection of sRNA Homologs From a Single Input Sequence

    Directory of Open Access Journals (Sweden)

    Steffen C. Lott

    2018-04-01

    Full Text Available Bacterial small RNAs (sRNAs are important post-transcriptional regulators of gene expression. The functional and evolutionary characterization of sRNAs requires the identification of homologs, which is frequently challenging due to their heterogeneity, short length and partly, little sequence conservation. We developed the GLobal Automatic Small RNA Search go (GLASSgo algorithm to identify sRNA homologs in complex genomic databases starting from a single sequence. GLASSgo combines an iterative BLAST strategy with pairwise identity filtering and a graph-based clustering method that utilizes RNA secondary structure information. We tested the specificity, sensitivity and runtime of GLASSgo, BLAST and the combination RNAlien/cmsearch in a typical use case scenario on 40 bacterial sRNA families. The sensitivity of the tested methods was similar, while the specificity of GLASSgo and RNAlien/cmsearch was significantly higher than that of BLAST. GLASSgo was on average ∼87 times faster than RNAlien/cmsearch, and only ∼7.5 times slower than BLAST, which shows that GLASSgo optimizes the trade-off between speed and accuracy in the task of finding sRNA homologs. GLASSgo is fully automated, whereas BLAST often recovers only parts of homologs and RNAlien/cmsearch requires extensive additional bioinformatic work to get a comprehensive set of homologs. GLASSgo is available as an easy-to-use web server to find homologous sRNAs in large databases.

  14. Srna - Monte Carlo codes for proton transport simulation in combined and voxelized geometries

    Directory of Open Access Journals (Sweden)

    Ilić Radovan D.

    2002-01-01

    Full Text Available This paper describes new Monte Carlo codes for proton transport simulations in complex geometrical forms and in materials of different composition. The SRNA codes were developed for three dimensional (3D dose distribution calculation in proton therapy and dosimetry. The model of these codes is based on the theory of proton multiple scattering and a simple model of compound nucleus decay. The developed package consists of two codes: SRNA-2KG and SRNA-VOX. The first code simulates proton transport in combined geometry that can be described by planes and second order surfaces. The second one uses the voxelized geometry of material zones and is specifically adopted for the application of patient computer tomography data. Transition probabilities for both codes are given by the SRNADAT program. In this paper, we will present the models and algorithms of our programs, as well as the results of the numerical experiments we have carried out applying them, along with the results of proton transport simulation obtained through the PETRA and GEANT programs. The simulation of the proton beam characterization by means of the Multi-Layer Faraday Cup and spatial distribution of positron emitters obtained by our program indicate the imminent application of Monte Carlo techniques in clinical practice.

  15. The Monte Carlo SRNA-VOX code for 3D proton dose distribution in voxelized geometry using CT data

    International Nuclear Information System (INIS)

    Ilic, Radovan D; Spasic-Jokic, Vesna; Belicev, Petar; Dragovic, Milos

    2005-01-01

    This paper describes the application of the SRNA Monte Carlo package for proton transport simulations in complex geometry and different material compositions. The SRNA package was developed for 3D dose distribution calculation in proton therapy and dosimetry and it was based on the theory of multiple scattering. The decay of proton induced compound nuclei was simulated by the Russian MSDM model and our own using ICRU 63 data. The developed package consists of two codes: the SRNA-2KG, which simulates proton transport in combinatorial geometry and the SRNA-VOX, which uses the voxelized geometry using the CT data and conversion of the Hounsfield's data to tissue elemental composition. Transition probabilities for both codes are prepared by the SRNADAT code. The simulation of the proton beam characterization by multi-layer Faraday cup, spatial distribution of positron emitters obtained by the SRNA-2KG code and intercomparison of computational codes in radiation dosimetry, indicate immediate application of the Monte Carlo techniques in clinical practice. In this paper, we briefly present the physical model implemented in the SRNA package, the ISTAR proton dose planning software, as well as the results of the numerical experiments with proton beams to obtain 3D dose distribution in the eye and breast tumour

  16. SRNA-2K5, Proton Transport Using 3-D by Monte Carlo Techniques

    International Nuclear Information System (INIS)

    Ilic, Radovan D.

    2005-01-01

    1 - Description of program or function: SRNA-2K5 performs Monte Carlo transport simulation of proton in 3D source and 3D geometry of arbitrary materials. The proton transport based on condensed history model, and on model of compound nuclei decays that creates in nonelastic nuclear interaction by proton absorption. 2 - Methods: The SRNA-2K5 package is developed for time independent simulation of proton transport by Monte Carlo techniques for numerical experiments in complex geometry, using PENGEOM from PENELOPE with different material compositions, and arbitrary spectrum of proton generated from the 3D source. This package developed for 3D proton dose distribution in proton therapy and dosimetry, and it was based on the theory of multiple scattering. The compound nuclei decay was simulated by our and Russian MSDM models using ICRU 49 and ICRU 63 data. If protons trajectory is divided on great number of steps, protons passage can be simulated according to Berger's Condensed Random Walk model. Conditions of angular distribution and fluctuation of energy loss determinate step length. Physical picture of these processes is described by stopping power, Moliere's angular distribution, Vavilov's distribution with Sulek's correction per all electron orbits, and Chadwick's cross sections for nonelastic nuclear interactions, obtained by his GNASH code. According to physical picture of protons passage and with probabilities of protons transition from previous to next stage, which is prepared by SRNADAT program, simulation of protons transport in all SRNA codes runs according to usual Monte Carlo scheme: (i) proton from the spectrum prepared for random choice of energy, position and space angle is emitted from the source; (ii) proton is loosing average energy on the step; (iii) on that step, proton experience a great number of collisions, and it changes direction of movement randomly chosen from angular distribution; (iv) random fluctuation is added to average energy loss; (v

  17. Klebsiella pneumoniae asparagine tDNAs are integration hotspots for different genomic islands encoding microcin E492 production determinants and other putative virulence factors present in hypervirulent strains

    Directory of Open Access Journals (Sweden)

    Andrés Esteban Marcoleta

    2016-06-01

    Full Text Available Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492 and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized.In this work we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mytomicin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least 7 protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we

  18. Comprehensive processing of high-throughput small RNA sequencing data including quality checking, normalization, and differential expression analysis using the UEA sRNA Workbench.

    Science.gov (United States)

    Beckers, Matthew; Mohorianu, Irina; Stocks, Matthew; Applegate, Christopher; Dalmay, Tamas; Moulton, Vincent

    2017-06-01

    Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this. © 2017 Beckers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. Characterization of an Hfq dependent antisense sRNA in the Gram-positive human pathogen Listeria monocytogenes

    DEFF Research Database (Denmark)

    Nielsen, Jesper Sejrup; Lei Kristensen, Lisbeth; Hanghøj Chrisitansen, Mie

    between sRNA and target mRNA rely on the RNA chaperone Hfq. Hfq is a ubiquitous protein found in almost all genres of bacterial life. However, so far its role as an RNA chaperone has only been described in Gram-negative species such as Escherichia coli and Salmonella (Vogel, J. 2009). We previously...... identified several Hfq-binding sRNAs in the Gram-positive human pathogen L. monocytogenes (Christiansen et al 2006). Through bioinformatics, we have identified a number of candidate targets for one of these sRNAs (LhrA). Here, we present the characterization of one of these targets. Our results suggest...

  20. The novel sRNA s015 improves nisin yield by increasing acid tolerance of Lactococcus lactis F44.

    Science.gov (United States)

    Qi, Jiakun; Caiyin, Qinggele; Wu, Hao; Tian, Kairen; Wang, Binbin; Li, Yanni; Qiao, Jianjun

    2017-08-01

    Nisin, a polycyclic antibacterial peptide produced by Lactococcus lactis, is stable at low pH. Improving the acid tolerance of L. lactis could thus enhance nisin yield. Small non-coding RNAs (sRNAs) play essential roles in acid tolerance by regulating their target mRNAs at the post-transcriptional level. In this study, a novel sRNA, s015, was identified in L. lactis F44 via the use of RNA sequencing, qRT-PCR analysis, and Northern blotting. s015 improved the acid tolerance of L. lactis and boosted nisin yield at low pH. In silico predictions enabled us to construct a library of possible s015 target mRNAs. Statistical analysis and validation suggested that s015 contains a highly conserved region (5'-GAAAAAAAC-3') that likely encompasses the regulatory core of the sRNA. atpG, busAB, cysD, ilvB, tcsR, ung, yudD, and ywdA were verified as direct targets of s015, and the interactions between s015 and its target genes were elucidated. This work provided new insight into the adaptation mechanism of L. lactis under acid stress.

  1. Determination of sRNA expressions by RNA-seq in Yersinia pestis grown in vitro and during infection.

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    Yanfeng Yan

    Full Text Available BACKGROUND: Small non-coding RNAs (sRNAs facilitate host-microbe interactions. They have a central function in the post-transcriptional regulation during pathogenic lifestyles. Hfq, an RNA-binding protein that many sRNAs act in conjunction with, is required for Y. pestis pathogenesis. However, information on how Yersinia pestis modulates the expression of sRNAs during infection is largely unknown. METHODOLOGY AND PRINCIPAL FINDINGS: We used RNA-seq technology to identify the sRNA candidates expressed from Y. pestis grown in vitro and in the infected lungs of mice. A total of 104 sRNAs were found, including 26 previously annotated sRNAs, by searching against the Rfam database with 78 novel sRNA candidates. Approximately 89% (93/104 of these sRNAs from Y. pestis are shared with its ancestor Y. pseudotuberculosis. Ninety-seven percent of these sRNAs (101/104 are shared among more than 80 sequenced genomes of 135 Y. pestis strains. These 78 novel sRNAs include 62 intergenic and 16 antisense sRNAs. Fourteen sRNAs were selected for verification by independent Northern blot analysis. Results showed that nine selected sRNA transcripts were Hfq-dependent. Interestingly, three novel sRNAs were identified as new members of the transcription factor CRP regulon. Semi-quantitative analysis revealed that Y. pestis from the infected lungs induced the expressions of six sRNAs including RyhB1, RyhB2, CyaR/RyeE, 6S RNA, RybB and sR039 and repressed the expressions of four sRNAs, including CsrB, CsrC, 4.5S RNA and sR027. CONCLUSIONS AND SIGNIFICANCE: This study is the first attempt to subject RNA from Y. pestis-infected samples to direct high-throughput sequencing. Many novel sRNAs were identified and the expression patterns of relevant sRNAs in Y. pestis during in vitro growth and in vivo infection were revealed. The annotated sRNAs accounted for the most abundant sRNAs either expressed in bacteria grown in vitro or differentially expressed in the infected lungs

  2. The UEA sRNA Workbench (version 4.4): a comprehensive suite of tools for analyzing miRNAs and sRNAs.

    Science.gov (United States)

    Stocks, Matthew B; Mohorianu, Irina; Beckers, Matthew; Paicu, Claudia; Moxon, Simon; Thody, Joshua; Dalmay, Tamas; Moulton, Vincent

    2018-05-02

    RNA interference, a highly conserved regulatory mechanism, is mediated via small RNAs. Recent technical advances enabled the analysis of larger, complex datasets and the investigation of microRNAs and the less known small interfering RNAs. However, the size and intricacy of current data requires a comprehensive set of tools, able to discriminate the patterns from the low-level, noise-like, variation; numerous and varied suggestions from the community represent an invaluable source of ideas for future tools, the ability of the community to contribute to this software is essential. We present a new version of the UEA sRNA Workbench, reconfigured to allow an easy insertion of new tools/workflows. In its released form, it comprises of a suite of tools in a user-friendly environment, with enhanced capabilities for a comprehensive processing of sRNA-seq data e.g. tools for an accurate prediction of sRNA loci (CoLIde) and miRNA loci (miRCat2), as well as workflows to guide the users through common steps such as quality checking of the input data, normalization of abundances or detection of differential expression represent the first step in sRNA-seq analyses. The UEA sRNA Workbench is available at: http://srna-workbench.cmp.uea.ac.uk The source code is available at: https://github.com/sRNAworkbenchuea/UEA_sRNA_Workbench. v.moulton@uea.ac.uk.

  3. EsrE-A yigP Locus-Encoded Transcript-Is a 3′ UTR sRNA Involved in the Respiratory Chain of E. coli

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    Hui Xia

    2017-08-01

    Full Text Available The yigP locus is widely conserved among γ-proteobacteria. Mutation of the yigP locus impacts aerobic growth of Gram-negative bacteria. However, the underlying mechanism of how the yigP locus influences aerobic growth remains largely unknown. Here, we demonstrated that the yigP locus in Escherichia coli encodes two transcripts; the mRNA of ubiquinone biosynthesis protein, UbiJ, and the 3′ untranslated region small regulatory RNA (sRNA, EsrE. EsrE is an independent transcript that is transcribed using an internal promoter of the yigP locus. Surprisingly, we found that both the EsrE sRNA and UbiJ protein were required for Q8 biosynthesis, and were sufficient to rescue the growth defect ascribed to deletion of the yigP locus. Moreover, our data showed that EsrE targeted multiple mRNAs involved in several cellular processes including murein biosynthesis and the tricarboxylic acid cycle. Among these targets, sdhD mRNA that encodes one subunit of succinate dehydrogenase (SDH, was significantly activated. Our findings provided an insight into the important function of EsrE in bacterial adaptation to various environments, as well as coordinating different aspects of bacterial physiology.

  4. An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA.

    Science.gov (United States)

    Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O; Linne, Uwe; Albaum, Stefan P; Jiménez-Zurdo, José I; Becker, Anke

    2018-01-01

    Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans- encoded sRNA ( trans- sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving

  5. Effect of Crc and Hfq proteins on the transcription, processing, and stability of the Pseudomonas putida CrcZ sRNA.

    Science.gov (United States)

    Hernández-Arranz, Sofía; Sánchez-Hevia, Dione; Rojo, Fernando; Moreno, Renata

    2016-12-01

    In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN σ factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ΔcrcZΔcrcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3'-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases. © 2016 Hernández-Arranz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  6. Two Sets of Piwi Proteins Are Involved in Distinct sRNA Pathways Leading to Elimination of Germline-Specific DNA

    Directory of Open Access Journals (Sweden)

    Dominique I. Furrer

    2017-07-01

    Full Text Available Piwi proteins and piRNAs protect eukaryotic germlines against the spread of transposons. During development in the ciliate Paramecium, two Piwi-dependent sRNA classes are involved in the elimination of transposons and transposon-derived DNA: scan RNAs (scnRNAs, associated with Ptiwi01 and Ptiwi09, and iesRNAs, whose binding partners we now identify as Ptiwi10 and Ptiwi11. scnRNAs derive from the maternal genome and initiate DNA elimination during development, whereas iesRNAs continue DNA targeting until the removal process is complete. Here, we show that scnRNAs and iesRNAs are processed by distinct Dicer-like proteins and bind Piwi proteins in a mutually exclusive manner, suggesting separate biogenesis pathways. We also demonstrate that the PTIWI10 gene is transcribed from the developing nucleus and that its transcription depends on prior DNA excision, suggesting a mechanism of gene expression control triggered by the removal of short DNA segments interrupting the gene.

  7. Genomic Variability of O Islands Encoding Tellurite Resistance in Enterohemorrhagic Escherichia coli O157:H7 Isolates

    OpenAIRE

    Taylor, Diane E.; Rooker, Michelle; Keelan, Monika; Ng, Lai-King; Martin, Irene; Perna, Nicole T.; Burland, N. T. Valerie; Blattner, Fredrick R.

    2002-01-01

    Strains of Escherichia coli causing enterohemorrhagic colitis belonging to the O157:H7 lineage are reported to be highly related. Fifteen strains of E. coli O157:H7 and 1 strain of E. coli O46:H− (nonflagellated) were examined for the presence of potassium tellurite resistance (Ter). Ter genes comprising terABCDEF were shown previously to be part of a pathogenicity island also containing integrase, phage, and urease genes. PCR analysis, both conventional and light cycler based, demonstrated t...

  8. Genomic Variability of O Islands Encoding Tellurite Resistance in Enterohemorrhagic Escherichia coli O157:H7 Isolates

    Science.gov (United States)

    Taylor, Diane E.; Rooker, Michelle; Keelan, Monika; Ng, Lai-King; Martin, Irene; Perna, Nicole T.; Burland, N. T. Valerie; Blattner, Fredrick R.

    2002-01-01

    Strains of Escherichia coli causing enterohemorrhagic colitis belonging to the O157:H7 lineage are reported to be highly related. Fifteen strains of E. coli O157:H7 and 1 strain of E. coli O46:H− (nonflagellated) were examined for the presence of potassium tellurite resistance (Ter). Ter genes comprising terABCDEF were shown previously to be part of a pathogenicity island also containing integrase, phage, and urease genes. PCR analysis, both conventional and light cycler based, demonstrated that about one-half of the Ter E. coli O157:H7 strains (6 of 15), including the Sakai strain, which has been sequenced, carried a single copy of the Ter genes. Five of the strains, including EDL933, which has also been sequenced, contained two copies. Three other O157:H7 strains and the O46:H− strain did not contain the Ter genes. In strains containing two copies, the Ter genes were associated with the serW and serX tRNA genes. Five O157:H7 strains resembled the O157 Sakai strain whose sequence contained one copy, close to serX, whereas in one isolate the single copy was associated with serW. There was no correlation between Ter and the ability to produce Shiga toxin ST1 or ST2. The Ter MIC for most strains, containing either one or two copies, was 1,024 μg/ml, although for a few the MIC was intermediate, 64 to 128 μg/ml, which could be increased to 512 μg/ml by pregrowth of strains in subinhibitory concentrations of potassium tellurite. Reverse transcriptase PCR analysis confirmed that in most strains Ter was constitutive but that in the rest it was inducible and involved induction of terB and terC genes. Only the terB, -C, -D, and -E genes are required for Ter. The considerable degree of homology between the ter genes on IncH12 plasmid R478, which originated in Serratia marcescens, and pTE53, from an E. coli clinical isolate, suggests that the pathogenicity island was acquired from a plasmid. This work demonstrates diversity among E. coli O157:H7 isolates, at least as far as the presence of Ter genes is concerned. PMID:12169592

  9. Lymphoblast-derived integration-free ISRM-CON9 iPS cell line from a 75 year old female

    Directory of Open Access Journals (Sweden)

    Soraia Martins

    2018-01-01

    Full Text Available Human lymphoblast cells were used to generate integration-free induced pluripotent stem cells (iPSCs employing episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The derived iPSCs were defined as pluripotent based on (i expression of pluripotency-associated markers, (ii embryoid body-based differentiation into cell types representative of the three germ layers and (iii the similarity between the transcriptomes of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.95.

  10. A broad range quorum sensing inhibitor working through sRNA inhibition

    DEFF Research Database (Denmark)

    Jakobsen, Tim H.; Warming, Anders N.; Vejborg, Rebecca M.

    2017-01-01

    For the last decade, chemical control of bacterial virulence has received considerable attention. Ajoene, a sulfur-rich molecule from garlic has been shown to reduce expression of key quorum sensing regulated virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. Here we show...

  11. Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria

    Czech Academy of Sciences Publication Activity Database

    Hnilicová, Jarmila; Jirát-Matějčková, Jitka; Šiková, Michaela; Pospíšil, Jiří; Halada, Petr; Pánek, Josef; Krásný, Libor

    2014-01-01

    Roč. 42, č. 18 (2014), s. 11763-11776 ISSN 0305-1048 R&D Projects: GA ČR(CZ) GBP305/12/G034; GA ČR GP13-27150P Grant - others:Magistrát hl. m. P.(CZ) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388971 Keywords : ESCHERICHIA-COLI * 6S RNA * NONCODING RNA Subject RIV: EE - Microbiology, Virology Impact factor: 9.112, year: 2014

  12. The influence of prenatal X-irradiation on the activity of SRNA-aminoacyl synthetases in the developing rabbit brain

    International Nuclear Information System (INIS)

    Wender, M.; Zgorzalewicz, B.

    1976-01-01

    The activities of sRNA-aminoacyl synthetases were investigated in the cerebral white and grey matter of rabbits subjected during their prenatal life to a single x-ray dose of 150 rad. The results of investigations have shown that ionizing radiation acting during intrauterine development of the experimental animal brings about a distinct depression of all sRNA-aminoacyl synthetase activities in the newborn irradiated litter. During the postnatal development of these animals the activities of some of the synthetases further decreased and even at adulthood, where they are normally very low, their activities were below the control values. The activities of some other synthetases, after the initial depression, showed no further decrease and at adulthood had values comparable to controls. The results indicate clearly that prenatal exposure to ionizing radiation also affects the steps of protein biosynthesis which depend on the activity of sRNA-aminoacyl synthetases. (author)

  13. Cloacal microbiome structure in a long-distance migratory bird assessed using deep 16sRNA pyrosequencing

    Czech Academy of Sciences Publication Activity Database

    Kreisinger, Jakub; Čížková, Dagmar; Kropáčková, L.; Albrecht, Tomáš

    2015-01-01

    Roč. 10, č. 9 (2015), e0137401 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GAP506/12/2472; GA MŠk EE2.3.20.0303 Institutional support: RVO:68081766 Keywords : gastrointestinal microbiota * gut microbiome * sexual selection * immune-system * barn swallows * bacteria * evolution * communities * diversity * taxonomy Subject RIV: EG - Zoology Impact factor: 3.057, year: 2015

  14. The KL24 gene cluster and a genomic island encoding a Wzy polymerase contribute genes needed for synthesis of the K24 capsular polysaccharide by the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51.

    Science.gov (United States)

    Kenyon, Johanna J; Kasimova, Anastasiya A; Shneider, Mikhail M; Shashkov, Alexander S; Arbatsky, Nikolay P; Popova, Anastasiya V; Miroshnikov, Konstantin A; Hall, Ruth M; Knirel, Yuriy A

    2017-03-01

    The whole-genome sequence of the multiply antibiotic resistant Acinetobacter baumannii isolate RCH51 belonging to sequence type ST103 (Institut Pasteur scheme) revealed that the set of genes at the capsule locus, KL24, includes four genes predicted to direct the synthesis of 3-acetamido-3,6-dideoxy-d-galactose (d-Fuc3NAc), and this sugar was found in the capsular polysaccharide (CPS). One of these genes, fdtE, encodes a novel bifunctional protein with an N-terminal FdtA 3,4-ketoisomerase domain and a C-terminal acetyltransferase domain. KL24 lacks a gene encoding a Wzy polymerase to link the oligosaccharide K units to form the CPS found associated with isolate RCH51, and a wzy gene was found in a small genomic island (GI) near the cpn60 gene. This GI is in precisely the same location as another GI carrying wzy and atr genes recently found in several A. baumannii isolates, but it does not otherwise resemble it. The CPS isolated from RCH51, studied by sugar analysis and 1D and 2D 1H and 13C NMR spectroscopy, revealed that the K unit has a branched pentasaccharide structure made up of Gal, GalNAc and GlcNAc residues with d-Fuc3NAc as a side branch, and the K units are linked via a β-d-GlcpNAc-(1→3)-β-d-Galp linkage formed by the Wzy encoded by the GI. The functions of the glycosyltransferases encoded by KL24 were assigned to formation of specific bonds. A correspondence between the order of the genes in KL24 and other KL and the order of the linkages they form was noted, and this may be useful in future predictions of glycosyltransferase specificities.

  15. Gene Expressing and sRNA Sequencing Show That Gene Differentiation Associates with a Yellow Acer palmatum Mutant Leaf in Different Light Conditions.

    Science.gov (United States)

    Li, Shu-Shun; Li, Qian-Zhong; Rong, Li-Ping; Tang, Ling; Zhang, Bo

    2015-01-01

    Acer palmatum Thunb., like other maples, is a widely ornamental-use small woody tree for leaf shapes and colors. Interestingly, we found a yellow-leaves mutant "Jingling Huangfeng" turned to green when grown in shade or low-density light condition. In order to study the potential mechanism, we performed high-throughput sequencing and obtained 1,082 DEGs in leaves grown in different light conditions that result in A. palmatum significant morphological and physiological changes. A total of 989 DEGs were annotated and clustered, of which many DEGs were found associating with the photosynthesis activity and pigment synthesis. The expression of CHS and FDR gene was higher while the expression of FLS gene was lower in full-sunlight condition; this may cause more colorful substance like chalcone and anthocyanin that were produced in full-light condition, thus turning the foliage to yellow. Moreover, this is the first available miRNA collection which contains 67 miRNAs of A. palmatum, including 46 conserved miRNAs and 21 novel miRNAs. To get better understanding of which pathways these miRNAs involved, 102 Unigenes were found to be potential targets of them. These results will provide valuable genetic resources for further study on the molecular mechanisms of Acer palmatum leaf coloration.

  16. In Situ Redox Manipulation Field Injection Test Report - Hanford 100-H Area

    International Nuclear Information System (INIS)

    Fruchter, J.S.; Amonette, J.E.; Cole, C.R.

    1996-11-01

    This report presents results of an In Situ Redox Manipulation (ISRM) Field Injection Withdrawal Test performed at the 100-H Area of the US. Department of Energy's (DOE's) Hanford Site in Washington State in Fiscal Year 1996 by researchers at Pacific Northwest National Laboratory (PNNL). The test is part of the overall ISRM project, the purpose of which is to determine the potential for remediating contaminated groundwater with a technology based on in situ manipulation of subsurface reduction-oxidation (redox) conditions. The ISRM technology would be used to treat subsurface contaminants in groundwater zones at DOE sites

  17. Duplex formation between the sRNA DsrA and rpoS mRNA is not sufficient for efficient RpoS synthesis at low temperature

    Czech Academy of Sciences Publication Activity Database

    Hämmerle, H.; Večerek, Branislav; Resch, A.; Bläsi, U.

    2013-01-01

    Roč. 10, č. 12 (2013), s. 1834-1841 ISSN 1547-6286 Institutional support: RVO:61388971 Keywords : DsrA * Hfq * Riboregulation Subject RIV: EE - Microbiology, Virology Impact factor: 5.377, year: 2013 http://www.ncbi.nlm.nih.gov/pubmed/24448230

  18. Predictive modelling of noise level generated during sawing of rocks ...

    Indian Academy of Sciences (India)

    This paper presents an experimental and statistical study on noise level generated .... hardness were determined according to related ISRM (1981) suggested methods. Thin section ..... tistical Package for the Social Sciences). Additionally, the ...

  19. Project Work Plan: Hanford 100-D Area Treatability Demonstration - In Situ Biostimulation for Reducing Barrier

    Energy Technology Data Exchange (ETDEWEB)

    Fruchter, Jonathan S.; Truex, Michael J.; Vermeul, Vince R.; Long, Philip E.

    2006-05-31

    This work plan supports a new, integrated approach to accelerate cleanup of chromium in the Hanford 100 Areas. This new approach will provide supplemental treatment upgradient of the ISRM barrier by directly treating chromium and other oxidizing species in groundwater (i.e., nitrate and dissolved oxygen), thereby increasing the longevity of the ISRM barrier and protecting the ecological receptors and human health at the river boundary.

  20. Trans-kingdom cross-talk

    DEFF Research Database (Denmark)

    Knip, Marijn; Constantin, Maria-Ermioni; Thordal-Christensen, Hans

    2014-01-01

    This review focuses on the mobility of small RNA (sRNA) molecules from the perspective of trans-kingdom gene silencing. Mobility of sRNA molecules within organisms is a well-known phenomenon, facilitating gene silencing between cells and tissues. sRNA signals are also transmitted between organism...

  1. Evaluation of fall chinook salmon spawning adjacent to the In-Situ Redox Manipulation treatability test site, Hanford Site, Washington

    International Nuclear Information System (INIS)

    Mueller, R.P.; Geist, D.R.

    1998-10-01

    The In Situ Redox Manipulation (ISRM) experiment is being evaluated as a potential method to remove contaminants from groundwater adjacent to the Columbia River near the 100-D Area. The ISRM experiment involves using sodium dithionate (Na 2 O 6 S 2 ) to precipitate chromate from the groundwater. The treatment will likely create anoxic conditions in the groundwater down-gradient of the ISRM treatability test site; however, the spatial extent of this anoxic plume is not exactly known. Surveys were conducted in November 1997, following the peak spawning of fall chinook salmon. Aerial surveys documented 210 redds (spawning nests) near the downstream island in locations consistent with previous surveys. Neither aerial nor underwater surveys documented fall chinook spawning in the vicinity of the ISRM treatability test site. Based on measurements of depth, velocity, and substrate, less than 1% of the study area contained suitable fall chinook salmon spawning habitat, indicating low potential for fall chinook salmon to spawn in the vicinity of the ISRM experiment

  2. TargetRNA: a tool for predicting targets of small RNA action in bacteria

    OpenAIRE

    Tjaden, Brian

    2008-01-01

    Many small RNA (sRNA) genes in bacteria act as posttranscriptional regulators of target messenger RNAs. Here, we present TargetRNA, a web tool for predicting mRNA targets of sRNA action in bacteria. TargetRNA takes as input a genomic sequence that may correspond to an sRNA gene. TargetRNA then uses a dynamic programming algorithm to search each annotated message in a specified genome for mRNAs that evince basepair-binding potential to the input sRNA sequence. Based on the calculated basepair-...

  3. Plodnost i veličina legla kod europske srne (capreolus capreolus, L.) u šumi Haljevo

    OpenAIRE

    Nikolandić, Đuro; Degmečić, Dražen

    2007-01-01

    U razdoblju od 1968. do 1972. godine vršena su ekološka istraživanja srna u šumama Baranje. Iz toga doba jednim projektom istraživanja postavljen je cilj ustanoviti plodnost populacije srna u šumi Haljevo. Redovito parenje srna počinjalo je u drugoj polovici srpnja a završavalo oko sredine kolovoza. Zametak (embrij) oplođenih srna je zbog postojanja embriotenije u fazi mirovanja, odnosno zbog neprimjetne diobe stanica zametka, sve do kraja prosinca, prostim okom čovjeka nije primjetan. Iz tog...

  4. “caracterización de bacterias metalofijadoras de mercurío, a través de la subunidad 16srna, mediante la técnica de pcr-dgge del rio gala (aguas abajo en el recinto San Rafael) en la parroquia tenguel”

    OpenAIRE

    Llivisaca, Susana Alexandra; Burgos, Felix Adolfo; Vargas, Jeffrey David

    2011-01-01

    El proyecto sobre el cual tratamos, se refiere el uso de la técnica molecular PCR-DGGE, para la caracterización de bacterias, y su potencial existencia en las aguas del río Gala de la parroquia Tenguel. La selección del sitio se basó al alto grado de contaminación con metales pesados en la que este se encuentra este rio. La probabilidad sobre la presencia de microorganismos con capacidad metalofijadora, es alta dada sus características biológicas y bioquímicas con las que cuenta esta bacteria...

  5. Developing and exploiting a unique seismic dataset from South African gold mines for source characterization and wave propagation

    CSIR Research Space (South Africa)

    Julia, J

    2008-09-01

    Full Text Available peaking and yield estimation, submitted to Bull. Seism. Soc. Am. Spottiswoode, S. and L. Linzer (2003). Improved seismic event locations, ISRM 2003: Technology Roadmap for Rock Mechanics 1–6. Trifu, C. I., D. Angus, and V. Shumila (2000). A fast...

  6. Teaching an Interdisciplinary Graduate-Level Methods Course in an Openly-Networked Connected Learning Environment: A Glass Half-Full

    Science.gov (United States)

    Secret, Mary; Bryant, Nita L.; Cummings, Cory R.

    2017-01-01

    Our paper describes the design and delivery of an online interdisciplinary social science research methods course (ISRM) for graduate students in sociology, education, social work, and public administration. Collaborative activities and learning took place in two types of computer-mediated learning environments: a closed Blackboard course…

  7. 100-D Area In Situ Redox Treatability Test for Chromate-Contaminated Groundwater

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Mark D.; Vermeul, Vincent R.; Szecsody, James E.; Fruchter, Jonathan S.

    2000-10-12

    A treatability test was conducted for the In Situ Redox Manipulation (ISRM) technology at the 100 D Area of the U. S. Department of Energy's Hanford Site. The target contaminant was dissolved chromate in groundwater. The ISRM technology creates a permeable subsurface treatment zone to reduce mobile chromate in groundwater to an insoluble form. The ISRM permeable treatment zone is created by reducing ferric iron to ferrous iron within the aquifer sediments, which is accomplished by injecting aqueous sodium dithionite into the aquifer and then withdrawing the reaction products. The goal of the treatability test was to create a linear ISRM barrier by injecting sodium dithionite into five wells. Well installation and site characterization activities began in spring 1997; the first dithionite injection took place in September 1997. The results of this first injection were monitored through the spring of 1998. The remaining four dithionite injections were carried out in May through July of 1998.These five injections created a reduced zone in the Hanford unconfined aquifer approximately 150 feet in length (perpendicular to groundwater flow) and 50 feet wide. The reduced zone extended over the thickness of the unconfined zone. Analysis of post-emplacement groundwater samples showed concentrations of chromate, in the reduced zone decreased from approximately 1.0 mg/L before the tests to below analytical detection limits (<0.007 mg/L). Chromate concentrations also declined in downgradient monitoring wells to as low as 0.020 mg/L. These data, in addition to results from pre-test reducible iron characterization, indicate the barrier should be effective for 20 to 25 years. The 100-D Area ISRM barrier is being expanded to a length of up to 2,300 ft to capture a larger portion of the chromate plume.

  8. Identification and expression profiles of sRNAs and their biogenesis and action-related genes in male and female cones of Pinus tabuliformis.

    Science.gov (United States)

    Niu, Shi-Hui; Liu, Chang; Yuan, Hu-Wei; Li, Pei; Li, Yue; Li, Wei

    2015-09-15

    Small RNA (sRNA) play pivotal roles in reproductive development, and their biogenesis and action mechanisms are well characterised in angiosperm plants; however, corresponding studies in conifers are very limited. To improve our understanding of the roles of sRNA pathways in the reproductive development of conifers, the genes associated with sRNA biogenesis and action pathways were identified and analysed, and sRNA sequencing and parallel analysis of RNA ends (PARE) were performed in male and female cones of the Chinese pine (Pinus tabuliformis). Based on high-quality reference transcriptomic sequences, 21 high-confidence homologues involved in sRNA biogenesis and action in P. tabuliformis were identified, including two different DCL3 genes and one AGO4 gene. More than 75 % of genes involved in sRNA biogenesis and action have higher expression levels in female than in male cones. Twenty-six microRNA (miRNA) families and 74 targets, including 46 24-nt sRNAs with a 5' A, which are specifically expressed in male cones or female cones and probably bind to AGO4, were identified. The sRNA pathways have higher activity in female than in male cones, and the miRNA pathways are the main sRNA pathways in P. tabuliformis. The low level of 24-nt short-interfering RNAs in conifers is not caused by the absence of biogenesis-related genes or AGO-binding proteins, but most likely caused by the low accumulation of these key components. The identification of sRNAs and their targets, as well as genes associated with sRNA biogenesis and action, will provide a good starting point for investigations into the roles of sRNA pathways in cone development in conifers.

  9. Geologic, geochemical, microbiologic, and hydrologic characterization at the In Situ Redox Manipulation Test Site

    International Nuclear Information System (INIS)

    Vermeul, V.R.; Teel, S.S.; Amonette, J.E.

    1995-07-01

    This report documents results from characterization activities at the In Situ Redox Manipulation (ISRM) Field Test Site which is located within the 100-HR-3 Operable Unit of the US Department of Energy's (DOE's) Hanford Site in Richland, Washington. Information obtained during hydrogeologic characterization of the site included sediment physical properties, geochemical properties, microbiologic population data, and aquifer hydraulic properties. The purpose of obtaining this information was to improve the conceptual understanding of the hydrogeology beneath the ISRM test site and provide detailed, site specific hydrogeologic parameter estimates. The resulting characterization data will be incorporated into a numerical model developed to simulate the physical and chemical processes associated with the field experiment and aid in experiment design and interpretation

  10. Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli

    DEFF Research Database (Denmark)

    Boysen, Anders; Møller-Jensen, Jakob; Kallipolitis, Birgitte H.

    2010-01-01

    Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post...... that adaptation to anaerobic growth involves the action of a small regulatory RNA....... of at least one sRNA regulator. Here, we extend this view by the identification and characterization of a highly conserved, anaerobically induced small sRNA in E. coli, whose expression is strictly dependent on the anaerobic transcriptional fumarate and nitrate reductase regulator (FNR). The sRNA, named Fnr...

  11. Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1-ribonucleoprotein complex.

    Science.gov (United States)

    Ham, Byung-Kook; Li, Gang; Jia, Weitao; Leary, Julie A; Lucas, William J

    2014-11-01

    In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  12. Influence of Nitrate on the Hanford 100D Area In Situ Redox Manipulation Barrier Longevity

    International Nuclear Information System (INIS)

    Szecsody, Jim E.; Phillips, Jerry L.; Vermeul, Vince R.; Fruchter, Jonathan S.; Williams, Mark D.

    2005-01-01

    The purpose of this laboratory study is to determine the influence of nitrate on the Hanford 100D Area in situ redox manipulation (ISRM) barrier longevity. There is a wide spread groundwater plume of 60 mg/L nitrate upgradient of the ISRM barrier with lower nitrate concentrations downgradient, suggestive of nitrate reduction occurring. Batch and 1-D column experiments showed that nitrate is being slowly reduced to nitrite and ammonia. These nitrate reduction reactions are predominantly abiotic, as experiments with and without bactericides present showed no difference in nitrate degradation rates. Nitrogen species transformation rates determined in experiments covered a range of ferrous iron/nitrate ratios such that the data can be used to predict rates in field scale conditions. Field scale reaction rate estimates for 100% reduced sediment (16 C) are: (a) nitrate degradation = 202 ± 50 h (half-life), (b) nitrite production = 850 ± 300 h, and (c) ammonia production = 650 ± 300 h. Calculation of the influence of nitrate reduction on the 100D Area reductive capacity requires consideration of mass balance and reaction rate effects. While dissolved oxygen and chromate reduction rates are rapid and essentially at equilibrium in the aquifer, nitrate transformation reactions are slow (100s of hours). In the limited (20-40 day) residence time in the ISRM barrier, only a portion of the nitrate will be reduced, whereas dissolved oxygen and chromate are reduced to completion. Assuming a groundwater flow rate of 1 ft/day, it is estimated that the ISRM barrier reductive capacity is 160 pore volumes (with no nitrate), and 85 pore volumes if 60 mg/L nitrate is present (i.e., a 47% decrease in the ISRM barrier longevity). Zones with more rapid groundwater flow will be less influenced by nitrate reduction. For example, a zone with a groundwater flow rate of 3 ft/day and 60 mg/L nitrate will have a reductive capacity of 130 pore volumes. Finally, long-term column experiments

  13. Genome-wide Annotation, Identification, and Global Transcriptomic Analysis of Regulatory or Small RNA Gene Expression in Staphylococcus aureus.

    Science.gov (United States)

    Carroll, Ronan K; Weiss, Andy; Broach, William H; Wiemels, Richard E; Mogen, Austin B; Rice, Kelly C; Shaw, Lindsey N

    2016-02-09

    In Staphylococcus aureus, hundreds of small regulatory or small RNAs (sRNAs) have been identified, yet this class of molecule remains poorly understood and severely understudied. sRNA genes are typically absent from genome annotation files, and as a consequence, their existence is often overlooked, particularly in global transcriptomic studies. To facilitate improved detection and analysis of sRNAs in S. aureus, we generated updated GenBank files for three commonly used S. aureus strains (MRSA252, NCTC 8325, and USA300), in which we added annotations for >260 previously identified sRNAs. These files, the first to include genome-wide annotation of sRNAs in S. aureus, were then used as a foundation to identify novel sRNAs in the community-associated methicillin-resistant strain USA300. This analysis led to the discovery of 39 previously unidentified sRNAs. Investigating the genomic loci of the newly identified sRNAs revealed a surprising degree of inconsistency in genome annotation in S. aureus, which may be hindering the analysis and functional exploration of these elements. Finally, using our newly created annotation files as a reference, we perform a global analysis of sRNA gene expression in S. aureus and demonstrate that the newly identified tsr25 is the most highly upregulated sRNA in human serum. This study provides an invaluable resource to the S. aureus research community in the form of our newly generated annotation files, while at the same time presenting the first examination of differential sRNA expression in pathophysiologically relevant conditions. Despite a large number of studies identifying regulatory or small RNA (sRNA) genes in Staphylococcus aureus, their annotation is notably lacking in available genome files. In addition to this, there has been a considerable lack of cross-referencing in the wealth of studies identifying these elements, often leading to the same sRNA being identified multiple times and bearing multiple names. In this work

  14. StarScan: a web server for scanning small RNA targets from degradome sequencing data.

    Science.gov (United States)

    Liu, Shun; Li, Jun-Hao; Wu, Jie; Zhou, Ke-Ren; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

    2015-07-01

    Endogenous small non-coding RNAs (sRNAs), including microRNAs, PIWI-interacting RNAs and small interfering RNAs, play important gene regulatory roles in animals and plants by pairing to the protein-coding and non-coding transcripts. However, computationally assigning these various sRNAs to their regulatory target genes remains technically challenging. Recently, a high-throughput degradome sequencing method was applied to identify biologically relevant sRNA cleavage sites. In this study, an integrated web-based tool, StarScan (sRNA target Scan), was developed for scanning sRNA targets using degradome sequencing data from 20 species. Given a sRNA sequence from plants or animals, our web server performs an ultrafast and exhaustive search for potential sRNA-target interactions in annotated and unannotated genomic regions. The interactions between small RNAs and target transcripts were further evaluated using a novel tool, alignScore. A novel tool, degradomeBinomTest, was developed to quantify the abundance of degradome fragments located at the 9-11th nucleotide from the sRNA 5' end. This is the first web server for discovering potential sRNA-mediated RNA cleavage events in plants and animals, which affords mechanistic insights into the regulatory roles of sRNAs. The StarScan web server is available at http://mirlab.sysu.edu.cn/starscan/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Genome-Wide Identification, Characterization, and Expression Analysis of Small RNA Biogenesis Purveyors Reveal Their Role in Regulation of Biotic Stress Responses in Three Legume Crops

    Directory of Open Access Journals (Sweden)

    Rajeev K. Varshney

    2017-04-01

    Full Text Available Biotic stress in legume crops is one of the major threats to crop yield and productivity. Being sessile organisms, plants have evolved a myriad of mechanisms to combat different stresses imposed on them. One such mechanism, deciphered in the last decade, is small RNA (sRNA mediated defense in plants. Small RNAs (sRNAs have emerged as one of the major players in gene expression regulation in plants during developmental stages and under stress conditions. They are known to act both at transcriptional and post-transcriptional levels. Dicer-like (DCL, Argonaute (AGO, and RNA dependent RNA polymerase (RDR constitute the major components of sRNA biogenesis machinery and are known to play a significant role in combating biotic and abiotic stresses. This study is, therefore, focused on identification and characterization of sRNA biogenesis proteins in three important legume crops, namely chickpea, pigeonpea, and groundnut. Phylogenetic analysis of these proteins between legume species classified them into distinct clades and suggests the evolutionary conservation of these genes across the members of Papillionidoids subfamily. Variable expression of sRNA biogenesis genes in response to the biotic stresses among the three legumes indicate the possible existence of specialized regulatory mechanisms in different legumes. This is the first ever study to understand the role of sRNA biogenesis genes in response to pathogen attacks in the studied legumes.

  16. Analysis of small RNA production patterns among the two potato spindle tuber viroid variants in tomato plants

    Directory of Open Access Journals (Sweden)

    Charith Raj Adkar-Purushothama

    2015-12-01

    Full Text Available In order to analyze the production of small RNA (sRNA by viroids upon infecting the plants, the tomato plants (Solanum lycopersicum cultivar Rutgers were inoculated with the variants of Potato spindle tuber viroid (PSTVd. After 21-days of postinoculation, total RNA was extracted and subjected for deep-sequencing using Illumina HiSeq platform. The primers were trimmed and only 21- to 24-nt long sRNAs were filtered after quality check of the raw data. The filtered sRNA population was then mapped against both the genomic (+ and antigenomic (− strands of the respective PSTVd variants using standard pattern-matching algorithm. The profiling of viroid derived sRNA (vd-sRNA revealed that the viroids are susceptible to host RNA silencing mechanism. High-throughput sequence data linked to this project have been deposited in the Gene Expression Omnibus (GEO database under accession number GSE69225.

  17. Risk management of energy system for identifying optimal power mix with financial-cost minimization and environmental-impact mitigation under uncertainty

    International Nuclear Information System (INIS)

    Nie, S.; Li, Y.P.; Liu, J.; Huang, Charley Z.

    2017-01-01

    An interval-stochastic risk management (ISRM) method is launched to control the variability of the recourse cost as well as to capture the notion of risk in stochastic programming. The ISRM method can examine various policy scenarios that are associated with economic penalties under uncertainties presented as probability distributions and interval values. An ISRM model is then formulated to identify the optimal power mix for the Beijing's energy system. Tradeoffs between risk and cost are evaluated, indicating any change in targeted cost and risk level would yield different expected costs. Results reveal that the inherent uncertainty of system components and risk attitude of decision makers have significant effects on the city's energy-supply and electricity-generation schemes as well as system cost and probabilistic penalty. Results also disclose that import electricity as a recourse action to compensate the local shortage would be enforced. The import electricity would increase with a reduced risk level; under every risk level, more electricity would be imported with an increased demand. The findings can facilitate the local authority in identifying desired strategies for the city's energy planning and management in association with financial-cost minimization and environmental-impact mitigation. - Highlights: • Interval-stochastic risk management method is launched to identify optimal power mix. • It is advantageous in capturing the notion of risk in stochastic programming. • Results reveal that risk attitudes can affect optimal power mix and financial cost. • Developing renewable energies would enhance the sustainability of energy management. • Import electricity as an action to compensate the local shortage would be enforced.

  18. Construction of new synthetic biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002.

    Science.gov (United States)

    Zess, Erin K; Begemann, Matthew B; Pfleger, Brian F

    2016-02-01

    Predictive control of gene expression is an essential tool for developing synthetic biological systems. The current toolbox for controlling gene expression in cyanobacteria is a barrier to more in-depth genetic analysis and manipulation. Towards relieving this bottleneck, this work describes the use of synthetic biology to construct an anhydrotetracycline-based induction system and adapt a trans-acting small RNA (sRNA) system for use in the cyanobacterium Synechococcus sp. strain PCC 7002. An anhydrotetracycline-inducible promoter was developed to maximize intrinsic strength and dynamic range. The resulting construct, PEZtet , exhibited tight repression and a maximum 32-fold induction upon addition of anhydrotetracycline. Additionally, a sRNA system based on the Escherichia coli IS10 RNA-IN/OUT regulator was adapted for use in Synechococcus sp. strain PCC 7002. This system exhibited 70% attenuation of target gene expression, providing a demonstration of the use of sRNAs for differential gene expression in cyanobacteria. These systems were combined to produce an inducible sRNA system, which demonstrated 59% attenuation of target gene expression. Lastly, the role of Hfq, a critical component of sRNA systems in E. coli, was investigated. Genetic studies showed that the Hfq homolog in Synechococcus sp. strain PCC 7002 did not impact repression by the engineered sRNA system. In summary, this work describes new synthetic biology tools that can be applied to physiological studies, metabolic engineering, or sRNA platforms in Synechococcus sp. strain PCC 7002. © 2015 Wiley Periodicals, Inc.

  19. CoLIde

    Science.gov (United States)

    Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

    2013-01-01

    Small RNAs (sRNAs) are 20–25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the genomic location of the constituent sRNAs, hindering existing approaches to identify sRNA loci.   To infer the location of significant biological units, we propose an approach for sRNA loci detection called CoLIde (Co-expression based sRNA Loci Identification) that combines genomic location with the analysis of other information such as variation in expression levels (expression pattern) and size class distribution. For CoLIde, we define a locus as a union of regions sharing the same pattern and located in close proximity on the genome. Biological relevance, detected through the analysis of size class distribution, is also calculated for each locus. CoLIde can be applied on ordered (e.g., time-dependent) or un-ordered (e.g., organ, mutant) series of samples both with or without biological/technical replicates. The method reliably identifies known types of loci and shows improved performance on sequencing data from both plants (e.g., A. thaliana, S. lycopersicum) and animals (e.g., D. melanogaster) when compared with existing locus detection techniques. CoLIde is available for use within the UEA Small RNA Workbench which can be downloaded from: http://srna-workbench.cmp.uea.ac.uk. PMID:23851377

  20. CoLIde: a bioinformatics tool for CO-expression-based small RNA Loci Identification using high-throughput sequencing data.

    Science.gov (United States)

    Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

    2013-07-01

    Small RNAs (sRNAs) are 20-25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the genomic location of the constituent sRNAs, hindering existing approaches to identify sRNA loci. To infer the location of significant biological units, we propose an approach for sRNA loci detection called CoLIde (Co-expression based sRNA Loci Identification) that combines genomic location with the analysis of other information such as variation in expression levels (expression pattern) and size class distribution. For CoLIde, we define a locus as a union of regions sharing the same pattern and located in close proximity on the genome. Biological relevance, detected through the analysis of size class distribution, is also calculated for each locus. CoLIde can be applied on ordered (e.g., time-dependent) or un-ordered (e.g., organ, mutant) series of samples both with or without biological/technical replicates. The method reliably identifies known types of loci and shows improved performance on sequencing data from both plants (e.g., A. thaliana, S. lycopersicum) and animals (e.g., D. melanogaster) when compared with existing locus detection techniques. CoLIde is available for use within the UEA Small RNA Workbench which can be downloaded from: http://srna-workbench.cmp.uea.ac.uk.

  1. CoLIde: A bioinformatics tool for CO-expression based small RNA Loci Identification using high-throughput sequencing data

    OpenAIRE

    Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

    2013-01-01

    Small RNAs (sRNAs) are 20–25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the...

  2. 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

  3. Hanford 100-D Area Biostimulation Treatability Test Results

    Energy Technology Data Exchange (ETDEWEB)

    Truex, Michael J.; Vermeul, Vincent R.; Fritz, Brad G.; Mackley, Rob D.; Mendoza, Donaldo P.; Elmore, Rebecca P.; Mitroshkov, Alexandre V.; Sklarew, Deborah S.; Johnson, Christian D.; Oostrom, Martinus; Newcomer, Darrell R.; Brockman, Fred J.; Bilskis, Christina L.; Hubbard, Susan S.; Peterson, John E.; Williams, Kenneth H.; Gasperikova, E.; Ajo-Franklin, J.

    2009-09-30

    Pacific Northwest National Laboratory conducted a treatability test designed to demonstrate that in situ biostimulation can be applied to help meet cleanup goals in the Hanford Site 100-D Area. In situ biostimulation has been extensively researched and applied for aquifer remediation over the last 20 years for various contaminants. In situ biostimulation, in the context of this project, is the process of amending an aquifer with a substrate that induces growth and/or activity of indigenous bacteria for the purpose of inducing a desired reaction. For application at the 100-D Area, the purpose of biostimulation is to induce reduction of chromate, nitrate, and oxygen to remove these compounds from the groundwater. The in situ biostimulation technology is intended to provide supplemental treatment upgradient of the In Situ Redox Manipulation (ISRM) barrier previously installed in the Hanford 100-D Area and thereby increase the longevity of the ISRM barrier. Substrates for the treatability test were selected to provide information about two general approaches for establishing and maintaining an in situ permeable reactive barrier based on biological reactions, i.e., a biobarrier. These approaches included 1) use of a soluble (miscible) substrate that is relatively easy to distribute over a large areal extent, is inexpensive, and is expected to have moderate longevity; and 2) use of an immiscible substrate that can be distributed over a reasonable areal extent at a moderate cost and is expected to have increased longevity.

  4. Enhancing the design of in situ chemical barriers with multicomponent reactive transport modeling

    International Nuclear Information System (INIS)

    Sevougian, S.D.; Steefel, C.I.; Yabusaki, S.B.

    1994-11-01

    This paper addresses the need for systematic control of field-scale performance in the emplacement and operation of in situ chemical treatment barriers; in particular, it addresses the issue of how the local coupling of reaction kinetics and material heterogeneities at the laboratory or bench scale can be accurately upscaled to the field. The authors have recently developed modeling analysis tools that can explicitly account for all relevant chemical reactions that accompany the transport of reagents and contaminants through a chemically and physically heterogeneous subsurface rock or soil matrix. These tools are incorporated into an enhanced design methodology for in situ chemical treatment technologies, and the new methodology is demonstrated in the ongoing design of a field experiment for the In Situ Redox Manipulation (ISRM) project at the U.S. Department of Energy (DOE) Hanford Site. The ISRM design approach, which systematically integrates bench-scale and site characterization information, provides an ideal test for the new reactive transport techniques. The need for the enhanced chemistry capability is demonstrated by an example that shows how intra-aqueous redox kinetics can affect the transport of reactive solutes. Simulations are carried out on massively parallel computer architectures to resolve the influence of multiscale heterogeneities on multicomponent, multidimensional reactive transport. The technology will soon be available to design larger-scale remediation schemes

  5. Micron-Size Zero-Valent Iron Emplacement in Porous Media Using Polymer Additives: Column and Flow Cell Ex-periments

    Energy Technology Data Exchange (ETDEWEB)

    Oostrom, Mart; Wietsma, Thomas W.; Covert, Matthew A.; Vermeul, Vince R.

    2006-03-20

    At the Hanford Site, an extensive In Situ Redox Manipulation (ISRM) permeable reactive barrier was installed to prevent chromate from reaching the Columbia River. However, chromium has been detected in several wells, indicating a premature loss of the reductive capacity in the aquifer. Laboratory experiments have been conducted to investigate whether barrier reductive capacity can be enhanced by adding micron-scale zero-valent iron to the high-permeability zones within the aquifer using shear-thinning fluids containing polymers. Porous media were packed in a wedge-shaped flow cell to create either a heterogeneous layered system with a high-permeability zone between two low-permeability zones or a high-permeability channel sur-rounded by low-permeability materials. The injection flow rate, polymer type, polymer concentration, and injected pore volumes were determined based on preliminary short- and long-column experiments. The flow cell experiments indicated that iron concentration enhancements of at least 0.6% (w/w) could be obtained using moderate flow rates and injection of 30 pore volumes. The 0.6% amended Fe0 concentration would provide approximately 20 times the average reductive capacity that is provided by the dithionite-reduced iron in the ISRM barrier. Calculations show that a 1-m-long Fe0 amended zone with an average concentration of 0.6% w/w iron subject to a groundwater velocity of 1 m/day will have an estimated longevity of 7.2 years.

  6. Theoretical studies on sRNA-mediated regulation in bacteria

    Science.gov (United States)

    Chang, Xiao-Xue; Xu, Liu-Fang; Shi, Hua-Lin

    2015-12-01

    Small RNA(sRNA)-mediated post-transcriptional regulation differs from protein-mediated regulation. Through base-pairing, sRNA can regulate the target mRNA in a catalytic or stoichiometric manner. Some theoretical models were built for comparison of the protein-mediated and sRNA-mediated modes in the steady-state behaviors and noise properties. Many experiments demonstrated that a single sRNA can regulate several mRNAs, which causes crosstalk between the targets. Here, we focus on some models in which two target mRNAs are silenced by the same sRNA to discuss their crosstalk features. Additionally, the sequence-function relationship of sRNA and its role in the kinetic process of base-pairing have been highlighted in model building. Project supported by the National Basic Research Program of China (Grant No. 2013CB834100), the National Natural Science Foundation of China (Grant Nos. 11121403 and 11274320), the Open Project Program of State Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Sciences, China (Grant No. Y4KF171CJ1), the National Natural Science Foundation for Young Scholar of China (Grant No. 11304115), and the China Postdoctoral Science Foundation (Grant No. 2013M541282).

  7. Novel meiotic miRNAs and indications for a role of phasiRNAs in meiosis

    Science.gov (United States)

    Small RNAs (sRNA) add additional layers to the regulation of gene expression, with siRNAs directing gene silencing at the DNA level by RdDM (RNA-directed DNA methylation), and miRNAs directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolate...

  8. SoMART, a web server for miRNA, tasiRNA and target gene analysis in Solanaceae plants

    Science.gov (United States)

    Plant micro(mi)RNAs and trans-acting small interfering (tasi)RNAs mediate posttranscriptional silencing of genes and play important roles in a variety of biological processes. Although bioinformatics prediction and small (s)RNA cloning are the key approaches used for identification of miRNAs, tasiRN...

  9. An integrated expression atlas of miRNAs and their promoters in human and mouse

    DEFF Research Database (Denmark)

    de Rie, Derek; Abugessaisa, Imad; Alam, Tanvir

    2017-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libr...

  10. Genome-wide identification of novel small RNAs in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Gómez Lozano, María

    and RNA sequencing (RNAseq) technologies. The latter approach, in particular, has revolutionized sRNA discovery by enabling interrogation of the transcriptome at unprecedented depths. The size and complexity of the P. aeruginosa genome suggests that it encodes many hitherto undetected sRNAs. In this study...

  11. Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2.

    Science.gov (United States)

    Gyula, Péter; Baksa, Ivett; Tóth, Tamás; Mohorianu, Irina; Dalmay, Tamás; Szittya, György

    2018-06-01

    Plants substantially alter their developmental program upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15, 21 and 27 °C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive miRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants. This article is protected by copyright. All rights reserved.

  12. 6S RNA modulates growth and antibiotic production in Streptomyces coelicolor

    Czech Academy of Sciences Publication Activity Database

    Mikulík, Karel; Bobek, Jan; Zídková, J.; Felsberg, Jürgen

    2014-01-01

    Roč. 98, č. 16 (2014), s. 7185-7197 ISSN 0175-7598 R&D Projects: GA ČR GAP302/10/0468 Institutional support: RVO:61388971 Keywords : 6SRNA * Secondary structure * pRNAs Subject RIV: EE - Microbiology, Virology Impact factor: 3.337, year: 2014

  13. An efficient and high fidelity method for amplification, cloning and sequencing of complete tospovirus genomic RNA segments

    Science.gov (United States)

    Amplification and sequencing of the complete M- and S-RNA segments of Tomato spotted wilt virus and Impatiens necrotic spot virus as a single fragment is useful for whole genome sequencing of tospoviruses co-infecting a single host plant. It avoids issues associated with overlapping amplicon-based ...

  14. Rational Modular RNA Engineering Based on In Vivo Profiling of Structural Accessibility.

    Science.gov (United States)

    Leistra, Abigail N; Amador, Paul; Buvanendiran, Aishwarya; Moon-Walker, Alex; Contreras, Lydia M

    2017-12-15

    Bacterial small RNAs (sRNAs) have been established as powerful parts for controlling gene expression. However, development and application of engineered sRNAs has primarily focused on regulating novel synthetic targets. In this work, we demonstrate a rational modular RNA engineering approach that uses in vivo structural accessibility measurements to tune the regulatory activity of a multisubstrate sRNA for differential control of its native target network. Employing the CsrB global sRNA regulator as a model system, we use published in vivo structural accessibility data to infer the contribution of its local structures (substructures) to function and select a subset for engineering. We then modularly recombine the selected substructures, differentially representing those of presumed high or low functional contribution, to build a library of 21 CsrB variants. Using fluorescent translational reporter assays, we demonstrate that the CsrB variants achieve a 5-fold gradient of control of well-characterized Csr network targets. Interestingly, results suggest that less conserved local structures within long, multisubstrate sRNAs may represent better targets for rational engineering than their well-conserved counterparts. Lastly, mapping the impact of sRNA variants on a signature Csr network phenotype indicates the potential of this approach for tuning the activity of global sRNA regulators in the context of metabolic engineering applications.

  15. Dual function of the McaS small RNA in controlling biofilm formation

    DEFF Research Database (Denmark)

    Jørgensen, Mikkel Girke; Thomason, Maureen K.; Havelund, Johannes

    2013-01-01

    , and biofilm formation. Moreover, ectopic McaS expression leads to induction of two additional CsrA-repressed genes encoding diguanylate cyclases. Collectively, our study shows that McaS is a dual-function sRNA with roles in the two major post-transcriptional regulons controlled by the RNA-binding proteins Hfq...

  16. Mycoplasma non-coding RNA: identification of small RNAs and targets

    Directory of Open Access Journals (Sweden)

    Franciele Maboni Siqueira

    2016-10-01

    Full Text Available Abstract Background Bacterial non-coding RNAs act by base-pairing as regulatory elements in crucial biological processes. We performed the identification of trans-encoded small RNAs (sRNA from the genomes of Mycoplama hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis, which are Mycoplasma species that have been identified in the porcine respiratory system. Results A total of 47, 15 and 11 putative sRNAs were predicted in M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively. A comparative genomic analysis revealed the presence of species or lineage specific sRNA candidates. Furthermore, the expression profile of some M. hyopneumoniae sRNAs was determined by a reverse transcription amplification approach, in three different culture conditions. All tested sRNAs were transcribed in at least one condition. A detailed investigation revealed a differential expression profile for two M. hyopneumoniae sRNAs in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. Moreover, we analyzed sRNA-mRNA hybrids and accessed putative target genes for the novel sRNA candidates. The majority of the sRNAs showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity. Conclusion This study contributes to our knowledge of Mycoplasma sRNAs and their response to environmental changes. Furthermore, the mRNA target prediction provides a perspective for the characterization and comprehension of the function of the sRNA regulatory mechanisms.

  17. Comparative analysis among the small RNA populations of source, sink and conductive tissues in two different plant-virus pathosystems.

    Science.gov (United States)

    Herranz, Mari Carmen; Navarro, Jose Antonio; Sommen, Evelien; Pallas, Vicente

    2015-02-22

    In plants, RNA silencing plays a fundamental role as defence mechanism against viruses. During last years deep-sequencing technology has allowed to analyze the sRNA profile of a large variety of virus-infected tissues. Nevertheless, the majority of these studies have been restricted to a unique tissue and no comparative analysis between phloem and source/sink tissues has been conducted. In the present work, we compared the sRNA populations of source, sink and conductive (phloem) tissues in two different plant virus pathosystems. We chose two cucurbit species infected with two viruses very different in genome organization and replication strategy; Melon necrotic spot virus (MNSV) and Prunus necrotic ringspot virus (PNRSV). Our findings showed, in both systems, an increase of the 21-nt total sRNAs together with a decrease of those with a size of 24-nt in all the infected tissues, except for the phloem where the ratio of 21/24-nt sRNA species remained constant. Comparing the vsRNAs, both PNRSV- and MNSV-infected plants share the same vsRNA size distribution in all the analyzed tissues. Similar accumulation levels of sense and antisense vsRNAs were observed in both systems except for roots that showed a prevalence of (+) vsRNAs in both pathosystems. Additionally, the presence of overrepresented discrete sites along the viral genome, hot spots, were identified and validated by stem-loop RT-PCR. Despite that in PNRSV-infected plants the presence of vsRNAs was scarce both viruses modulated the host sRNA profile. We compare for the first time the sRNA profile of four different tissues, including source, sink and conductive (phloem) tissues, in two plant-virus pathosystems. Our results indicate that antiviral silencing machinery in melon and cucumber acts mainly through DCL4. Upon infection, the total sRNA pattern in phloem remains unchanged in contrast to the rest of the analyzed tissues indicating a certain tissue-tropism to this polulation. Independently of the

  18. Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

    Science.gov (United States)

    Chen, Jiandong

    2016-01-01

    ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression. PMID:27849174

  19. Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays

    Directory of Open Access Journals (Sweden)

    Patenge Nadja

    2012-10-01

    Full Text Available Abstract Background Small non-coding RNAs (sRNAs have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49, employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases. Results We identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of these, 42 were novel. Some of the newly-identified sRNAs belonged to one of the common non-coding RNA families described in the Rfam database. Comparison of the results of our screen with the outcome of two recently published bioinformatics tools showed a low level of overlap between putative sRNA genes. Previously, 40 potential sRNAs have been reported to be expressed in a GAS M1T1 serotype, as detected by a whole genome intergenic tiling array approach. Our screen detected 12 putative sRNA genes that were expressed in both strains. Twenty sRNA candidates appeared to be regulated in a medium-dependent fashion, while eight sRNA genes were regulated throughout growth in chemically defined medium. Expression of candidate genes was verified by reverse transcriptase-qPCR. For a subset of sRNAs, the transcriptional start was determined by 5′ rapid amplification of cDNA ends-PCR (RACE-PCR analysis. Conclusions In accord with the results of previous studies, we found little overlap between different screening methods, which underlines the fact that a comprehensive analysis of s

  20. 4th IFToMM International Symposium on Robotics and Mechatronics

    CERN Document Server

    Laribi, Med; Gazeau, Jean-Pierre

    2016-01-01

    This volume contains papers that have been selected after review for oral presentation at ISRM 2015, the Fourth IFToMM International Symposium on Robotics and Mechatronics held in Poitiers, France 23-24 June 2015. These papers  provide a vision of the evolution of the disciplines of robotics and mechatronics, including but not limited to: mechanism design; modeling and simulation; kinematics and dynamics of multibody systems; control methods; navigation and motion planning; sensors and actuators; bio-robotics; micro/nano-robotics; complex robotic systems; walking machines, humanoids-parallel kinematic structures: analysis and synthesis; smart devices; new design; application and prototypes. The book can be used by researchers and engineers in the relevant areas of robotics and mechatronics.

  1. Well Completion Report for the Fiscal Year 1999 Drilling Within the Chromium Plume West of the 100-D/DR Reactors

    International Nuclear Information System (INIS)

    Ford, B. H.

    1999-01-01

    This report describes the fiscal year (FY) 1999 field activities associated with installing 12 groundwater monitoring wells in the vicinity of the 100-D Area chromium plume west of the 100-D/DR Reactors (100-HR-3 Operable Unit [OU]). The wells were installed to further investigate the extent of the hexavalent chromium hot spot west of the 100-D/DR Reactors and to support future remedial action decisions associated with the 100-HR-3 OU. These wells were designed for multi-purpose use (i.e., monitoring, extraction, and injection). In addition, one of the wells was installed to support the initial deployment of the In Situ Redox Manipulation (ISRM) technology to remediate the chromium plume

  2. Experimental investigation of the mechanical properties of Alfas stone

    Directory of Open Access Journals (Sweden)

    Konstas N. Kaklis

    2017-04-01

    Full Text Available This paper focuses on the experimental investigation of the mechanical properties of the Alfas natural building stone. Two series of uniaxial compression tests and indirect tensile tests (Brazilian tests were performed in order to determine the uniaxial compressive strength and the indirect tensile strength respectively. Different sets of cylindrical specimens and circular discs were prepared by varying their geometry in order to examine the size effect on the respective strength values. Also, the size effect was investigated with respect to the calculated intact rock modulus and Poisson’s ratio. All specimens were prepared by following the ISRM suggested methods and the load was applied using a stiff 1600 kN MTS hydraulic testing machine and a 500 kN load cell. Strain was measured using biaxial 0/90 stacked rosettes appropriately attached on each specimen.

  3. Engineering a Functional Small RNA Negative Autoregulation Network with Model-Guided Design.

    Science.gov (United States)

    Hu, Chelsea Y; Takahashi, Melissa K; Zhang, Yan; Lucks, Julius B

    2018-05-22

    RNA regulators are powerful components of the synthetic biology toolbox. Here, we expand the repertoire of synthetic gene networks built from these regulators by constructing a transcriptional negative autoregulation (NAR) network out of small RNAs (sRNAs). NAR network motifs are core motifs of natural genetic networks, and are known for reducing network response time and steady state signal. Here we use cell-free transcription-translation (TX-TL) reactions and a computational model to design and prototype sRNA NAR constructs. Using parameter sensitivity analysis, we design a simple set of experiments that allow us to accurately predict NAR function in TX-TL. We transfer successful network designs into Escherichia coli and show that our sRNA transcriptional network reduces both network response time and steady-state gene expression. This work broadens our ability to construct increasingly sophisticated RNA genetic networks with predictable function.

  4. Intercomparison on the usage of computational codes in radiation dosimetry

    International Nuclear Information System (INIS)

    Ilic, R.; Pesic, M.; Pavlovic, R.

    2003-01-01

    SRNA-2KG software package was modified for this work to include necessary input and output data and for predicted voxelized geometry and dosimetry. SRNA is a Monte Carlo code developed for applications in proton transport, radiotherapy and dosimetry. Protons within energy range from 100 keV to 250 MeV with predefined spectra are transported in 3D geometry through material zones confined by planes and second order surfaces or in 3D voxelized geometry. The code can treat proton transport in a few hundred different materials including elements from Z=1 to Z=98. Simulation of proton transport is based on the multiple scattering theory of charged particles and on the model for compound nucleus decay

  5. Final report for ER65039, The Role of Small RNA in Biomass Deposition

    Energy Technology Data Exchange (ETDEWEB)

    Hudson, Matthew E. [Univ. of Illinois, Urbana, IL (United States)

    2015-03-12

    Our objective in this project was to discover the role of sRNA in regulating both biomass biosynthesis and perenniality in the Andropogoneae feedstock grasses. Our central hypothesis was that there is a time-and space specific sRNA network playing a crucial role in regulating processes associated with cell wall biosynthesis, flowering time control, overwintering/juvenility, and nutrient sequestration in the feedstock grasses. To address this, we performed a large scale biological project consisting of the growth of material, generation of Illumina libraries, sequencing and analysis for small RNA, mRNA and Degradome / cmRNA. Our subsidiary objectives included analysis of the biology of small RNAs and the cell wall composition of Miscanthus. These objectives have all been completed, one publication is in print, one is submitted and several more are in progress.

  6. Genome‐wide identification of novel small RNAs in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Gómez Lozano, María; Marvig, Rasmus Lykke; Molin, Søren

    2012-01-01

    Bacterial small regulatory RNAs (sRNAs) function in post‐transcriptional control of gene expression and control a variety of processes including metabolic reactions, stress responses and pathogenesis in response to environmental signals. A variety of approaches have been used previously to identify...... with this approach. Although the use of three libraries increased the number of novel transcripts identified, there were significant differences in the subset of transcripts detected in each library, underscoring the importance of library preparation strategy and relative sRNA abundance for successful sRNA detection...... and that the approach described here may be applied to identify sRNAs in any bacterium under different growth and stress conditions....

  7. The influence of jaw's curvature on the results of the Brazilian disc test

    Directory of Open Access Journals (Sweden)

    Ch.F. Markides

    2016-04-01

    Full Text Available The general contact problem of a disc squeezed between jaws of arbitrary curvature is considered employing Muskhelishvili's complex potentials. Taking advantage of the general solution introduced, the closed-form expressions for the stresses along strategic loci (loaded rim, loaded diameter, disc's center are obtained, in terms of the ratio ρ of the disc's to the jaw's curvature. Then, the effect of ρ (as well as that of the relative stiffness of the disc's and jaw's materials dictating the contact arc on the stress distribution along these loci is explored. It is concluded that, for both smooth contact (zero friction and contact with friction, the role of the jaw's curvature is significant not only along the disc-jaw contact arc (as it could be expected, but also all along the loaded diameter. On the other hand, it is indicated that the stress field at the disc's center is more or less insensitive to the jaw's curvature assuming that ρ lies within the range (0, 0.67 or in other words within the limits defined by the two standardized suggestions, i.e. that of American Society for Testing and Materials (ASTM (plane loading platens with ρ = 0 and that of International Society for Rock Mechanics (ISRM (curved jaws with ρ = 0.67. The upper limit of this range is a kind of compromise between the need to make the stress field at the disc's center independent of the boundary conditions while keeping at the same time the contact angle large enough to reduce the stress concentration and the risk for premature fracture initiation far from the disc's center. For jaws with radius of curvature exceeded by that suggested by ISRM, the stress field at the disc's center is significantly influenced. Especially for jaws with radius approaching that of the disc, the stress field at the disc's center is dramatically distorted rendering Hondros' formula inapplicable and the test results erroneous.

  8. Biotechnological uses of RNAi in plants: risk assessment considerations.

    Science.gov (United States)

    Casacuberta, Josep M; Devos, Yann; du Jardin, Patrick; Ramon, Matthew; Vaucheret, Hervé; Nogué, Fabien

    2015-03-01

    RNAi offers opportunities to generate new traits in genetically modified (GM) plants. Instead of expressing novel proteins, RNAi-based GM plants reduce target gene expression. Silencing of off-target genes may trigger unintended effects, and identifying these genes would facilitate risk assessment. However, using bioinformatics alone is not reliable, due to the lack of genomic data and insufficient knowledge of mechanisms governing mRNA-small (s)RNA interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Genome-wide identification of microRNA and siRNA responsive to endophytic beneficial diazotrophic bacteria in maize.

    Science.gov (United States)

    Thiebaut, Flávia; Rojas, Cristian A; Grativol, Clícia; Motta, Mariana Romeiro; Vieira, Tauan; Regulski, Michael; Martienssen, Robert A; Farinelli, Laurent; Hemerly, Adriana S; Ferreira, Paulo C G

    2014-09-06

    Small RNA (sRNA) has been described as a regulator of gene expression. In order to understand the role of maize sRNA (Zea mays-hybrid UENF 506-8) during association with endophytic nitrogen-fixing bacteria, we analyzed the sRNA regulated by its association with two diazotrophic bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. Deep sequencing analysis was done with RNA extracted from plants inoculated with H. seropedicae, allowing the identification of miRNA and siRNA. A total of 25 conserved miRNA families and 15 novel miRNAs were identified. A dynamic regulation in response to inoculation was also observed. A hypothetical model involving copper-miRNA is proposed, emphasizing the fact that the up-regulation of miR397, miR398, miR408 and miR528, which is followed by inhibition of their targets, can facilitate association with diazotrophic bacteria. Similar expression patterns were observed in samples inoculated with A. brasilense. Moreover, novel miRNA and siRNA were classified in the Transposable Elements (TE) database, and an enrichment of siRNA aligned with TE was observed in the inoculated samples. In addition, an increase in 24-nt siRNA mapping to genes was observed, which was correlated with an increase in methylation of the coding regions and a subsequent reduction in transcription. Our results show that maize has RNA-based silencing mechanisms that can trigger specific responses when plants interact with beneficial endophytic diazotrophic bacteria. Our findings suggest important roles for sRNA regulation in maize, and probably in other plants, during association with diazotrophic bacteria, emphasizing the up-regulation of Cu-miRNA.

  10. Next generation sequencing (NGS)technologies and applications

    Energy Technology Data Exchange (ETDEWEB)

    Vuyisich, Momchilo [Los Alamos National Laboratory

    2012-09-11

    NGS technology overview: (1) NGS library preparation - Nucleic acids extraction, Sample quality control, RNA conversion to cDNA, Addition of sequencing adapters, Quality control of library; (2) Sequencing - Clonal amplification of library fragments, (except PacBio), Sequencing by synthesis, Data output (reads and quality); and (3) Data analysis - Read mapping, Genome assembly, Gene expression, Operon structure, sRNA discovery, and Epigenetic analyses.

  11. SRD: a Staphylococcus regulatory RNA database.

    Science.gov (United States)

    Sassi, Mohamed; Augagneur, Yoann; Mauro, Tony; Ivain, Lorraine; Chabelskaya, Svetlana; Hallier, Marc; Sallou, Olivier; Felden, Brice

    2015-05-01

    An overflow of regulatory RNAs (sRNAs) was identified in a wide range of bacteria. We designed and implemented a new resource for the hundreds of sRNAs identified in Staphylococci, with primary focus on the human pathogen Staphylococcus aureus. The "Staphylococcal Regulatory RNA Database" (SRD, http://srd.genouest.org/) compiled all published data in a single interface including genetic locations, sequences and other features. SRD proposes novel and simplified identifiers for Staphylococcal regulatory RNAs (srn) based on the sRNA's genetic location in S. aureus strain N315 which served as a reference. From a set of 894 sequences and after an in-depth cleaning, SRD provides a list of 575 srn exempt of redundant sequences. For each sRNA, their experimental support(s) is provided, allowing the user to individually assess their validity and significance. RNA-seq analysis performed on strains N315, NCTC8325, and Newman allowed us to provide further details, upgrade the initial annotation, and identified 159 RNA-seq independent transcribed sRNAs. The lists of 575 and 159 sRNAs sequences were used to predict the number and location of srns in 18 S. aureus strains and 10 other Staphylococci. A comparison of the srn contents within 32 Staphylococcal genomes revealed a poor conservation between species. In addition, sRNA structure predictions obtained with MFold are accessible. A BLAST server and the intaRNA program, which is dedicated to target prediction, were implemented. SRD is the first sRNA database centered on a genus; it is a user-friendly and scalable device with the possibility to submit new sequences that should spread in the literature. © 2015 Sassi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  12. DeAnnIso: a tool for online detection and annotation of isomiRs from small RNA sequencing data.

    Science.gov (United States)

    Zhang, Yuanwei; Zang, Qiguang; Zhang, Huan; Ban, Rongjun; Yang, Yifan; Iqbal, Furhan; Li, Ao; Shi, Qinghua

    2016-07-08

    Small RNA (sRNA) Sequencing technology has revealed that microRNAs (miRNAs) are capable of exhibiting frequent variations from their canonical sequences, generating multiple variants: the isoforms of miRNAs (isomiRs). However, integrated tool to precisely detect and systematically annotate isomiRs from sRNA sequencing data is still in great demand. Here, we present an online tool, DeAnnIso (Detection and Annotation of IsomiRs from sRNA sequencing data). DeAnnIso can detect all the isomiRs in an uploaded sample, and can extract the differentially expressing isomiRs from paired or multiple samples. Once the isomiRs detection is accomplished, detailed annotation information, including isomiRs expression, isomiRs classification, SNPs in miRNAs and tissue specific isomiR expression are provided to users. Furthermore, DeAnnIso provides a comprehensive module of target analysis and enrichment analysis for the selected isomiRs. Taken together, DeAnnIso is convenient for users to screen for isomiRs of their interest and useful for further functional studies. The server is implemented in PHP + Perl + R and available to all users for free at: http://mcg.ustc.edu.cn/bsc/deanniso/ and http://mcg2.ustc.edu.cn/bsc/deanniso/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. A mathematical model and quantitative comparison of the small RNA circuit in the Vibrio harveyi and Vibrio cholerae quorum sensing systems

    International Nuclear Information System (INIS)

    Hunter, G A M; Vasquez, F Guevara; Keener, J P

    2013-01-01

    Quorum sensing is the process by which bacteria regulate their gene expression based on the local cell-population density. The quorum sensing systems of Vibrio harveyi and Vibrio cholerae are comprised of a phosphorelay cascade coupled to a small RNA (sRNA) circuit. The sRNA circuit contains multiple quorum regulated small RNA (Qrr) that regulate expression of the homologous master transcriptional regulators LuxR (in V. harveyi) and HapR (in V. cholerae). Their quorum sensing systems are topologically similar and homologous thereby making it difficult to understand why repression of HapR is more robust than LuxR to changes in Qrr. In this work we formulate and parameterize a novel mathematical model of the V. harveyi and V. cholerae sRNA circuit. We parameterize the model by fitting it to a variety of empirical data from both species. We show that we can distinguish all of the parameters and that the parameterizations (one for each species) are robust to errors in the data. We then use our model to propose some experiments to identify and explain kinetic differences between the species. We find that V. cholerae Qrr are more abundant and more sensitive to changes in LuxO than V. harveyi Qrr and argue that this is why expression of HapR is more robust than LuxR to changes in Qrr. (paper)

  14. Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

    Science.gov (United States)

    Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

    2017-10-01

    Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

  15. The Regulatory Small RNA MarS Supports Virulence of Streptococcus pyogenes.

    Science.gov (United States)

    Pappesch, Roberto; Warnke, Philipp; Mikkat, Stefan; Normann, Jana; Wisniewska-Kucper, Aleksandra; Huschka, Franziska; Wittmann, Maja; Khani, Afsaneh; Schwengers, Oliver; Oehmcke-Hecht, Sonja; Hain, Torsten; Kreikemeyer, Bernd; Patenge, Nadja

    2017-09-25

    Small regulatory RNAs (sRNAs) play a role in the control of bacterial virulence gene expression. In this study, we investigated an sRNA that was identified in Streptococcus pyogenes (group A Streptococcus, GAS) but is conserved throughout various streptococci. In a deletion strain, expression of mga, the gene encoding the multiple virulence gene regulator, was reduced. Accordingly, transcript and proteome analyses revealed decreased expression of several Mga-activated genes. Therefore, and because the sRNA was shown to interact with the 5' UTR of the mga transcript in a gel-shift assay, we designated it MarS for m ga-activating regulatory sRNA. Down-regulation of important virulence factors, including the antiphagocytic M-protein, led to increased susceptibility of the deletion strain to phagocytosis and reduced adherence to human keratinocytes. In a mouse infection model, the marS deletion mutant showed reduced dissemination to the liver, kidney, and spleen. Additionally, deletion of marS led to increased tolerance towards oxidative stress. Our in vitro and in vivo results indicate a modulating effect of MarS on virulence gene expression and on the pathogenic potential of GAS.

  16. A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response.

    Science.gov (United States)

    Chao, Yanjie; Vogel, Jörg

    2016-02-04

    Small RNAs (sRNAs) from conserved noncoding genes are crucial regulators in bacterial signaling pathways but have remained elusive in the Cpx response to inner membrane stress. Here we report that an alternative biogenesis pathway releasing the conserved mRNA 3' UTR of stress chaperone CpxP as an ∼60-nt sRNA provides the noncoding arm of the Cpx response. This so-called CpxQ sRNA, generated by general mRNA decay through RNase E, acts as an Hfq-dependent repressor of multiple mRNAs encoding extracytoplasmic proteins. Both CpxQ and the Cpx pathway are required for cell survival under conditions of dissipation of membrane potential. Our discovery of CpxQ illustrates how the conversion of a transcribed 3' UTR into an sRNA doubles the output of a single mRNA to produce two factors with spatially segregated functions during inner membrane stress: a chaperone that targets problematic proteins in the periplasm and a regulatory RNA that dampens their synthesis in the cytosol. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    Science.gov (United States)

    Mars, Ruben A T; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Mäder, Ulrike; Völker, Uwe; van Dijl, Jan Maarten; Denham, Emma L

    2015-03-01

    Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions.

  18. The Use of Backscattered Electron Imaging and Transmission Electron Microscopy to Assess Bone Architecture and Mineral Loci: Effect of Intermittent Slow-Release Sodium Fluoride Therapy

    Science.gov (United States)

    Zerwekh, Joseph E.; Bellotto, Dennis; Prostak, Kenneth S.; Hagler, Herbert K.; Pak, Charles Y. C.

    1996-04-01

    Backscattered electron imaging (BEI) and transmission electron microscopy (TEM) were used to examine the effects of treatment with intermittent slow-release sodium fluoride (SRNaF) and continuous calcium citrate on bone architecture and crystallinity. Examination was performed in nondecalcified biopsies obtained from patients following up to four years of therapy (placebo or SRNaF) and compared to pretreatment biopsies from each patient, as well as to bone from young, normal subjects. BEI images disclosed increased areas of recent bone formation following fluoride administration. There was no evidence of a mineralization defect in any biopsy and both cortical and trabecular architecture remained normal. TEM analysis demonstrated intrafibrillar platelike crystals and extrafibrillar needlelike crystals for both the pre- and post-treatment biopsies as well as for the bone from young normal subjects. There was no evidence of increased crystal size or of an increase in extrafibrillar mineral deposition. These observations suggest that intermittent SRNaF and continuous calcium therapy exerts an anabolic action on the skeleton not accompanied by a mineralization defect or an alteration of bone mineral deposition. The use of BEI and TEM holds promise for the study of the pathophysiology and treatment of metabolic bone diseases.

  19. Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines

    Science.gov (United States)

    Tosar, Juan Pablo; Gámbaro, Fabiana; Sanguinetti, Julia; Bonilla, Braulio; Witwer, Kenneth W.; Cayota, Alfonso

    2015-01-01

    Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19–60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5′ tRNA halves and 5′ RNA Y4-derived fragments of 31–33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space. PMID:25940616

  20. The UEA Small RNA Workbench: A Suite of Computational Tools for Small RNA Analysis.

    Science.gov (United States)

    Mohorianu, Irina; Stocks, Matthew Benedict; Applegate, Christopher Steven; Folkes, Leighton; Moulton, Vincent

    2017-01-01

    RNA silencing (RNA interference, RNAi) is a complex, highly conserved mechanism mediated by short, typically 20-24 nt in length, noncoding RNAs known as small RNAs (sRNAs). They act as guides for the sequence-specific transcriptional and posttranscriptional regulation of target mRNAs and play a key role in the fine-tuning of biological processes such as growth, response to stresses, or defense mechanism.High-throughput sequencing (HTS) technologies are employed to capture the expression levels of sRNA populations. The processing of the resulting big data sets facilitated the computational analysis of the sRNA patterns of variation within biological samples such as time point experiments, tissue series or various treatments. Rapid technological advances enable larger experiments, often with biological replicates leading to a vast amount of raw data. As a result, in this fast-evolving field, the existing methods for sequence characterization and prediction of interaction (regulatory) networks periodically require adapting or in extreme cases, a complete redesign to cope with the data deluge. In addition, the presence of numerous tools focused only on particular steps of HTS analysis hinders the systematic parsing of the results and their interpretation.The UEA small RNA Workbench (v1-4), described in this chapter, provides a user-friendly, modular, interactive analysis in the form of a suite of computational tools designed to process and mine sRNA datasets for interesting characteristics that can be linked back to the observed phenotypes. First, we show how to preprocess the raw sequencing output and prepare it for downstream analysis. Then we review some quality checks that can be used as a first indication of sources of variability between samples. Next we show how the Workbench can provide a comparison of the effects of different normalization approaches on the distributions of expression, enhanced methods for the identification of differentially expressed

  1. MENDING THE IN SITU MANIPULATION BARRIER

    Energy Technology Data Exchange (ETDEWEB)

    PETERSEN, S.W.

    2006-02-06

    In early 2004, the U.S. Department of Energy (DOE) Richland and Fluor Hanford requested technical assistance from the DOE Headquarters EM-23 Technical Assistance Program to provide a team of technical experts to develop recommendations for mending the In Situ Redox Manipulation (ISRM) Barrier in the 100-D Area of the Hanford Site in Washington State. To accommodate this request, EM-23 provided support to convene a group of technical experts from industry, a national laboratory, and a DOE site to participate in a 2 1/2-day workshop with the objective of identifying and recommending options to enhance the performance of the 100-D Area reactive barrier and of a planned extension to the northeast. This report provides written documentation of the team's findings and recommendations. In 1995, a plume of dissolved hexavalent chromium [Cr(VI)], which resulted from operation of the D/DR Reactors at the Hanford site, was discovered along the Columbia River shoreline and in the 100-D Area. Between 1999 and 2003, a reactive barrier using the In Situ Redox Manipulation (ISRM) technology, was installed a distance of 680 meters along the river to reduce the Cr(VI) in the groundwater. The ISRM technology creates a treatment zone within the aquifer by injection of sodium dithionite, a strong reducing agent that scavenges dissolved oxygen (DO) from the aquifer and reduces ferric iron [Fe(III)], related metals, and oxy-ions. The reduction of Fe(III) to ferrous [Fe(II)] iron provides the primary reduction capacity to reduce Cr(VI) to the +3 state, which is less mobile and less toxic. Bench-scale and field-scale treatability tests were initially conducted to demonstrate proof-of principle and to provide data for estimation of barrier longevity. These calculations estimated barrier longevity in excess of twenty years. However, several years after initial and secondary treatment, groundwater in a number of wells has been found to contain elevated chromium (Cr) concentrations

  2. Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization.

    Science.gov (United States)

    Locati, Mauro D; Terpstra, Inez; de Leeuw, Wim C; Kuzak, Mateusz; Rauwerda, Han; Ensink, Wim A; van Leeuwen, Selina; Nehrdich, Ulrike; Spaink, Herman P; Jonker, Martijs J; Breit, Timo M; Dekker, Rob J

    2015-08-18

    There is an increasing interest in complementing RNA-seq experiments with small-RNA (sRNA) expression data to obtain a comprehensive view of a transcriptome. Currently, two main experimental challenges concerning sRNA-seq exist: how to check the size distribution of isolated sRNAs, given the sensitive size-selection steps in the protocol; and how to normalize data between samples, given the low complexity of sRNA types. We here present two separate sets of synthetic RNA spike-ins for monitoring size-selection and for performing data normalization in sRNA-seq. The size-range quality control (SRQC) spike-in set, consisting of 11 oligoribonucleotides (10-70 nucleotides), was tested by intentionally altering the size-selection protocol and verified via several comparative experiments. We demonstrate that the SRQC set is useful to reproducibly track down biases in the size-selection in sRNA-seq. The external reference for data-normalization (ERDN) spike-in set, consisting of 19 oligoribonucleotides, was developed for sample-to-sample normalization in differential-expression analysis of sRNA-seq data. Testing and applying the ERDN set showed that it can reproducibly detect differential expression over a dynamic range of 2(18). Hence, biological variation in sRNA composition and content between samples is preserved while technical variation is effectively minimized. Together, both spike-in sets can significantly improve the technical reproducibility of sRNA-seq. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

    Science.gov (United States)

    Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

    2012-10-02

    The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

  4. Identification and validation of sRNAs in Edwardsiella tarda S08.

    Directory of Open Access Journals (Sweden)

    Yuying Sun

    Full Text Available Bacterial small non-coding RNAs (sRNAs are known as novel regulators involved in virulence, stress responsibility, and so on. Recently, a lot of new researches have highlighted the critical roles of sRNAs in fine-tune gene regulation in both prokaryotes and eukaryotes. Edwardsiella tarda (E. tarda is a gram-negative, intracellular pathogen that causes edwardsiellosis in fish. Thus far, no sRNA has been reported in E. tarda. The present study represents the first attempt to identify sRNAs in E. tarda S08. Ten sRNAs were validated by RNA sequencing and quantitative PCR (qPCR. ET_sRNA_1 and ET_sRNA_2 were homolous to tmRNA and GcvB, respectively. However, the other candidate sRNAs have not been reported till now. The cellular abundance of 10 validated sRNA was detected by qPCR at different growth phases to monitor their biosynthesis. Nine candidate sRNAs were expressed in the late-stage of exponential growth and stationary stages of growth (36~60 h. And the expression of the nine sRNAs was growth phase-dependent. But ET_sRNA_10 was almost expressed all the time and reached the highest peak at 48 h. Their targets were predicted by TargetRNA2 and each sRNA target contains some genes that directly or indirectly relate to virulence. These results preliminary showed that sRNAs probably play a regulatory role of virulence in E. tarda.

  5. High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L..

    Directory of Open Access Journals (Sweden)

    Livia Donaire

    Full Text Available Small RNAs (sRNAs of 20 to 25 nucleotides (nt in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.. sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive.

  6. Differential impact of the HEN1 homolog HENN-1 on 21U and 26G RNAs in the germline of Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Leonie M Kamminga

    Full Text Available RNA interference (RNAi-related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3' ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA-methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3'-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1-bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4-bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.

  7. DNA conformational analysis in solution by uranyl mediated photocleavage

    DEFF Research Database (Denmark)

    Nielsen, Peter E.; Møllegaard, N E; Jeppesen, C

    1990-01-01

    Uranyl mediated photocleavage of double stranded DNA is proposed as a general probing for DNA helix conformation in terms of minor groove width/electronegative potential. Specifically, it is found that A/T-tracts known to constitute strong distamycin binding sites are preferentially photocleaved ......, uranyl photocleavage of the internal control region (ICR) of the 5S-RNA gene yields a cleavage modulation pattern fully compatible with that obtained by DNase I which also--in a more complex way--senses DNA minor groove width....

  8. Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida.

    Science.gov (United States)

    Bojanovič, Klara; D'Arrigo, Isotta; Long, Katherine S

    2017-04-01

    Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one

  9. Dynamic evolution and biogenesis of small RNAs during sex reversal

    OpenAIRE

    Liu, Jie; Luo, Majing; Sheng, Yue; Hong, Qiang; Cheng, Hanhua; Zhou, Rongjia

    2015-01-01

    Understanding origin, evolution and functions of small RNA (sRNA) genes has been a great challenge in the past decade. Molecular mechanisms underlying sexual reversal in vertebrates, particularly sRNAs involved in this process, are largely unknown. By deep-sequencing of small RNA transcriptomes in combination with genomic analysis, we identified a large amount of piRNAs and miRNAs including over 1,000 novel miRNAs, which were differentially expressed during gonad reversal from ovary to testis...

  10. A genome-wide analysis of the RNA-guided silencing pathway in coffee reveals insights into its regulatory mechanisms.

    Directory of Open Access Journals (Sweden)

    Christiane Noronha Fernandes-Brum

    Full Text Available microRNAs (miRNAs are derived from self-complementary hairpin structures, while small-interfering RNAs (siRNAs are derived from double-stranded RNA (dsRNA or hairpin precursors. The core mechanism of sRNA production involves DICER-like (DCL in processing the smallRNAs (sRNAs and ARGONAUTE (AGO as effectors of silencing, and siRNA biogenesis also involves action of RNA-Dependent RNA Polymerase (RDR, Pol IV and Pol V in biogenesis. Several other proteins interact with the core proteins to guide sRNA biogenesis, action, and turnover. We aimed to unravel the components and functions of the RNA-guided silencing pathway in a non-model plant species of worldwide economic relevance. The sRNA-guided silencing complex members have been identified in the Coffea canephora genome, and they have been characterized at the structural, functional, and evolutionary levels by computational analyses. Eleven AGO proteins, nine DCL proteins (which include a DCL1-like protein that was not previously annotated, and eight RDR proteins were identified. Another 48 proteins implicated in smallRNA (sRNA pathways were also identified. Furthermore, we identified 235 miRNA precursors and 317 mature miRNAs from 113 MIR families, and we characterized ccp-MIR156, ccp-MIR172, and ccp-MIR390. Target prediction and gene ontology analyses of 2239 putative targets showed that significant pathways in coffee are targeted by miRNAs. We provide evidence of the expansion of the loci related to sRNA pathways, insights into the activities of these proteins by domain and catalytic site analyses, and gene expression analysis. The number of MIR loci and their targeted pathways highlight the importance of miRNAs in coffee. We identified several roles of sRNAs in C. canephora, which offers substantial insight into better understanding the transcriptional and post-transcriptional regulation of this major crop.

  11. From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen

    KAUST Repository

    Weynberg, Karen D.

    2015-12-08

    Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.

  12. From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen

    KAUST Repository

    Weynberg, Karen D.; Voolstra, Christian R.; Neave, Matthew J.; Buerger, Patrick; van Oppen, Madeleine J. H.

    2015-01-01

    Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.

  13. The Leeb Hardness Test for Rock: An Updated Methodology and UCS Correlation

    Science.gov (United States)

    Corkum, A. G.; Asiri, Y.; El Naggar, H.; Kinakin, D.

    2018-03-01

    The Leeb hardness test (LHT with test value of L D ) is a rebound hardness test, originally developed for metals, that has been correlated with the Unconfined Compressive Strength (test value of σ c ) of rock by several authors. The tests can be carried out rapidly, conveniently and nondestructively on core and block samples or on rock outcrops. This makes the relatively small LHT device convenient for field tests. The present study compiles test data from literature sources and presents new laboratory testing carried out by the authors to develop a substantially expanded database with wide-ranging rock types. In addition, the number of impacts that should be averaged to comprise a "test result" was revisited along with the issue of test specimen size. Correlation for L D and σ c for various rock types is provided along with recommended testing methodology. The accuracy of correlated σ c estimates was assessed and reasonable correlations were observed between L D and σ c . The study findings show that LHT can be useful particularly for field estimation of σ c and offers a significant improvement over the conventional field estimation methods outlined by the ISRM (e.g., hammer blows). This test is rapid and simple, with relatively low equipment costs, and provides a reasonably accurate estimate of σ c .

  14. U.S. National Committee for Rock Mechanics; and Conceptual model of fluid infiltration in fractured media. Project summary, July 28, 1997--July 27, 1998

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-09-01

    The title describes the two tasks summarized in this report. The remainder of the report contains information on meetings held or to be held on the subjects. The US National Committee for Rock Mechanics (USNC/RM) provides for US participation in international activities in rock mechanics, principally through adherence to the International Society for Rock Mechanics (ISRM). It also keeps the US rock mechanics community informed about new programs directed toward major areas of national concern in which rock mechanics problems represent critical or limiting factors, such as energy resources, excavation, underground storage and waste disposal, and reactor siting. The committee also guides or produces advisory studies and reports on problem areas in rock mechanics. A new panel under the auspices of the US National Committee for Rock Mechanics has been appointed to conduct a study on Conceptual Models of Fluid Infiltration in Fractured Media. The study has health and environmental applications related to the underground flow of pollutants through fractured rock in and around mines and waste repositories. Support of the study has been received from the US Nuclear Regulatory Commission and the Department of Energy`s Yucca Mountain Project Office. The new study builds on the success of a recent USNC/RM report entitled Rock Fractures and Fluid Flow: Contemporary Understanding and Applications (National Academy Press, 1996, 551 pp.). A summary of the new study is provided.

  15. Experimental Study on Properties of Methane Diffusion of Coal Block under Triaxial Compressive Stress

    Science.gov (United States)

    Zhao, Hong-Bao

    2014-01-01

    Taking the standard size coal block samples defined by ISRM as research objects, both properties of methane diffusion of coal block under triaxial compressive stress and characteristic influences caused by methane pressure were systematically studied with thermo-fluid-solid coupling with triaxial servocontrolled seepage equipment of methane-containing coal. The result shows the methane diffusion property of coal block under triaxial compressive stress was shown in four-stage as follow, first is sharply reduce stage, second is hyperbolic reduce stage, third is close to a fixed value stage, fourth stage is 0. There is a special point making the reduced rate of characteristic curve of methane diffusion speed become sharply small; the influences of shape of methane diffusion speed characteristic curve caused by methane pressure are not obvious, which only is shown in numerical size of methane diffusion speed. Test time was extended required by appear of the special point makes the reduce rate of methane diffusion speed become sharply small. The fitting four-phase relation of methane diffusion of coal block under triaxial compressive stress was obtained, and the idea is proposed that influences of the fitting four-phase relation caused by methane pressure were only shown in value of fitting parameters. PMID:25531000

  16. Analytical Solution for Stress Field and Intensity Factor in CSTBD under Mixed Mode Conditions

    Directory of Open Access Journals (Sweden)

    Najaf Ali Ghavidel

    2014-06-01

    Full Text Available Considering the fact that rocks fail faster under tensile stress, rock tensile strength is of greatimportance in applications such as blasting, rock fragmentation, slope stability, hydraulic fracturing,caprock integrity, and geothermal energy extraction. There are two direct and indirect methods tomeasure tensile strength. Since direct methods always encompass difficulties in test setup, indirectmethods, specifically the Brazilian test, have often been employed for tensile strength measurement.Tensile failure is technically attributed to crack propagation in rock. Fracture mechanics hassignificant potential for the determination of crack behaviour as well as propagation pattern. To applyBrazilian tests, cracked disc geometry has been suggested by the International Society for RockMechanics ISRM. Accordingly, a comprehensive study is necessary to evaluate stress field and stressintensity factor (SIF around the crack in the centre of the specimen. In this paper, superpositionprinciple is employed to solve the problem of cracked straight-through Brazilian disc (CSTBD, usingtwo methods of dislocation and complex stress function. Stress field and SIF in the vicinity of thecrack tip are then calculated. With the proposed method, the magnitude of critical load for crackinitiation in structures can be predicted. This method is valid for any crack of any arbitrary length andangle. In addition, numerical modelling has been carried out for the Brazilian disc. Finally, theanalytical solution has been compared with numerical modelling results showing the same outcomefor both methods.

  17. Alteration dependent physical-mechanical properties of quartz-diorite building stones

    Directory of Open Access Journals (Sweden)

    Masoud Torkan

    2016-12-01

    Full Text Available The microscopic and geomechanical properties of igneous building stones include the level of alteration, presence of micro cracks, peak strength, porosity, proportion of detrimental minerals, etc. Porosity is reportedly of a devastating impact on the peak strength of igneous rocks. The quartz diorite rock samples in this study were selected from five quarries in Natanz, Iran and subject to microscopic and geomechanical investigations. The level of alteration and the minerals detrimental to the strength of the samples were identified from thin sections. Therefore, the geomechanical tests upon density, porosity, durability index, the Brazilian, and triaxial tests were conducted as per ISRM standards. The findings from microscopic studies reveal that alteration is of more intense impact on rock peak strength compared to that of porosity. The results were compared to standard values and a qualitative correlation between strength and microscopic properties was detected accentuating the importance of microscopic studies on construction stones. The correlation thereupon may be adopted in the exploration, exploitation, and process of construction stones to avoid heavy expenditures and damage to the environment.

  18. U.S. National Committee for Rock Mechanics and conceptual model of fluid infiltration in fractured media. Project summary, July 28, 1997 - July 27, 1998

    International Nuclear Information System (INIS)

    1998-01-01

    The title describes the two tasks summarized in this report. The remainder of the report contains information on meetings held or to be held on the subjects. The US National Committee for Rock Mechanics (USNC/RM) provides for US participation in international activities in rock mechanics, principally through adherence to the International Society for Rock Mechanics (ISRM). It also keeps the US rock mechanics community informed about new programs directed toward major areas of national concern in which rock mechanics problems represent critical or limiting factors, such as energy resources, excavation, underground storage and waste disposal, and reactor siting. The committee also guides or produces advisory studies and reports on problem areas in rock mechanics. A new panel under the auspices of the US National Committee for Rock Mechanics has been appointed to conduct a study on Conceptual Models of Fluid Infiltration in Fractured Media. The study has health and environmental applications related to the underground flow of pollutants through fractured rock in and around mines and waste repositories. Support of the study has been received from the US Nuclear Regulatory Commission and the Department of Energy's Yucca Mountain Project Office. The new study builds on the success of a recent USNC/RM report entitled Rock Fractures and Fluid Flow: Contemporary Understanding and Applications (National Academy Press, 1996, 551 pp.). A summary of the new study is provided

  19. Determining the REV for Fracture Rock Mass Based on Seepage Theory

    Directory of Open Access Journals (Sweden)

    Lili Zhang

    2017-01-01

    Full Text Available Seepage problems of the fractured rock mass have always been a heated topic within hydrogeology and engineering geology. The equivalent porous medium model method is the main method in the study of the seepage of the fractured rock mass and its engineering application. The key to the method is to determine a representative elementary volume (REV. The FractureToKarst software, that is, discrete element software, is a main analysis tool in this paper and developed by a number of authors. According to the standard of rock classification established by ISRM, this paper aims to discuss the existence and the size of REV of fractured rock masses with medium tractility and provide a general method to determine the existence of REV. It can be gleaned from the study that the existence condition of fractured rock mass with medium tractility features average fracture spacing smaller than 0.6 m. If average fracture spacing is larger than 0.6 m, there is no existence of REV. The rationality of the model is verified by a case study. The present research provides a method for the simulation of seepage field in fissured rocks.

  20. psRNATarget: a plant small RNA target analysis server (2017 release).

    Science.gov (United States)

    Dai, Xinbin; Zhuang, Zhaohong; Zhao, Patrick Xuechun

    2018-04-30

    Plant regulatory small RNAs (sRNAs), which include most microRNAs (miRNAs) and a subset of small interfering RNAs (siRNAs), such as the phased siRNAs (phasiRNAs), play important roles in regulating gene expression. Although generated from genetically distinct biogenesis pathways, these regulatory sRNAs share the same mechanisms for post-translational gene silencing and translational inhibition. psRNATarget was developed to identify plant sRNA targets by (i) analyzing complementary matching between the sRNA sequence and target mRNA sequence using a predefined scoring schema and (ii) by evaluating target site accessibility. This update enhances its analytical performance by developing a new scoring schema that is capable of discovering miRNA-mRNA interactions at higher 'recall rates' without significantly increasing total prediction output. The scoring procedure is customizable for the users to search both canonical and non-canonical targets. This update also enables transmitting and analyzing 'big' data empowered by (a) the implementation of multi-threading chunked file uploading, which can be paused and resumed, using HTML5 APIs and (b) the allocation of significantly more computing nodes to its back-end Linux cluster. The updated psRNATarget server has clear, compelling and user-friendly interfaces that enhance user experiences and present data clearly and concisely. The psRNATarget is freely available at http://plantgrn.noble.org/psRNATarget/.

  1. Oasis 2: improved online analysis of small RNA-seq data.

    Science.gov (United States)

    Rahman, Raza-Ur; Gautam, Abhivyakti; Bethune, Jörn; Sattar, Abdul; Fiosins, Maksims; Magruder, Daniel Sumner; Capece, Vincenzo; Shomroni, Orr; Bonn, Stefan

    2018-02-14

    Small RNA molecules play important roles in many biological processes and their dysregulation or dysfunction can cause disease. The current method of choice for genome-wide sRNA expression profiling is deep sequencing. Here we present Oasis 2, which is a new main release of the Oasis web application for the detection, differential expression, and classification of small RNAs in deep sequencing data. Compared to its predecessor Oasis, Oasis 2 features a novel and speed-optimized sRNA detection module that supports the identification of small RNAs in any organism with higher accuracy. Next to the improved detection of small RNAs in a target organism, the software now also recognizes potential cross-species miRNAs and viral and bacterial sRNAs in infected samples. In addition, novel miRNAs can now be queried and visualized interactively, providing essential information for over 700 high-quality miRNA predictions across 14 organisms. Robust biomarker signatures can now be obtained using the novel enhanced classification module. Oasis 2 enables biologists and medical researchers to rapidly analyze and query small RNA deep sequencing data with improved precision, recall, and speed, in an interactive and user-friendly environment. Oasis 2 is implemented in Java, J2EE, mysql, Python, R, PHP and JavaScript. It is freely available at https://oasis.dzne.de.

  2. Two small RNAs, CrcY and CrcZ, act in concert to sequester the Crc global regulator in Pseudomonas putida, modulating catabolite repression.

    Science.gov (United States)

    Moreno, Renata; Fonseca, Pilar; Rojo, Fernando

    2012-01-01

    The Crc protein is a translational repressor that recognizes a specific target at some mRNAs, controlling catabolite repression and co-ordinating carbon metabolism in pseudomonads. In Pseudomonas aeruginosa, the levels of free Crc protein are controlled by CrcZ, a sRNA that sequesters Crc, acting as an antagonist. We show that, in Pseudomonas putida, the levels of free Crc are controlled by CrcZ and by a novel 368 nt sRNA named CrcY. CrcZ and CrcY, which contain six potential targets for Crc, were able to bind Crc specifically in vitro. The levels of CrcZ and CrcY were low under conditions generating a strong catabolite repression, and increased strongly when catabolite repression was absent. Deletion of either crcZ or crcY had no effect on catabolite repression, but the simultaneous absence of both sRNAs led to constitutive catabolite repression that compromised growth on some carbon sources. Overproduction of CrcZ or CrcY significantly reduced repression. We propose that CrcZ and CrcY act in concert, sequestering and modulating the levels of free Crc according to metabolic conditions. The CbrA/CbrB two-component system activated crcZ transcription, but had little effect on crcY. CrcY was detected in P. putida, Pseudomonas fluorescens and Pseudomonas syringae, but not in P. aeruginosa. © 2011 Blackwell Publishing Ltd.

  3. Plant Responses to Pathogen Attack: Small RNAs in Focus.

    Science.gov (United States)

    Islam, Waqar; Noman, Ali; Qasim, Muhammad; Wang, Liande

    2018-02-08

    Small RNAs (sRNA) are a significant group of gene expression regulators for multiple biological processes in eukaryotes. In plants, many sRNA silencing pathways produce extensive array of sRNAs with specialized roles. The evidence on record advocates for the functions of sRNAs during plant microbe interactions. Host sRNAs are reckoned as mandatory elements of plant defense. sRNAs involved in plant defense processes via different pathways include both short interfering RNA (siRNA) and microRNA (miRNA) that actively regulate immunity in response to pathogenic attack via tackling pathogen-associated molecular patterns (PAMPs) and other effectors. In response to pathogen attack, plants protect themselves with the help of sRNA-dependent immune systems. That sRNA-mediated plant defense responses play a role during infections is an established fact. However, the regulations of several sRNAs still need extensive research. In this review, we discussed the topical advancements and findings relevant to pathogen attack and plant defense mediated by sRNAs. We attempted to point out diverse sRNAs as key defenders in plant systems. It is hoped that sRNAs would be exploited as a mainstream player to achieve food security by tackling different plant diseases.

  4. Micropathogen Community Analysis in Hyalomma rufipes via High-Throughput Sequencing of Small RNAs

    Science.gov (United States)

    Luo, Jin; Liu, Min-Xuan; Ren, Qiao-Yun; Chen, Ze; Tian, Zhan-Cheng; Hao, Jia-Wei; Wu, Feng; Liu, Xiao-Cui; Luo, Jian-Xun; Yin, Hong; Wang, Hui; Liu, Guang-Yuan

    2017-01-01

    Ticks are important vectors in the transmission of a broad range of micropathogens to vertebrates, including humans. Because of the role of ticks in disease transmission, identifying and characterizing the micropathogen profiles of tick populations have become increasingly important. The objective of this study was to survey the micropathogens of Hyalomma rufipes ticks. Illumina HiSeq2000 technology was utilized to perform deep sequencing of small RNAs (sRNAs) extracted from field-collected H. rufipes ticks in Gansu Province, China. The resultant sRNA library data revealed that the surveyed tick populations produced reads that were homologous to St. Croix River Virus (SCRV) sequences. We also observed many reads that were homologous to microbial and/or pathogenic isolates, including bacteria, protozoa, and fungi. As part of this analysis, a phylogenetic tree was constructed to display the relationships among the homologous sequences that were identified. The study offered a unique opportunity to gain insight into the micropathogens of H. rufipes ticks. The effective control of arthropod vectors in the future will require knowledge of the micropathogen composition of vectors harboring infectious agents. Understanding the ecological factors that regulate vector propagation in association with the prevalence and persistence of micropathogen lineages is also imperative. These interactions may affect the evolution of micropathogen lineages, especially if the micropathogens rely on the vector or host for dispersal. The sRNA deep-sequencing approach used in this analysis provides an intuitive method to survey micropathogen prevalence in ticks and other vector species. PMID:28861401

  5. Bacteria of leg atheromatous arteries responsible for inflammation.

    Science.gov (United States)

    Olszewski, Waldemar Lech; Rutkowska, Joanna; Moscicka-Wesolowska, Maria; Swoboda-Kopec, Ewa; Stelmach, Ewa; Zaleska, Marzanna; Zagozda, Malgorzata

    2016-09-01

    Ischaemia of the lower limbs is frequently followed by inflammation and, in advanced cases, necrosis of peripheral tissues. Whether this is caused by arterial hypoperfusion only or by the presence of bacteria in the arterial walI as well remains unclear. The aim of the study was to prove the presence and source of bacteria in arterial specimens and evaluate their chemotactic properties resulting in the formation of periarterial cellular infiltrates. Bacterial culture and testing for 16sRNA were performed in fragments of popliteal artery harvested from amputated limbs. Carotid artery plaques served as controls. Fragments of arteries were transplanted into scid mice to evaluate their chemotactic activity for macrophages. a) higher prevalence of isolates and 16sRNA in atherosclerotic popliteal than carotid arteries, b) high density of plaque and periarterial infiltrates and mRNA level for pro-inflammatory cytokines in popliteal arteries, c) prevalent microbes were Staphylococcus aureus, S. epidermidis and Enterococci, d) foot skin and arterial bacterial phenotypes and DNA revealed evident similarities, and e) more intensive mouse macrophage accumulation in popliteal than carotid implants into scid mice. The presence of bacteria in the lower limb arterial wall was documented. They may predispose to inflammation secondary to ischaemic changes.

  6. Expression of matrix metalloproteinases 2 and 9 in human gastric cancer and superficial gastritis.

    Science.gov (United States)

    Sampieri, Clara Luz; de la Peña, Sol; Ochoa-Lara, Mariana; Zenteno-Cuevas, Roberto; León-Córdoba, Kenneth

    2010-03-28

    To assess expression of matrix metalloproteinases 2 (MMP2) and MMP9 in gastric cancer, superficial gastritis and normal mucosa, and to measure metalloproteinase activity. MMP2 and MMP9 mRNA expression was determined by quantitative real-time polymerase chain reaction. Normalization was carried out using three different factors. Proteins were analyzed by quantitative gelatin zymography (qGZ). 18S ribosomal RNA (18SRNA) was very highly expressed, while hypoxanthine ribosyltransferase-1 (HPRT-1) was moderately expressed. MMP2 was highly expressed, while MMP9 was not detected or lowly expressed in normal tissues, moderately or highly expressed in gastritis and highly expressed in cancer. Relative expression of 18SRNA and HPRT-1 showed no significant differences. Significant differences in MMP2 and MMP9 were found between cancer and normal tissue, but not between gastritis and normal tissue. Absolute quantification of MMP9 echoed this pattern, but differential expression of MMP2 proved conflictive. Analysis by qGZ indicated significant differences between cancer and normal tissue in MMP-2, total MMP-9, 250 and 110 kDa bands. MMP9 expression is enhanced in gastric cancer compared to normal mucosa; interpretation of differential expression of MMP2 is difficult to establish.

  7. Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs.

    Science.gov (United States)

    Chávez Montes, Ricardo A; de Fátima Rosas-Cárdenas, Flor; De Paoli, Emanuele; Accerbi, Monica; Rymarquis, Linda A; Mahalingam, Gayathri; Marsch-Martínez, Nayelli; Meyers, Blake C; Green, Pamela J; de Folter, Stefan

    2014-04-23

    Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies.

  8. Characterization of ribonuclease III from Brucella.

    Science.gov (United States)

    Wu, Chang-Xian; Xu, Xian-Jin; Zheng, Ke; Liu, Fang; Yang, Xu-Dong; Chen, Chuang-Fu; Chen, Huan-Chun; Liu, Zheng-Fei

    2016-04-01

    Bacterial ribonuclease III (RNase III) is a highly conserved endonuclease, which plays pivotal roles in RNA maturation and decay pathways by cleaving double-stranded structure of RNAs. Here we cloned rncS gene from the genomic DNA of Brucella melitensis, and analyzed the cleavage properties of RNase III from Brucella. We identified Brucella-encoding small RNA (sRNA) by high-throughput sequencing and northern blot, and found that sRNA of Brucella and Homo miRNA precursor (pre-miRNA) can be bound and cleaved by B.melitensis ribonuclease III (Bm-RNase III). Cleavage activity of Bm-RNase III is bivalent metal cations- and alkaline buffer-dependent. We constructed several point mutations in Bm-RNase III, whose cleavage activity indicated that the 133th Glutamic acid residue was required for catalytic activity. Western blot revealed that Bm-RNase III was differently expressed in Brucella virulence strain 027 and vaccine strain M5-90. Collectively, our data suggest that Brucella RNase III can efficiently bind and cleave stem-loop structure of small RNA, and might participate in regulation of virulence in Brucella. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    tang, T. H.; Polacek, N.; Zywicki, M.

    2005-01-01

    By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense...... elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites...... on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted...

  10. Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes.

    Science.gov (United States)

    Sittka, Alexandra; Sharma, Cynthia M; Rolle, Katarzyna; Vogel, Jörg

    2009-01-01

    The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small non-coding RNAs (sRNAs) in Escherichia coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus jannaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.

  11. Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

    Science.gov (United States)

    Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

    2014-07-01

    Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.

  12. Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing

    DEFF Research Database (Denmark)

    Svenningsen, Sine L; Tu, Kimberly C; Bassler, Bonnie L

    2009-01-01

    the quorum regulatory RNAs 1-4 (Qrr1-4). The four Qrr sRNAs are functionally redundant. That is, expression of any one of them is sufficient for wild-type quorum-sensing behaviour. Here, we show that the combined action of two feedback loops, one involving the sRNA-activator LuxO and one involving the sRNA......Quorum sensing is a mechanism of cell-to-cell communication that allows bacteria to coordinately regulate gene expression in response to changes in cell-population density. At the core of the Vibrio cholerae quorum-sensing signal transduction pathway reside four homologous small RNAs (sRNAs), named......-target HapR, promotes gene dosage compensation between the four qrr genes. Gene dosage compensation adjusts the total Qrr1-4 sRNA pool and provides the molecular mechanism underlying sRNA redundancy. The dosage compensation mechanism is exquisitely sensitive to small perturbations in Qrr levels. Precisely...

  13. A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics.

    Science.gov (United States)

    Feng, Lihui; Rutherford, Steven T; Papenfort, Kai; Bagert, John D; van Kessel, Julia C; Tirrell, David A; Wingreen, Ned S; Bassler, Bonnie L

    2015-01-15

    Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes

    Science.gov (United States)

    Yu, Dongliang; Meng, Yijun; Zuo, Ziwei; Xue, Jie; Wang, Huizhong

    2016-01-01

    Nat-siRNAs (small interfering RNAs originated from natural antisense transcripts) are a class of functional small RNA (sRNA) species discovered in both plants and animals. These siRNAs are highly enriched within the annealed regions of the NAT (natural antisense transcript) pairs. To date, great research efforts have been taken for systematical identification of the NATs in various organisms. However, developing a freely available and easy-to-use program for NAT prediction is strongly demanded by researchers. Here, we proposed an integrative pipeline named NATpipe for systematical discovery of NATs from de novo assembled transcriptomes. By utilizing sRNA sequencing data, the pipeline also allowed users to search for phase-distributed nat-siRNAs within the perfectly annealed regions of the NAT pairs. Additionally, more reliable nat-siRNA loci could be identified based on degradome sequencing data. A case study on the non-model plant Dendrobium officinale was performed to illustrate the utility of NATpipe. Finally, we hope that NATpipe would be a useful tool for NAT prediction, nat-siRNA discovery, and related functional studies. NATpipe is available at www.bioinfolab.cn/NATpipe/NATpipe.zip. PMID:26858106

  15. A transcriptome-wide, organ-specific regulatory map of Dendrobium officinale, an important traditional Chinese orchid herb

    Science.gov (United States)

    Meng, Yijun; Yu, Dongliang; Xue, Jie; Lu, Jiangjie; Feng, Shangguo; Shen, Chenjia; Wang, Huizhong

    2016-01-01

    Dendrobium officinale is an important traditional Chinese herb. Here, we did a transcriptome-wide, organ-specific study on this valuable plant by combining RNA, small RNA (sRNA) and degradome sequencing. RNA sequencing of four organs (flower, root, leaf and stem) of Dendrobium officinale enabled us to obtain 536,558 assembled transcripts, from which 2,645, 256, 42 and 54 were identified to be highly expressed in the four organs respectively. Based on sRNA sequencing, 2,038, 2, 21 and 24 sRNAs were identified to be specifically accumulated in the four organs respectively. A total of 1,047 mature microRNA (miRNA) candidates were detected. Based on secondary structure predictions and sequencing, tens of potential miRNA precursors were identified from the assembled transcripts. Interestingly, phase-distributed sRNAs with degradome-based processing evidences were discovered on the long-stem structures of two precursors. Target identification was performed for the 1,047 miRNA candidates, resulting in the discovery of 1,257 miRNA--target pairs. Finally, some biological meaningful subnetworks involving hormone signaling, development, secondary metabolism and Argonaute 1-related regulation were established. All of the sequencing data sets are available at NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/). Summarily, our study provides a valuable resource for the in-depth molecular and functional studies on this important Chinese orchid herb. PMID:26732614

  16. The strontium-90 equilibrium parameters in Saclay soil; Les parametres d'equilibre du strontium 90 dans le sol de Saclay

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, P; Gailledreau, C [Commissariat a l' Energie Atomique, Saclay (France).Centre d' Etudes Nucleaires

    1960-07-01

    The equilibrium parameters of {sup 90}Sr in the presence of the following elements: Ca, Na, K, NH{sub 4} are measured in the soil at Saclay for each of the pairs Sr-Ca, Sr-Na, Sr-K, Sr-NH{sub 4}. The adsorption mechanism for Sr is studied in comparison with a synthetic resin which is a pure ion-exchanger. A preliminary study of a three component system; one being a tracer, is outlined. (author) [French] Les parametres d'equilibre du {sup 90}Sr en presence des elements suivants: Ca, Na, K, NH{sub 4} sont mesures sur le sol de Saclay pour chacun des couples Sr-Ca, Sr-Na, Sr-K, Sr-NH{sub 4}. Le mecanisme d'adsorption du Sr est etudie par comparaison avec une resine synthetique qui est un echangeur d'ions pur. Une etude preliminaire d'un systeme a trois composants, dont l'un est un traceur, est exposee. (auteur)

  17. Mal de Río Cuarto Virus Infection Triggers the Production of Distinctive Viral-Derived siRNA Profiles in Wheat and Its Planthopper Vector.

    Science.gov (United States)

    de Haro, Luis A; Dumón, Analía D; Mattio, María F; Argüello Caro, Evangelina Beatriz; Llauger, Gabriela; Zavallo, Diego; Blanc, Hervé; Mongelli, Vanesa C; Truol, Graciela; Saleh, María-Carla; Asurmendi, Sebastián; Del Vas, Mariana

    2017-01-01

    Plant reoviruses are able to multiply in gramineae plants and delphacid vectors encountering different defense strategies with unique features. This study aims to comparatively assess alterations of small RNA (sRNA) populations in both hosts upon virus infection. For this purpose, we characterized the sRNA profiles of wheat and planthopper vectors infected by Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae ) and quantified virus genome segments by quantitative reverse transcription PCR We provide evidence that plant and insect silencing machineries differentially recognize the viral genome, thus giving rise to distinct profiles of virus-derived small interfering RNAs (vsiRNAs). In plants, most of the virus genome segments were targeted preferentially within their upstream sequences and vsiRNAs mapped with higher density to the smaller genome segments than to the medium or larger ones. This tendency, however, was not observed in insects. In both hosts, vsiRNAs were equally derived from sense and antisense RNA strands and the differences in vsiRNAs accumulation did not correlate with mRNAs accumulation. We also established that the piwi-interacting RNA (piRNA) pathway was active in the delphacid vector but, contrary to what is observed in virus-infected mosquitoes, virus-specific piRNAs were not detected. This work contributes to the understanding of the silencing response in insect and plant hosts.

  18. smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni

    Science.gov (United States)

    2012-01-01

    Background MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. Results To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. Conclusions This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to

  19. Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

    Energy Technology Data Exchange (ETDEWEB)

    Ansong, Charles; Yoon, Hyunjin; Porwollik, Steffen; Mottaz-Brewer, Heather; Petritis, Brianne O.; Jaitly, Navdeep; Adkins, Joshua N.; Mcclelland, Michael; Heffron, Fred; Smith, Richard D.

    2009-03-11

    In recent years the profound importance of sRNA-mediated translational/post-transcriptional regulation has been increasingly appreciated. However, the global role played by translational regulation in control of gene expression has never been elucidated in any organism for the simple reason that global proteomics methods required to accurately characterize post-transcriptional processes and the knowledge of translational control mechanisms have only become available within the last few years. The proteins Hfq and SmpB are essential for the biological activity of a range of regulatory sRNAs and thus provide a means to identify potential targets of sRNA regulation. We performed a sample-matched global proteomics and transcriptional analysis to examine the role of Hfq and SmpB in global protein translation and virulence using the Salmonella typhimurium model system. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all Salmonella proteins, respectively, with limited correlation between transcription and protein expression. This is the first report suggesting that SmpB could be a general translational regulator. The broad spectrum of proteins modulated by Hfq was also surprising including central metabolism, LPS biosynthesis, two-component regulatory systems, quorum sensing, SP1-TTSS, oxidative stress, fatty acid metabolism, nucleoside and nucleotide metabolism, envelope stress, aminoacyl-tRNA synthetases, amino acid biosynthesis, peptide transport, and motility.. The extent of global regulation of translation by Hfq is unexpected, with profound effects in all stages of Salmonella’s life cycle. Our results represent the first global systems-level analysis of translational regulation; the elucidated potential targets of sRNA regulation from our analysis will

  20. The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus.

    Science.gov (United States)

    Danger, Jessica L; Makthal, Nishanth; Kumaraswami, Muthiah; Sumby, Paul

    2015-12-01

    The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection. More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small

  1. Mobile genetic elements, a key to microbial adaptation in extreme environments

    Science.gov (United States)

    van Houdt, Rob; Mijnendonckx, Kristel; Provoost, Ann; Monsieurs, Pieter; Mergeay, Max; Leys, Natalie

    hydrogenotrophy. Five transposons were identified in CH34. Two mercury transposons Tn4378 and Tn4380, respectively located on pMOL28 and pMOL30, were previously described. In addition, 3 novel transposons were identified. The large Tn6048 transposon contained 8 genes highly induced by zinc and lead. Transposon Tn6049 was found in twelve copies in the genome of strain CH34 and is seem-ingly often associated with genomic islands. Finally, Tn6050 was observed twice in the second chromosome with accessory genes not classically associated with transposons, namely a sulfate permease, a universal stress protein (UspA) and a DksA-like DnaK suppressor protein. The role of these genes is unclear but a functional association could be assumed. Finally, a set of 21 IS elements was found, counting in total for 57 copies dispersed over the genome. The number of copies ranged from 1 to 9 (IS1088 ) and 10 (ISRme3 ). The 21 IS elements could be divided into 10 different families. The elements ISRme5 and IS1071 were putatively involved in the recruitment of the genes for hydrogenotrophy and CO2 fixation, with mutants unable to grow on H2 and CO2 appeared to have lost these genes by IS1071 -mediated excision. These data clearly showed that the C. metallidurans CH34 model bacterium and some of it's Cupriavidus and Ralstonia relatives carry a multitude of tools that allowed them to genetically adapt to polluted and extreme soil environments, and that are a key in their success to adapt to the new anthropogenic spacecraft environments. Furthermore, these tools could be exploited to assess and measure genetic effects of spaceflight conditions. Acknowledgements This work was supported by the European Space Agency (ESA-PRODEX) and the Belgian Science Policy (Belspo) through the MISSEX and COMICS projects.

  2. The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism

    Science.gov (United States)

    Bækkedal, Cecilie; Haugen, Peik

    2015-01-01

    The Spot 42 RNA is a 109 nucleotide long (in Escherichia coli) noncoding small regulatory RNA (sRNA) encoded by the spf (spot fourty-two) gene. spf is found in gamma-proteobacteria and the majority of experimental work on Spot 42 RNA has been performed using E. coli, and recently Aliivibrio salmonicida. In the cell Spot 42 RNA plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex. Here we summarize the current knowledge on Spot 42, and present the natural distribution of spf, show family-specific secondary structural features of Spot 42, and link highly conserved structural regions to mRNA target binding. PMID:26327359

  3. Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

    Science.gov (United States)

    Xiong, Li-Ping; Ma, Yu-Qiang; Tang, Lei-Han

    2010-09-01

    Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology.

  4. Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

    International Nuclear Information System (INIS)

    Li-Ping, Xiong; Yu-Qiang, Ma; Lei-Han, Tang

    2010-01-01

    Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology

  5. Synthesis and biological incorporation of icons into macromolecules for NMR study. Final report, June 1, 1977--May 31, 1978

    International Nuclear Information System (INIS)

    Grant, D.M.; Horton, W.J.

    1978-01-01

    Carbon-13 enrichment synthesis and incorporation into three important biological systems have been carried out to provide materials for carbon-13 magnetic resonance studies. These systems include antibody-labeled haptens, labeled t-RNA and 5S-RNA molecules, and pyridoxal-5'-phosphate-labeled substrate mixtures. The synthesis phase of the work has been completed in all three cases, and the NMR studies completed on all but the antibody-hapten system which is still in process having been absorbed into other supported projects. Publications are now in preparation for the RNA and pyridoxal work. Preliminary results on the antibody-haptens work are encouraging as signals of antibody absorbed haptens have been observed but the results are still not yet conclusive

  6. Methods for Determination of 2′-O-Me in RNA

    DEFF Research Database (Denmark)

    Birkedal, Ulf; Krogh, Nicolai; Andersen, Kasper Langebjerg

    2016-01-01

    mechanism of ribose methylation is found in rRNA of Archaea and Eukarya where a methyltransferase (fibrillarin) use sRNA (Archaea) or box C/D snoRNA (Eukarya) as guide RNAs to specify the site of modification. The general function of these modifications is to promote ribosome biogenesis, in particular...... for mapping modifications and quantitating the fraction of RNA molecules modified in a population until recently remained poorly developed. Here, we review the methods that have been used to study 2′-O-Me in RNA starting with the original approach employing in vivo isotope labeling followed by paper......Ribose methylation is one of the most abundant RNA modifications and is found in all kingdoms of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. The most abundant...

  7. Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara; D'Arrigo, Isotta; Long, Katherine

    2017-01-01

    functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions...... intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous......Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification...

  8. A mixed incoherent feed-forward loop contributes to the regulation of bacterial photosynthesis genes.

    Science.gov (United States)

    Mank, Nils N; Berghoff, Bork A; Klug, Gabriele

    2013-03-01

    Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes. In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand. Recently, we identified a mixed incoherent feed-forward loop comprising the transcription factor PrrA, the sRNA PcrZ and photosynthesis target genes as part of this regulatory network. This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail.

  9. Identification of novel sRNAs in mycobacterial species.

    Directory of Open Access Journals (Sweden)

    Chen-Hsun Tsai

    Full Text Available Bacterial small RNAs (sRNAs are short transcripts that typically do not encode proteins and often act as regulators of gene expression through a variety of mechanisms. Regulatory sRNAs have been identified in many species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Here, we use a computational algorithm to predict sRNA candidates in the mycobacterial species M. smegmatis and M. bovis BCG and confirmed the expression of many sRNAs using Northern blotting. Thus, we have identified 17 and 23 novel sRNAs in M. smegmatis and M. bovis BCG, respectively. We have also applied a high-throughput technique (Deep-RACE to map the 5' and 3' ends of many of these sRNAs and identified potential regulators of sRNAs by analysis of existing ChIP-seq datasets. The sRNAs identified in this work likely contribute to the unique biology of mycobacteria.

  10. Integration of Bacterial Small RNAs in Regulatory Networks.

    Science.gov (United States)

    Nitzan, Mor; Rehani, Rotem; Margalit, Hanah

    2017-05-22

    Small RNAs (sRNAs) are central regulators of gene expression in bacteria, controlling target genes posttranscriptionally by base pairing with their mRNAs. sRNAs are involved in many cellular processes and have unique regulatory characteristics. In this review, we discuss the properties of regulation by sRNAs and how it differs from and combines with transcriptional regulation. We describe the global characteristics of the sRNA-target networks in bacteria using graph-theoretic approaches and review the local integration of sRNAs in mixed regulatory circuits, including feed-forward loops and their combinations, feedback loops, and circuits made of an sRNA and another regulator, both derived from the same transcript. Finally, we discuss the competition effects in posttranscriptional regulatory networks that may arise over shared targets, shared regulators, and shared resources and how they may lead to signal propagation across the network.

  11. Genome sequence and description of Paenibacillus ihuae strain GD6 sp. nov., isolated from the stool of a 62-year-old Frenchman

    Directory of Open Access Journals (Sweden)

    C. Al-Bayssari

    2018-05-01

    Full Text Available Paenibacillus ihuae strain GD6 (=CSUR P892 = DSMZ 45751T is the new type strain collected from the stool of a 69-year-old Frenchman admitted to an intensive care unit and receiving a 10-day course of imipenem at the time of stool collection. This is a Gram-positive, facultative anaerobic, rod-shaped bacterium. We describe here the features of this organism, together with its complete genome sequence and annotation. The genome size is 6 719 043 bp with 49.6% G+C content and contains 6211 protein-coding and 65 sRNA genes, including four 5S rRNA genes, one 16S rRNA gene and one 23S rRNA gene. Keywords: Culturomics, Paenibacillus ihuae, taxonogenomics

  12. Investigating membrane-bound Argonaute functions in Arabidopsis

    DEFF Research Database (Denmark)

    Barghetti, Andrea

    and how AGO1 membrane recruitment is mediated as well as its functional importance remain poorly characterized. Isoprenoid biogenesis was previously found to be required for both AGO1 activity and membrane association, but the mechanistic connection between the two pathways was not discovered. Since....... The key effectors of sRNA-guided gene regulation are ARGONAUTE (AGO) proteins. A group of Heat Shock Proteins of the HSP70/HSP90 chaperone machinery mediates the process, termed loading, that allow the functional association of sRNA with AGOs. Upon loading, Argonautes regulate complementary mRNA targets...... with the rough endoplasmic reticulum (rER). Membranelocalized argonaute functions include translational repression, production of secondary phased small interfering RNA (siRNA) and autophagy-mediated turnover. However proteins interacting with AGO1 specifically on membrane fractions have not been identified...

  13. The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism.

    Science.gov (United States)

    Bækkedal, Cecilie; Haugen, Peik

    2015-01-01

    The Spot 42 RNA is a 109 nucleotide long (in Escherichia coli) noncoding small regulatory RNA (sRNA) encoded by the spf (spot fourty-two) gene. spf is found in gamma-proteobacteria and the majority of experimental work on Spot 42 RNA has been performed using E. coli, and recently Aliivibrio salmonicida. In the cell Spot 42 RNA plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex. Here we summarize the current knowledge on Spot 42, and present the natural distribution of spf, show family-specific secondary structural features of Spot 42, and link highly conserved structural regions to mRNA target binding.

  14. Regulatory RNA-assisted genome engineering in microorganisms.

    Science.gov (United States)

    Si, Tong; HamediRad, Mohammad; Zhao, Huimin

    2015-12-01

    Regulatory RNAs are increasingly recognized and utilized as key modulators of gene expression in diverse organisms. Thanks to their modular and programmable nature, trans-acting regulatory RNAs are especially attractive in genome-scale applications. Here we discuss the recent examples in microbial genome engineering implementing various trans-acting RNA platforms, including sRNA, RNAi, asRNA and CRISRP-Cas. In particular, we focus on how the scalable and multiplex nature of trans-acting RNAs has been used to tackle the challenges in creating genome-wide and combinatorial diversity for functional genomics and metabolic engineering applications. Advances in computational design and context-dependent regulation are also discussed for their contribution in improving fine-tuning capabilities of trans-acting RNAs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. (Sr{sub 1-x}Na{sub x})(Cd{sub 1-x}Mn{sub x}){sub 2}As{sub 2}: A new charge and spin doping decoupled diluted magnetic semiconductors with CaAl{sub 2}Si{sub 2}-type structure

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Bijuan; Deng, Zheng; Li, Wenmin; Gao, Moran; Zhao, Guoqiang; Yu, Shuang; Wang, Xiancheng; Liu, Qingqing [Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China); Li, Zhi [School of Materials Science and Engineering, Hefei University of Technology, Hefei, Anhui 230009 (China); Jin, Changqing, E-mail: Jin@iphy.ac.cn [Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China); Collaborative Innovation Center of Quantum Matter, Beijing 100190 (China)

    2016-08-28

    We report the synthesis and characterization of a new bulk diluted ferromagnetic semiconductor via Na and Mn co-doping in SrCd{sub 2}As{sub 2} with a hexagonal CaAl{sub 2}Si{sub 2}-type structure. Together with carrier doping via (Sr,Na) substitution, spin doping via (Cd,Mn) substitution results in ferromagnetic order with Curie temperature of T{sub C} up to 13 K. Negative magnetoresistance is assigned to weak localization at low temperatures, where the magnetization of samples becomes saturated. The hexagonal structure of (Sr{sub 1−x}Na{sub x})(Cd{sub 1−x}Mn{sub x}){sub 2}As{sub 2} can be acted as a promising candidate for spin manipulations owing to its relatively small coercive field of less than 24 Oe.

  16. Identifying and characterizing Hfq-RNA interactions.

    Science.gov (United States)

    Faner, M A; Feig, A L

    2013-09-15

    To regulate stress responses and virulence, bacteria use small regulatory RNAs (sRNAs). These RNAs can up or down regulate target mRNAs through base pairing by influencing ribosomal access and RNA decay. A large class of these sRNAs, called trans-encoded sRNAs, requires the RNA binding protein Hfq to facilitate base pairing between the regulatory RNA and its target mRNA. The resulting network of regulation is best characterized in Escherichia coli and Salmonella typhimurium, but the importance of Hfq dependent sRNA regulation is recognized in a diverse population of bacteria. In this review we present the approaches and methods used to discover Hfq binding RNAs, characterize their interactions and elucidate their functions. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. sRNA-Mediated Regulation of P-Fimbriae Phase Variation in Uropathogenic Escherichia coli

    DEFF Research Database (Denmark)

    Khandige, Surabhi; Kronborg, Tina; Uhlin, Bernt Eric

    2015-01-01

    Uropathogenic Escherichia coli (UPEC) are capable of occupying physiologically distinct intracellular and extracellular niches within the urinary tract. This feat requires the timely regulation of gene expression and small RNAs (sRNAs) are known to mediate such rapid adjustments in response to ch...... to changing environmental cues. This study aimed to uncover sRNA-mediated gene regulation in the UPEC strain UTI89, during infection of bladder epithelial cells. Hfq is an RNA chaperone known to facilitate and stabilize sRNA and target mRNA interactions with bacterial cells. The co...... to the discovery of a novel virulence-associated trans-acting sRNA-PapR. Deletion of papR was found to enhance adhesion of UTI89 to both bladder and kidney cell lines in a manner independent of type-1 fimbriae. We demonstrate PapR mediated posttranscriptional repression of the P-fimbriae phase regulator gene pap...

  18. An Experimental Study of Micron-Size Zero-Valent Iron Emplacement in Permeable Porous Media Using Polymer-Enhanced Fluids

    Energy Technology Data Exchange (ETDEWEB)

    Oostrom, Mart; Wietsma, Thomas W.; Covert, Matthew A.; Vermeul, Vince R.

    2005-12-22

    At the Hanford Site, an extensive In Situ Redox Manipulation (ISRM) permeable reactive barrier was installed to prevent chromate from reaching the Columbia River. However, chromium has been detected in several wells, indicating a premature loss of the reductive capacity in the aquifer. One possible cause for premature chromate breakthrough is associated with the presence of high-permeability zones in the aquifer. In these zones, groundwater moves relatively fast and is able to oxidize iron more rapidly. There is also a possibility that the high-permeability flow paths are deficient in reducing equivalents (e.g. reactive iron), required for barrier performance. One way enhancement of the current barrier reductive capacity can be achieved is by the addition of micron-scale zero-valent iron to the high-permeability zones within the aquifer. The potential emplacement of zero-valent iron (Fe0) into high-permeability Hanford sediments (Ringold Unit E gravels) using shear-thinning fluids containing polymers was investigated in three-dimensional wedge-shaped aquifer models. Polymers were used to create a suspension viscous enough to keep the Fe0 in solution for extended time periods to improve colloid movement into the porous media without causing a permanent detrimental decrease in hydraulic conductivity. Porous media were packed in the wedge-shaped flow cell to create either a heterogeneous layered system with a high-permeability zone in between two low-permeability zones or a high-permeability channel surrounded by low-permeability materials. The injection flow rate, polymer type, polymer concentration, and injected pore volumes were determined based on preliminary short- and long-column experiments.

  19. Geo-Mechanical Characterization of Carbonate Rock Masses by Means of Laser Scanner Technique

    Science.gov (United States)

    Palma, Biagio; Parise, Mario; Ruocco, Anna

    2017-12-01

    Knowledge of the geometrical and structural setting of rock masses is crucial to evaluate the stability and to design the most suitable stabilization works. In this work we use the Terrestrial Laser Scanning (TLS) at the site of the Grave of the Castellana Caves, a famous show cave in southern Italy. The Grave is the natural access to the cave system, produced by collapse of the vault, due to upward progression of instabilities in the carbonate rock masses. It is about 55-m high, bell-shaped, with maximum width of 120 m. Aim of the work is the characterization of carbonate rock masses from the structural and geo-mechanical standpoints through the use of innovative survey techniques. TLS survey provides a product consisting of millions of geo-referenced points, to be managed in space, to become a suitable database for the morphological and geological-structural analysis. Studying by means of TLS a rock face, partly inaccessible or located in very complex environments, allows to investigate slopes in their overall areal extent, thus offering advantages both as regards safety of the workers and time needed for the survey. In addition to TLS, the traditional approach was also followed by performing scanlines surveys along the rims of the Grave, following the ISRM recommendations for characterization of discontinuity in rock masses. A quantitative comparison among the data obtained by TLS technique and those deriving from the classical geo-mechanical survey is eventually presented, to discuss potentiality of drawbacks of the different techniques used for surveying the rock masses.

  20. The phenotypic and molecular assessment of the non-conserved Arabidopsis MICRORNA163/S-ADENOSYL-METHYLTRANSFERASE regulatory module during biotic stress.

    Science.gov (United States)

    Litholdo, Celso Gaspar; Eamens, Andrew Leigh; Waterhouse, Peter Michael

    2018-04-01

    In plants, microRNAs (miRNAs) have evolved in parallel to the protein-coding genes that they target for expression regulation, and miRNA-directed gene expression regulation is central to almost every cellular process. MicroRNA, miR163, is unique to the Arabidopsis genus and is processed into a 24-nucleotide (nt) mature small regulatory RNA (sRNA) from a single precursor transcript transcribed from a single locus, the MIR163 gene. The MIR163 locus is a result of a recent inverted duplication event of one of the five closely related S-ADENOSYL-METHYLTRANSFERASE genes that the mature miR163 sRNA targets for expression regulation. Currently, however, little is known about the role of the miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in response to biotic stress. Here, we document the expression domains of MIR163 and the S-ADENOSYL-METHYLTRANSFERASE target genes following fusion of their putative promoter sequences to the β-glucuronidase (GUS) reporter gene and subsequent in planta expression. Further, we report on our phenotypic and molecular assessment of Arabidopsis thaliana plants with altered miR163 accumulation, namely the mir163-1 and mir163-2 insertion knockout mutants and the miR163 overexpression line, the MIR163-OE plant. Finally, we reveal miR163 accumulation and S-ADENOSYL-METHYLTRANSFERASE target gene expression post treatment with the defence elicitors, salicylic acid and jasmonic acid, and following Fusarium oxysporum infection, wounding, and herbivory attack. Together, the work presented here provides a comprehensive new biological insight into the role played by the Arabidopsis genus-specific miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in normal A. thaliana development and during the exposure of A. thaliana plants to biotic stress.

  1. Glucose uptake regulation in E. coli by the small RNA SgrS: comparative analysis of E. coli K-12 (JM109 and MG1655 and E. coli B (BL21

    Directory of Open Access Journals (Sweden)

    Ng Weng-Ian

    2010-09-01

    Full Text Available Abstract Background The effect of high glucose concentration on the transcription levels of the small RNA SgrS and the messenger RNA ptsG, (encoding the glucose transporter IICBGlc, was studied in both E. coli K-12 (MG1655 and JM109 and E. coli B (BL21. It is known that the transcription level of sgrS increases when E. coli K-12 (MG1655 and JM109 is exposed to the non-metabolized glucose alpha methyl glucoside (αMG or when the bacteria with a defective glycolysis pathway is grown in presence of glucose. The increased level of sRNA SgrS reduces the level of the ptsG mRNA and consequently lowers the level of the glucose transporter IICBGlc. The suggested trigger for this action is the accumulation of the corresponding phospho-sugars. Results In the course of the described work, it was found that E. coli B (BL21 and E. coli K-12 (JM109 and MG1655 responded similarly to αMG: both strains increased SgrS transcription and reduced ptsG transcription. However, the two strains reacted differently to high glucose concentration (40 g/L. E. coli B (BL21 reacted by increasing sgrS transcription and reducing ptsG transcription while E. coli K-12 (JM109 and MG1655 did not respond to the high glucose concentration, and, therefore, transcription of sgrS was not detected and ptsG mRNA level was not affected. Conclusions The results suggest that E. coli B (BL21 tolerates high glucose concentration not only by its more efficient central carbon metabolism, but also by controlling the glucose transport into the cells regulated by the sRNA SgrS, which may suggest a way to control glucose consumption and increase its efficient utilization.

  2. Small RNAs from Bemisia tabaci are transferred to Solanum lycopersicum phloem during feeding

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    Paula J.M. Van Kleeff

    2016-11-01

    Full Text Available The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling.Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.

  3. Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

    Science.gov (United States)

    Sonnleitner, Elisabeth; Valentini, Martina; Wenner, Nicolas; Haichar, Feth el Zahar; Haas, Dieter; Lapouge, Karine

    2012-01-01

    The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.

  4. Small RNA-Sequencing Links Physiological Changes and RdDM Process to Vegetative-to-Floral Transition in Apple

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    Xinwei Guo

    2017-05-01

    Full Text Available Transition from vegetative to floral buds is a critical physiological change during flower induction that determines fruit productivity. Small non-coding RNAs (sRNAs including microRNAs (miRNAs and small interfering RNAs (siRNAs are pivotal regulators of plant growth and development. Although the key role of sRNAs in flowering regulation has been well-described in Arabidopsis and some other annual plants, their relevance to vegetative-to-floral transition (hereafter, referred to floral transition in perennial woody trees remains under defined. Here, we performed Illumina sequencing of sRNA libraries prepared from vegetative and floral bud during flower induction of the apple trees. A large number of sRNAs exemplified by 33 previously annotated miRNAs and six novel members display significant differential expression (DE patterns. Notably, most of these DE-miRNAs in floral transition displayed opposite expression changes in reported phase transition in apple trees. Bioinformatics analysis suggests most of the DE-miRNAs targeted transcripts involved in SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL gene regulation, stress responses, and auxin and gibberellin (GA pathways, with further suggestion that there is an inherent link between physiological stress response and metabolism reprogramming during floral transition. We also observed significant changes in 24 nucleotide (nt sRNAs that are hallmarks for RNA-dependent DNA methylation (RdDM pathway, suggestive of the correlation between epigenetic modifications and the floral transition. The study not only provides new insight into our understanding of fundamental mechanism of poorly studied floral transition in apple and other woody plants, but also presents important sRNA resource for future in-depth research in the apple flowering physiology.

  5. Riboregulation of the bacterial actin-homolog MreB by DsrA small noncoding RNA.

    Science.gov (United States)

    Cayrol, Bastien; Fortas, Emilie; Martret, Claire; Cech, Grzegorz; Kloska, Anna; Caulet, Stephane; Barbet, Marion; Trépout, Sylvain; Marco, Sergio; Taghbalout, Aziz; Busi, Florent; Wegrzyn, Grzegorz; Arluison, Véronique

    2015-01-01

    The bacterial actin-homolog MreB is a key player in bacterial cell-wall biosynthesis and is required for the maintenance of the rod-like morphology of Escherichia coli. However, how MreB cellular levels are adjusted to growth conditions is poorly understood. Here, we show that DsrA, an E. coli small noncoding RNA (sRNA), is involved in the post-transcriptional regulation of mreB. DsrA is required for the downregulation of MreB cellular concentration during environmentally induced slow growth-rates, mainly growth at low temperature and during the stationary phase. DsrA interacts in an Hfq-dependent manner with the 5' region of mreB mRNA, which contains signals for translation initiation and thereby affects mreB translation and stability. Moreover, as DsrA is also involved in the regulation of two transcriptional regulators, σ(S) and the nucleoid associated protein H-NS, which negatively regulate mreB transcription, it also indirectly contributes to mreB transcriptional down-regulation. By using quantitative analyses, our results evidence the complexity of this regulation and the tangled interplay between transcriptional and post-transcriptional control. As transcription factors and sRNA-mediated post-transcriptional regulators use different timescales, we propose that the sRNA pathway helps to adapt to changes in temperature, but also indirectly mediates long-term regulation of MreB concentration. The tight regulation and fine-tuning of mreB gene expression in response to cellular stresses is discussed in regard to the effect of the MreB protein on cell elongation.

  6. A Genome-Wide Identification of the miRNAome in Response to Salinity Stress in Date palm (Phoenix dactylifera L.

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    Mahmoud W Yaish

    2015-11-01

    Full Text Available Although date palm is relatively salt-tolerant, little is known about the underlying molecular mechanisms that contribute to its salt tolerance. Only recently, investigators have uncovered microRNA-mediated post-transcriptional gene regulation, which is critical for typical plant development and adaptation to stress conditions such as salinity. To identify conserved and novel miRNAs in date palm and to characterise miRNAs that could play a role in salt tolerance, we have generated sRNA libraries from the leaves and roots of NaCl-treated and untreated seedlings of date palm. Deep sequencing of these four sRNA libraries yielded approximately 251 million reads. The bioinformatics analysis has identified 153 homologs of conserved miRNAs, 89 miRNA variants, and 180 putative novel miRNAs in date palm. Expression profiles under salinity revealed differential regulation of some miRNAs in date palm. In leaves, 54 of the identified miRNAs were significantly affected and the majority (70% of them were upregulated, whereas in roots, 25 of the identified miRNAs were significantly affected and 76% of them were upregulated by the salinity stress. The salt-responsiveness of some of these miRNAs was further validated using semi-quantitative PCR (qPCR. Some of the predicted targets for the identified miRNA include genes with known functions in plant salt tolerance, such as potassium channel AKT2-like proteins, vacuolar protein sorting-associated protein, calcium-dependent and mitogen-activated proteins. As one of the first cultivated trees in the world that can tolerate a wide range of abiotic stresses, date palm contains a large population of conserved and nonconserved miRNAs that function at the post-transcriptional level. This study provided insights into miRNA-mediated gene expression that are important for adaptation to salinity in date palms.

  7. A Regulatory Circuit Composed of a Transcription Factor, IscR, and a Regulatory RNA, RyhB, Controls Fe-S Cluster Delivery

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    Pierre Mandin

    2016-09-01

    Full Text Available Fe-S clusters are cofactors conserved through all domains of life. Once assembled by dedicated ISC and/or SUF scaffolds, Fe-S clusters are conveyed to their apo-targets via A-type carrier proteins (ATCs. Escherichia coli possesses four such ATCs. ErpA is the only ATC essential under aerobiosis. Recent studies reported a possible regulation of the erpA mRNA by the small RNA (sRNA RyhB, which controls the expression of many genes under iron starvation. Surprisingly, erpA has not been identified in recent transcriptomic analysis of the iron starvation response, thus bringing into question the actual physiological significance of the putative regulation of erpA by RyhB. Using an sRNA library, we show that among 26 sRNAs, only RyhB represses the expression of an erpA-lacZ translational fusion. We further demonstrate that this repression occurs during iron starvation. Using mutational analysis, we show that RyhB base pairs to the erpA mRNA, inducing its disappearance. In addition, IscR, the master regulator of Fe-S homeostasis, represses expression of erpA at the transcriptional level when iron is abundant, but depleting iron from the medium alleviates this repression. The conjunction of transcriptional derepression by IscR and posttranscriptional repression by RyhB under Fe-limiting conditions is best described as an incoherent regulatory circuit. This double regulation allows full expression of erpA at iron concentrations for which Fe-S biogenesis switches from the ISC to the SUF system. We further provide evidence that this regulatory circuit coordinates ATC usage to iron availability.

  8. Identification of isoforms of microRNAs in wheat (Triticum aestivum L. and their role in leaf rust pathogenesis

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    Summi Dutta

    2017-10-01

    Full Text Available Bread wheat, a type of grass under genus Triticum and species aestivum covers the largest land area when production of cereal crops is considered. Being an allohexaploid (2n=6x=42; AABBDD, its genome is contributed by three progenitors and is evolutionarily rich. Rust in leaves, caused by Puccinia triticina, severely affects grain quality. MicroRNAs are considered as major components of gene silencing and so have deep role to play during stress. Post transcriptional modification of miRNAs which generates isomiRNAs significantly affects target specificity especially when the modification occurs in 5′end. A total of four small RNA libraries were prepared through next-generation Illumina sequencing techniques from leaves of two wheat Near Isogenic Lines (NILs, HD2329 (susceptible and HD2329 + LR24 (resistant. Prior to this, one set of the two NILs was mock inoculated and considered as control (with sRNA library code named SM-mi and RM-mi while other was treated with urediniospores of leaf rust fungus (with sRNA library code named SPI-mi and RPI-mi. Clean reads in all four libraries were previously used for prediction of 559 novel miRNAs and in the current study it was used to detect isoforms of these miRNAs. A total of 237 isoforms were detected for 41 miRNAs. These isoforms included both 5′ and 3′ modifications of miRNAs. There were 27 miRNAs with 5′ modifications and five miRNAs with 3′ modifications while nine miRNAs showed both types of modifications.

  9. RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs.

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    Baoyan Bai

    Full Text Available Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA. Sequences from 47 sdRNAs were identified, which mapped to both 5' and 3' ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha-mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

  10. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

    Science.gov (United States)

    van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

    2016-01-01

    RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

  11. Evolutionary patterns of Escherichia coli small RNAs and their regulatory interactions.

    Science.gov (United States)

    Peer, Asaf; Margalit, Hanah

    2014-07-01

    Most bacterial small RNAs (sRNAs) are post-transcriptional regulators of gene expression, exerting their regulatory function by base-pairing with their target mRNAs. While it has become evident that sRNAs play central regulatory roles in the cell, little is known about their evolution and the evolution of their regulatory interactions. Here we used the prokaryotic phylogenetic tree to reconstruct the evolutionary history of Escherichia coli sRNAs and their binding sites on target mRNAs. We discovered that sRNAs currently present in E. coli mainly accumulated inside the Enterobacteriales order, succeeding the appearance of other types of noncoding RNAs and concurrently with the evolution of a variant of the Hfq protein exhibiting a longer C-terminal region. Our analysis of the evolutionary ages of sRNA-mRNA interactions revealed that while all sRNAs were evolutionarily older than most of their known binding sites on mRNA targets, for quite a few sRNAs there was at least one binding site that coappeared with or preceded them. It is conceivable that the establishment of these first interactions forced selective pressure on the sRNAs, after which additional targets were acquired by fitting a binding site to the active region of the sRNA. This conjecture is supported by the appearance of many binding sites on target mRNAs only after the sRNA gain, despite the prior presence of the target gene in ancestral genomes. Our results suggest a selective mechanism that maintained the sRNAs across the phylogenetic tree, and shed light on the evolution of E. coli post-transcriptional regulatory network. © 2014 Peer and Margalit; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  12. Patterns of gene flow define species of thermophilic Archaea.

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    Hinsby Cadillo-Quiroz

    2012-02-01

    Full Text Available Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.

  13. Patterns of gene flow define species of thermophilic Archaea.

    Science.gov (United States)

    Cadillo-Quiroz, Hinsby; Didelot, Xavier; Held, Nicole L; Herrera, Alfa; Darling, Aaron; Reno, Michael L; Krause, David J; Whitaker, Rachel J

    2012-02-01

    Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.

  14. Genetic variability of psychrotolerant Acidithiobacillus ferrivorans revealed by (meta)genomic analysis.

    Science.gov (United States)

    González, Carolina; Yanquepe, María; Cardenas, Juan Pablo; Valdes, Jorge; Quatrini, Raquel; Holmes, David S; Dopson, Mark

    2014-11-01

    Acidophilic microorganisms inhabit low pH environments such as acid mine drainage that is generated when sulfide minerals are exposed to air. The genome sequence of the psychrotolerant Acidithiobacillus ferrivorans SS3 was compared to a metagenome from a low temperature acidic stream dominated by an A. ferrivorans-like strain. Stretches of genomic DNA characterized by few matches to the metagenome, termed 'metagenomic islands', encoded genes associated with metal efflux and pH homeostasis. The metagenomic islands were enriched in mobile elements such as phage proteins, transposases, integrases and in one case, predicted to be flanked by truncated tRNAs. Cus gene clusters predicted to be involved in copper efflux and further Cus-like RND systems were predicted to be located in metagenomic islands and therefore, constitute part of the flexible gene complement of the species. Phylogenetic analysis of Cus clusters showed both lineage specificity within the Acidithiobacillus genus as well as niche specificity associated with an acidic environment. The metagenomic islands also contained a predicted copper efflux P-type ATPase system and a polyphosphate kinase potentially involved in polyphosphate mediated copper resistance. This study identifies genetic variability of low temperature acidophiles that likely reflects metal resistance selective pressures in the copper rich environment. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  15. Molecular Analysis of Asymptomatic Bacteriuria Escherichia coli Strain VR50 Reveals Adaptation to the Urinary Tract by Gene Acquisition

    DEFF Research Database (Denmark)

    Beatson, Scott A.; Ben Zakour, Nouri L.; Totsika, Makrina

    2015-01-01

    the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has...... a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50...... mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability...

  16. Genome wide identification of chilling responsive microRNAs in Prunus persica

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    Barakat Abdelali

    2012-09-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small RNAs (sRNAs approximately 21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. Within this context, miRNAs and siRNAs are coming to the forefront as molecular mediators of gene regulation in plant responses to annual temperature cycling and cold stress. For this reason, we chose to identify and characterize the conserved and non-conserved miRNA component of peach (Prunus persica (L. Batsch focusing our efforts on both the recently released whole genome sequence of peach and sRNA transcriptome sequences from two tissues representing non-dormant leaves and dormant leaf buds. Conserved and non-conserved miRNAs, and their targets were identified. These sRNA resources were used to identify cold-responsive miRNAs whose gene targets co-localize with previously described QTLs for chilling requirement (CR. Results Analysis of 21 million peach sRNA reads allowed us to identify 157 and 230 conserved and non-conserved miRNA sequences. Among the non-conserved miRNAs, we identified 205 that seem to be specific to peach. Comparative genome analysis between peach and Arabidopsis showed that conserved miRNA families, with the exception of miR5021, are similar in size. Sixteen of these conserved miRNA families are deeply rooted in land plant phylogeny as they are present in mosses and/or lycophytes. Within the other conserved miRNA families, five families (miR1446, miR473, miR479, miR3629, and miR3627 were reported only in tree species (Populustrichocarpa, Citrus trifolia, and Prunus persica. Expression analysis identified several up-regulated or down-regulated miRNAs in winter buds versus young leaves. A search of the peach proteome allowed the prediction of target genes for most of the conserved miRNAs and a large fraction of non-conserved miRNAs. A fraction of predicted targets in peach have not been previously reported in other

  17. A Regulatory Circuit Composed of a Transcription Factor, IscR, and a Regulatory RNA, RyhB, Controls Fe-S Cluster Delivery.

    Science.gov (United States)

    Mandin, Pierre; Chareyre, Sylvia; Barras, Frédéric

    2016-09-20

    Fe-S clusters are cofactors conserved through all domains of life. Once assembled by dedicated ISC and/or SUF scaffolds, Fe-S clusters are conveyed to their apo-targets via A-type carrier proteins (ATCs). Escherichia coli possesses four such ATCs. ErpA is the only ATC essential under aerobiosis. Recent studies reported a possible regulation of the erpA mRNA by the small RNA (sRNA) RyhB, which controls the expression of many genes under iron starvation. Surprisingly, erpA has not been identified in recent transcriptomic analysis of the iron starvation response, thus bringing into question the actual physiological significance of the putative regulation of erpA by RyhB. Using an sRNA library, we show that among 26 sRNAs, only RyhB represses the expression of an erpA-lacZ translational fusion. We further demonstrate that this repression occurs during iron starvation. Using mutational analysis, we show that RyhB base pairs to the erpA mRNA, inducing its disappearance. In addition, IscR, the master regulator of Fe-S homeostasis, represses expression of erpA at the transcriptional level when iron is abundant, but depleting iron from the medium alleviates this repression. The conjunction of transcriptional derepression by IscR and posttranscriptional repression by RyhB under Fe-limiting conditions is best described as an incoherent regulatory circuit. This double regulation allows full expression of erpA at iron concentrations for which Fe-S biogenesis switches from the ISC to the SUF system. We further provide evidence that this regulatory circuit coordinates ATC usage to iron availability. Regulatory small RNAs (sRNAs) have emerged as major actors in the control of gene expression in the last few decades. Relatively little is known about how these regulators interact with classical transcription factors to coordinate genetic responses. We show here how an sRNA, RyhB, and a transcription factor, IscR, regulate expression of an essential gene, erpA, in the bacterium E

  18. Some Challenges of Deep Mining†

    Directory of Open Access Journals (Sweden)

    Charles Fairhurst

    2017-08-01

    Full Text Available An increased global supply of minerals is essential to meet the needs and expectations of a rapidly rising world population. This implies extraction from greater depths. Autonomous mining systems, developed through sustained R&D by equipment suppliers, reduce miner exposure to hostile work environments and increase safety. This places increased focus on “ground control” and on rock mechanics to define the depth to which minerals may be extracted economically. Although significant efforts have been made since the end of World War II to apply mechanics to mine design, there have been both technological and organizational obstacles. Rock in situ is a more complex engineering material than is typically encountered in most other engineering disciplines. Mining engineering has relied heavily on empirical procedures in design for thousands of years. These are no longer adequate to address the challenges of the 21st century, as mines venture to increasingly greater depths. The development of the synthetic rock mass (SRM in 2008 provides researchers with the ability to analyze the deformational behavior of rock masses that are anisotropic and discontinuous—attributes that were described as the defining characteristics of in situ rock by Leopold Müller, the president and founder of the International Society for Rock Mechanics (ISRM, in 1966. Recent developments in the numerical modeling of large-scale mining operations (e.g., caving using the SRM reveal unanticipated deformational behavior of the rock. The application of massive parallelization and cloud computational techniques offers major opportunities: for example, to assess uncertainties in numerical predictions; to establish the mechanics basis for the empirical rules now used in rock engineering and their validity for the prediction of rock mass behavior beyond current experience; and to use the discrete element method (DEM in the optimization of deep mine design. For the first time, mining

  19. Compliant contact versus rigid contact: A comparison in the context of granular dynamics

    Science.gov (United States)

    Pazouki, Arman; Kwarta, Michał; Williams, Kyle; Likos, William; Serban, Radu; Jayakumar, Paramsothy; Negrut, Dan

    2017-10-01

    We summarize and numerically compare two approaches for modeling and simulating the dynamics of dry granular matter. The first one, the discrete-element method via penalty (DEM-P), is commonly used in the soft matter physics and geomechanics communities; it can be traced back to the work of Cundall and Strack [P. Cundall, Proc. Symp. ISRM, Nancy, France 1, 129 (1971); P. Cundall and O. Strack, Geotechnique 29, 47 (1979), 10.1680/geot.1979.29.1.47]. The second approach, the discrete-element method via complementarity (DEM-C), considers the grains perfectly rigid and enforces nonpenetration via complementarity conditions; it is commonly used in robotics and computer graphics applications and had two strong promoters in Moreau and Jean [J. J. Moreau, in Nonsmooth Mechanics and Applications, edited by J. J. Moreau and P. D. Panagiotopoulos (Springer, Berlin, 1988), pp. 1-82; J. J. Moreau and M. Jean, Proceedings of the Third Biennial Joint Conference on Engineering Systems and Analysis, Montpellier, France, 1996, pp. 201-208]. The DEM-P and DEM-C are manifestly unlike each other: They use different (i) approaches to model the frictional contact problem, (ii) sets of model parameters to capture the physics of interest, and (iii) classes of numerical methods to solve the differential equations that govern the dynamics of the granular material. Herein, we report numerical results for five experiments: shock wave propagation, cone penetration, direct shear, triaxial loading, and hopper flow, which we use to compare the DEM-P and DEM-C solutions. This exercise helps us reach two conclusions. First, both the DEM-P and DEM-C are predictive, i.e., they predict well the macroscale emergent behavior by capturing the dynamics at the microscale. Second, there are classes of problems for which one of the methods has an advantage. Unlike the DEM-P, the DEM-C cannot capture shock-wave propagation through granular media. However, the DEM-C is proficient at handling arbitrary grain

  20. Fracture toughness properties of rocks in Olkiluoto: Laboratory measurements 2008-2009

    Energy Technology Data Exchange (ETDEWEB)

    Siren, T.

    2012-05-15

    In Olkiluoto an underground rock characterization facility (ONKALO) for the final disposal site of spent nuclear fuel has been under thorough research many years, but further knowledge is needed on fracture toughness parameters. Fracture toughness parameters are important for example in fracture mechanics prediction for Posiva's Olkiluoto Spalling Experiment (POSE). This working report describes a laboratory campaign that was done between 2008 and 2009. The campaign aimed at determining the fracture mechanics parameters as well as density and ultrasonic velocities for Olkiluoto rocks. The specimens delivered were selected by Posiva; the core showed no damage and the quality of the delivered cores was good with varying sample diameter. Most of the test samples (9 out of 12) are gneissic rock. The Mode I fracture toughness was determined using two different methods to account for two different fracturing directions. The methods are the Chevron Bend (CB) test as proposed in the ISRM Suggested Method and a method based on the Brazilian Disk (BD) experiment. The Mode II fracture toughness was determined using the Punch-Through Shear with Confining Pressure experiment on the remaining pieces from the CB testing. The scatter in the results is very large, even within one piece of core sample. Usually the scatter of results is less than 5 %. The high scatter in the data at hand is believed to be due to the very inhomogeneous nature of the rock material. The magnitude of the determined Mode I fracture toughness compares well with available reported data for medium to coarse grained granitoide rocks. However the scatter of the mode II fracture toughness values is higher than experienced on other rock types, but the variability is reasonable for the inhomogeneous rock type. Distinguishing the fracture toughness values for different anisotropy directions would require more thorough testing with quality samples at different anisotropy directions. However since fracture

  1. Factors affecting the inter-annual to centennial timescale variability of Indian summer monsoon rainfall

    Science.gov (United States)

    Malik, Abdul; Brönnimann, Stefan

    2018-06-01

    The Modes of Ocean Variability (MOV) namely Atlantic Multidecadal Oscillation (AMO), Pacific Decadal Oscillation (PDO), and El Niño Southern Oscillation (ENSO) can have significant impacts on Indian Summer Monsoon Rainfall (ISMR) on different timescales. The timescales at which these MOV interacts with ISMR and the factors which may perturb their relationship with ISMR need to be investigated. We employ De-trended Cross-Correlation Analysis (DCCA), and De-trended Partial-Cross-Correlation Analysis (DPCCA) to study the timescales of interaction of ISMR with AMO, PDO, and ENSO using observational dataset (AD 1854-1999), and atmosphere-ocean-chemistry climate model simulations with SOCOL-MPIOM (AD 1600-1999). Further, this study uses De-trended Semi-Partial Cross-Correlation Analysis (DSPCCA) to address the relation between solar variability and the ISMR. We find statistically significant evidence of intrinsic correlations of ISMR with AMO, PDO, and ENSO on different timescales, consistent between model simulations and observations. However, the model fails to capture modulation in intrinsic relationship between ISRM and MOV due to external signals. Our analysis indicates that AMO is a potential source of non-stationary relationship between ISMR and ENSO. Furthermore, the pattern of correlation between ISMR and Total Solar Irradiance (TSI) is inconsistent between observations and model simulations. The observational dataset indicates statistically insignificant negative intrinsic correlation between ISMR and TSI on decadal-to-centennial timescales. This statistically insignificant negative intrinsic correlation is transformed to statistically significant positive extrinsic by AMO on 61-86-year timescale. We propose a new mechanism for Sun-monsoon connection which operates through AMO by changes in summer (June-September; JJAS) meridional gradient of tropospheric temperatures (ΔTTJJAS). There is a negative (positive) intrinsic correlation between ΔTTJJAS (AMO) and

  2. Permeability evolution due to dissolution of natural shale fractures reactivated by fracking

    Science.gov (United States)

    Kwiatkowski, Kamil; Kwiatkowski, Tomasz; Szymczak, Piotr

    2015-04-01

    Shale and their importance for hydraulic fracture treatments. AAPG bulletin, 91(4), 603-622. [2] Walton, I., & McLennan, J. (2013, May). The Role of Natural Fractures in Shale Gas Production. In ISRM International Conference for Effective and Sustainable Hydraulic Fracturing. International Society for Rock Mechanics. [3] Grieser, W. et al. "Surface Reactive Fluid's Effect on Shale." Proceedings of the Production and Operations Symposium, 31 March-3 April 2007, Oklahoma City (SPE-106815-MS) [4] Khosrokhavar, R., Griffiths, S., & Wolf, K. H. (2014). Shale Gas Formations and Their Potential for Carbon Storage: Opportunities and Outlook. Environmental Processes, 1(4), 595-611.

  3. Identification and functional analysis of flowering related microRNAs in common wild rice (Oryza rufipogon Griff.).

    Science.gov (United States)

    Chen, Zongxiang; Li, Fuli; Yang, Songnan; Dong, Yibo; Yuan, Qianhua; Wang, Feng; Li, Weimin; Jiang, Ying; Jia, Shirong; Pei, Xinwu

    2013-01-01

    MicroRNAs (miRNAs) is a class of non-coding RNAs involved in post- transcriptional control of gene expression, via degradation and/or translational inhibition. Six-hundred sixty-one rice miRNAs are known that are important in plant development. However, flowering-related miRNAs have not been characterized in Oryza rufipogon Griff. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. rufipogon using high-throughput sequencing (deep sequencing) to understand the changes that occurred during rice domestication, and to elucidate their functions in flowering. Three O. rufipogon sRNA libraries, two vegetative stage (CWR-V1 and CWR-V2) and one flowering stage (CWR-F2) were sequenced using Illumina deep sequencing. A total of 20,156,098, 21,531,511 and 20,995,942 high quality sRNA reads were obtained from CWR-V1, CWR-V2 and CWR-F2, respectively, of which 3,448,185, 4,265,048 and 2,833,527 reads matched known miRNAs. We identified 512 known rice miRNAs in 214 miRNA families and predicted 290 new miRNAs. Targeted functional annotation, GO and KEGG pathway analyses predicted that 187 miRNAs regulate expression of flowering-related genes. Differential expression analysis of flowering-related miRNAs showed that: expression of 95 miRNAs varied significantly between the libraries, 66 are flowering-related miRNAs, such as oru-miR97, oru-miR117, oru-miR135, oru-miR137, et al. 17 are early-flowering -related miRNAs, including osa-miR160f, osa-miR164d, osa-miR167d, osa-miR169a, osa-miR172b, oru-miR4, et al., induced during the floral transition. Real-time PCR revealed the same expression patterns as deep sequencing. miRNAs targets were confirmed for cleavage by 5'-RACE in vivo, and were negatively regulated by miRNAs. This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and lays a foundation for further study of phase change

  4. The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis

    Directory of Open Access Journals (Sweden)

    De Paola Domenico

    2012-01-01

    Full Text Available Abstract Background Plant microRNAs (miRNAs are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA libraries can detect even poorly expressed miRNAs. No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots. Results Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1. Conclusions The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant

  5. Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia.

    Directory of Open Access Journals (Sweden)

    Jeremiah Tattersall

    Full Text Available The developmental cycle of the obligate intracellular pathogen Chlamydia trachomatis serovar L2 is controlled in part by the small non-coding RNA (sRNA, IhtA. All Chlamydia alternate in a regulated fashion between the infectious elementary body (EB and the replicative reticulate body (RB which asynchronously re-differentiates back to the terminal EB form at the end of the cycle. The histone like protein HctA is central to RB:EB differentiation late in the cycle as it binds to and occludes the genome, thereby repressing transcription and translation. The sRNA IhtA is a critical component of this regulatory loop as it represses translation of hctA until late in infection at which point IhtA transcription decreases, allowing HctA expression to occur and RB to EB differentiation to proceed. It has been reported that IhtA is expressed during infection by the human pathogens C. trachomatis serovars L2, D and L2b and C. pneumoniae. We show in this work that IhtA is also expressed by the animal pathogens C. caviae and C. muridarum. Expression of HctA in E. coli is lethal and co-expression of IhtA relieves this phenotype. To determine if regulation of HctA by IhtA is a conserved mechanism across pathogenic chlamydial species, we cloned hctA and ihtA from C. trachomatis serovar D, C. muridarum, C. caviae and C. pneumoniae and assayed for rescue of growth repression in E. coli co-expression studies. In each case, co-expression of ihtA with the cognate hctA resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of C. trachomatis serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with hctA message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.

  6. Identification and functional analysis of flowering related microRNAs in common wild rice (Oryza rufipogon Griff..

    Directory of Open Access Journals (Sweden)

    Zongxiang Chen

    Full Text Available BACKGROUND: MicroRNAs (miRNAs is a class of non-coding RNAs involved in post- transcriptional control of gene expression, via degradation and/or translational inhibition. Six-hundred sixty-one rice miRNAs are known that are important in plant development. However, flowering-related miRNAs have not been characterized in Oryza rufipogon Griff. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. rufipogon using high-throughput sequencing (deep sequencing to understand the changes that occurred during rice domestication, and to elucidate their functions in flowering. RESULTS: Three O. rufipogon sRNA libraries, two vegetative stage (CWR-V1 and CWR-V2 and one flowering stage (CWR-F2 were sequenced using Illumina deep sequencing. A total of 20,156,098, 21,531,511 and 20,995,942 high quality sRNA reads were obtained from CWR-V1, CWR-V2 and CWR-F2, respectively, of which 3,448,185, 4,265,048 and 2,833,527 reads matched known miRNAs. We identified 512 known rice miRNAs in 214 miRNA families and predicted 290 new miRNAs. Targeted functional annotation, GO and KEGG pathway analyses predicted that 187 miRNAs regulate expression of flowering-related genes. Differential expression analysis of flowering-related miRNAs showed that: expression of 95 miRNAs varied significantly between the libraries, 66 are flowering-related miRNAs, such as oru-miR97, oru-miR117, oru-miR135, oru-miR137, et al. 17 are early-flowering -related miRNAs, including osa-miR160f, osa-miR164d, osa-miR167d, osa-miR169a, osa-miR172b, oru-miR4, et al., induced during the floral transition. Real-time PCR revealed the same expression patterns as deep sequencing. miRNAs targets were confirmed for cleavage by 5'-RACE in vivo, and were negatively regulated by miRNAs. CONCLUSIONS: This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and

  7. Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia.

    Science.gov (United States)

    Tattersall, Jeremiah; Rao, Geeta Vittal; Runac, Justin; Hackstadt, Ted; Grieshaber, Scott S; Grieshaber, Nicole A

    2012-01-01

    The developmental cycle of the obligate intracellular pathogen Chlamydia trachomatis serovar L2 is controlled in part by the small non-coding RNA (sRNA), IhtA. All Chlamydia alternate in a regulated fashion between the infectious elementary body (EB) and the replicative reticulate body (RB) which asynchronously re-differentiates back to the terminal EB form at the end of the cycle. The histone like protein HctA is central to RB:EB differentiation late in the cycle as it binds to and occludes the genome, thereby repressing transcription and translation. The sRNA IhtA is a critical component of this regulatory loop as it represses translation of hctA until late in infection at which point IhtA transcription decreases, allowing HctA expression to occur and RB to EB differentiation to proceed. It has been reported that IhtA is expressed during infection by the human pathogens C. trachomatis serovars L2, D and L2b and C. pneumoniae. We show in this work that IhtA is also expressed by the animal pathogens C. caviae and C. muridarum. Expression of HctA in E. coli is lethal and co-expression of IhtA relieves this phenotype. To determine if regulation of HctA by IhtA is a conserved mechanism across pathogenic chlamydial species, we cloned hctA and ihtA from C. trachomatis serovar D, C. muridarum, C. caviae and C. pneumoniae and assayed for rescue of growth repression in E. coli co-expression studies. In each case, co-expression of ihtA with the cognate hctA resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of C. trachomatis serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with hctA message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.

  8. Evaluación del efecto de modificadores y refinadores en el comportamiento mecánico y magnitud del rechupe de aleaciones Al-Si-Mg. // Modifier and refiners effect evaluation in the magnitude and mechanical shrinkage behavior of Al-Si-Mg alloys.

    Directory of Open Access Journals (Sweden)

    V. Lavaert

    2002-05-01

    Full Text Available Se estudia la influencia del Na, Sr y Ti y sus combinaciones (Na-Ti y Sr-Na en las propiedades mecánicas y el rechupe(superior y lateral de la aleación hipoeutéctica AlSi7Mg Aunque todos estos elementos y combinaciones tienden aneutralizar la formación del rechupe substancialmente, el Sr se presenta como el más eficaz para disminuir el rechupesuperior mientras la combinación Na-Ti llevó a la menor formación de rechupe lateral.Se observó una acción modificadora excelente para 0.02% de Sr y 0.02% de Na, pero a diferencia del Sr, el efectomodificador del Na comienza a desvanecerse después de 30 min afectando el alargamiento notablemente. El estroncio, sinembargo, mostró un efecto de la modificación muy duradero (aproximadamente 3 h. Otro hallazgo interesante es laexistencia de un cierto periodo de incubación de aproximadamente 90 minutos después de agregar Sr. Contrariamente a loesperado el uso de titanio no mejoró las propiedades mecánicas a pesar de un eficaz refinamiento de grano.Palabras claves: Rechupe superior, acción modificadora, propiedades mecánicas, metalurgia no ferrosa._________________________________________________________________________AbstractThe influence of Na, Sr and Ti and their combinations (Na-Ti and Sr-Na on the mechanical properties and the shrinkage(top macro shrinkage and lateral macro shrinkage of hypoeutectic aluminium-silicon alloys (AlSi7xMg has been studied.Although all these elements and combinations tend to counteract substantially the shrinkage formation, Sr appeared to bethe most effective to decrease top macro shrinkage whereas the combination Na-Ti led to the least formation of lateralmacro shrinkage. An excellent modifying action was observed for 0.02% Sr and 0.02% Na, but unlike Sr the modifyingeffect provided by Na started fading after 30 min of holding which affected the elongation markedly. However, strontiumshowed a very lasting modification effect (about 3 h. Another interesting

  9. APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4 involved in RNA silencing.

    Directory of Open Access Journals (Sweden)

    Katia Marrocco

    Full Text Available Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome is a master ubiquitin protein ligase (E3 that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA. This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2 of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.

  10. Characterization of the Burkholderia thailandensis SOS response by using whole-transcriptome shotgun sequencing.

    Science.gov (United States)

    Ulrich, Ricky L; Deshazer, David; Kenny, Tara A; Ulrich, Melanie P; Moravusova, Anna; Opperman, Timothy; Bavari, Sina; Bowlin, Terry L; Moir, Donald T; Panchal, Rekha G

    2013-10-01

    The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.

  11. High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak

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    Trout-Yakel Keri M

    2010-02-01

    Full Text Available Abstract Background A large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis (PFGE revealed two similar but distinct AscI PFGE patterns. High-throughput pyrosequencing of two L. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during PFGE. Results The chromosomes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs and three indels, including a 33 kbp prophage that accounted for the observed difference in AscI PFGE patterns. The distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes. Conclusions High-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.

  12. Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori

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    Backert Steffen

    2011-11-01

    Full Text Available Abstract Helicobacter pylori is a highly successful pathogen uniquely adapted to colonize humans. Gastric infections with this bacterium can induce pathology ranging from chronic gastritis and peptic ulcers to gastric cancer. More virulent H. pylori isolates harbour numerous well-known adhesins (BabA/B, SabA, AlpA/B, OipA and HopZ and the cag (cytotoxin-associated genes pathogenicity island encoding a type IV secretion system (T4SS. The adhesins establish tight bacterial contact with host target cells and the T4SS represents a needle-like pilus device for the delivery of effector proteins into host target cells such as CagA. BabA and SabA bind to blood group antigen and sialylated proteins respectively, and a series of T4SS components including CagI, CagL, CagY and CagA have been shown to target the integrin β1 receptor followed by injection of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine may also play a role in the delivery process. While substantial progress has been made in our current understanding of many of the above factors, the host cell receptors for OipA, HopZ and AlpA/B during infection are still unknown. Here we review the recent progress in characterizing the interactions of the various adhesins and structural T4SS proteins with host cell factors. The contribution of these interactions to H. pylori colonization and pathogenesis is discussed.

  13. A designated centre for people with disabilities operated by Nua Healthcare Services, Clare

    LENUS (Irish Health Repository)

    Backert, Steffen

    2011-11-01

    Abstract Helicobacter pylori is a highly successful pathogen uniquely adapted to colonize humans. Gastric infections with this bacterium can induce pathology ranging from chronic gastritis and peptic ulcers to gastric cancer. More virulent H. pylori isolates harbour numerous well-known adhesins (BabA\\/B, SabA, AlpA\\/B, OipA and HopZ) and the cag (cytotoxin-associated genes) pathogenicity island encoding a type IV secretion system (T4SS). The adhesins establish tight bacterial contact with host target cells and the T4SS represents a needle-like pilus device for the delivery of effector proteins into host target cells such as CagA. BabA and SabA bind to blood group antigen and sialylated proteins respectively, and a series of T4SS components including CagI, CagL, CagY and CagA have been shown to target the integrin β1 receptor followed by injection of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine may also play a role in the delivery process. While substantial progress has been made in our current understanding of many of the above factors, the host cell receptors for OipA, HopZ and AlpA\\/B during infection are still unknown. Here we review the recent progress in characterizing the interactions of the various adhesins and structural T4SS proteins with host cell factors. The contribution of these interactions to H. pylori colonization and pathogenesis is discussed.

  14. Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori

    LENUS (Irish Health Repository)

    Backert, Steffen

    2011-11-01

    Abstract Helicobacter pylori is a highly successful pathogen uniquely adapted to colonize humans. Gastric infections with this bacterium can induce pathology ranging from chronic gastritis and peptic ulcers to gastric cancer. More virulent H. pylori isolates harbour numerous well-known adhesins (BabA\\/B, SabA, AlpA\\/B, OipA and HopZ) and the cag (cytotoxin-associated genes) pathogenicity island encoding a type IV secretion system (T4SS). The adhesins establish tight bacterial contact with host target cells and the T4SS represents a needle-like pilus device for the delivery of effector proteins into host target cells such as CagA. BabA and SabA bind to blood group antigen and sialylated proteins respectively, and a series of T4SS components including CagI, CagL, CagY and CagA have been shown to target the integrin β1 receptor followed by injection of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine may also play a role in the delivery process. While substantial progress has been made in our current understanding of many of the above factors, the host cell receptors for OipA, HopZ and AlpA\\/B during infection are still unknown. Here we review the recent progress in characterizing the interactions of the various adhesins and structural T4SS proteins with host cell factors. The contribution of these interactions to H. pylori colonization and pathogenesis is discussed.

  15. Toxin Mediates Sepsis Caused by Methicillin-Resistant Staphylococcus epidermidis.

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    Li Qin

    2017-02-01

    Full Text Available Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the psm-mec locus that have been reported in S. aureus strains, inasmuch as our findings suggest that the psm-mec locus may exert effects in the background of S. aureus strains that differ from its original role in the CNS environment due to originally "unintended" interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.

  16. Identification and bioinformatics analysis of microRNAs from the sporophyte and gametophyte of Pyropia haitanensis

    Science.gov (United States)

    Huang, Aiyou; Wang, Guangce

    2016-05-01

    Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata ( Porphyra haitanensis) is an economically important genus that is cultured widely in China. P. haitanensis is cultured on a larger scale than Pyropia yezoensis, making up an important part of the total production of cultivated Pyropia in China. However, the majority of molecular mechanisms underlying the physiological processes of P. haitanensis remain unknown. P. haitanensis could utilize inorganic carbon and the sporophytes of P. haitanensis might possess a PCK-type C4-like carbon-fixation pathway. To identify microRNAs and their probable roles in sporophyte and gametophyte development, we constructed and sequenced small RNA libraries from sporophytes and gametophytes of P. haitanensis. Five microRNAs were identified that shared no sequence homology with known microRNAs. Our results indicated that P. haitanensis might posses a complex sRNA processing system in which the novel microRNAs act as important regulators of the development of different generations of P. haitanensis.

  17. Overexpression of MicA induces production of OmpC-enriched outer membrane vesicles that protect against Salmonella challenge.

    Science.gov (United States)

    Choi, Hyun-Il; Kim, Moonjeong; Jeon, Jinseong; Han, Jin Kwan; Kim, Kwang-Sun

    2017-08-26

    Outer membrane vesicles (OMVs) derived from bacteria are promising candidates for subunit vaccines. Stresses that modulate the composition of outer membrane proteins (OMPs) are important for OMV synthesis. Small RNAs (sRNAs) expressed in response to stress regulate OMPs, although the mechanism underlying sRNA-mediated OMV biogenesis and its utility for developing vaccine platforms remains to be elucidated. Here, we characterized the role of a sRNA, MicA, which regulates OmpA, a major OMP involved in both production of OMVs and reactive immunity against Salmonella challenge. A Salmonella strain overexpressing MicA generated more OMVs than a control strain. In addition, OmpC was the major component of MicA-derived OMV proteins. MicA-derived OMVs induced Th1- and Th17-type immune responses in vitro and reduced Salmonella-mediated lethality in a mouse model. Thus, OmpA-regulatory sRNA-derived OMVs may facilitate production of Salmonella-protective vaccines. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of Escherichia coli

    Science.gov (United States)

    Smith, Daniel R.; Price, Janet E.; Burby, Peter E.; Blanco, Luz P.; Chamberlain, Justin; Chapman, Matthew R.

    2017-01-01

    Curli amyloid fibers are the major protein component of the extracellular matrix produced by Enterobacteriaceae during biofilm formation. Curli are required for proper biofilm development and environmental persistence by Escherichia coli. Here, we present a complete and vetted genetic analysis of functional amyloid fiber biogenesis. The Keio collection of single gene deletions was screened on Congo red indicator plates to identify E. coli mutants that had defective amyloid production. We discovered that more than three hundred gene products modulated curli production. These genes were involved in fundamental cellular processes such as regulation, environmental sensing, respiration, metabolism, cell envelope biogenesis, transport, and protein turnover. The alternative sigma factors, σS and σE, had opposing roles in curli production. Mutations that induced the σE or Cpx stress response systems had reduced curli production, while mutant strains with increased σS levels had increased curli production. Mutations in metabolic pathways, including gluconeogenesis and the biosynthesis of lipopolysaccharide (LPS), produced less curli. Regulation of the master biofilm regulator, CsgD, was diverse, and the screen revealed several proteins and small RNAs (sRNA) that regulate csgD messenger RNA (mRNA) levels. Using previously published studies, we found minimal overlap between the genes affecting curli biogenesis and genes known to impact swimming or swarming motility, underlying the distinction between motile and sessile lifestyles. Collectively, the diversity and number of elements required suggest curli production is part of a highly regulated and complex developmental pathway in E. coli. PMID:29088115

  19. Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

    Science.gov (United States)

    Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

    2014-01-01

    MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

  20. Small RNA profiling reveals important roles for miRNAs in Arabidopsis response to Bacillus velezensis FZB42.

    Science.gov (United States)

    Xie, Shanshan; Jiang, Haiyang; Xu, Zhilan; Xu, Qianqian; Cheng, Beijiu

    2017-09-20

    Bacillus velezensis FZB42 (previously classified as Bacillus amyloliquefaciens FZB42) has been confirmed to successfully colonize plant roots and enhance defense response against pathogen infection. This study indicated that FZB42 inoculation enhanced Arabidopsis defense response against Pseudomonas syringae DC3000 through inducing the expression of PR1, PDF1.2 and stomata closure. To further clarify the induced defense response at miRNA level, sRNA libraries from Arabidopsis roots inoculated with FZB42 and control were constructed and sequenced. The reads of 21nt and 24nt in length were the most abundant groups in FZB42-treated library and control library, respectively. 234 known miRNAs and 16 novel miRNAs were identified. Among them, 11 known miRNAs and 4 novel miRNAs were differentially expressed after FZB42 inoculation. Moreover cis-elements (TC-rich repeats, TCA-element and CGTCA-motif) associated with plant defense were also found in the promoters of these miRNAs. Additionally, 141 mRNAs were predicted as potential targets of these differentially expressed miRNAs. GO annotations of the target genes indicated their potential roles in polyamine biosynthetic process and intracellular protein transport biological process, which may contribute to increased defense response. Our findings indicated that Bacillus velezensis FZB42 inoculation altered the expression of Arabidopsis miRNAs and their target genes, which were associated with defense response. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

    Science.gov (United States)

    Lemus-Diaz, Nicolas; Böker, Kai O.; Rodriguez-Polo, Ignacio; Mitter, Michael; Preis, Jasmin; Arlt, Maximilian; Gruber, Jens

    2017-01-01

    Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. PMID:28338079

  2. Determination of the number of copies of genes coding for 5s-rRNA and tRNA in the genomes of 43 species of wheat and Aegilops

    International Nuclear Information System (INIS)

    Vakhitov, V.A.; Gimalov, F.R.; Nikonorov, Yu.M.

    1986-01-01

    The number of 5s-rRNA and tRNA genes has been studied in 43 species of wheat and Aegilops differing in ploidy level, genomic composition and origin. It has been demonstrated that the repeatability of the 5s-rRNA and tRNA genes increases in wheat with increasing ploidy level, but not in proportion to the genome size. In Aegilops, in distinction from wheat, the relative as well as absolute number of 5s-RNA genes increases with increasing ploidy level. The proportion of the sequences coding for tRNA in the dipoloid and polyploid Aegilops species is practically similar, while the number of tRNA genes increases almost 2-3 times with increasing ploidy level. Large variability has been recorded between the species with similar genomic composition and ploidy level in respect of the number of the 5s-rRNA and tRNA genes. It has been demonstrated that integration of the initial genomes of the amphidiploids is accompanied by elimination of a particular part of these genomes. It has been concluded that the mechanisms of establishment and evolution of genomes in the intra- and intergeneric allopolyploids are not identical

  3. Sequence recombination and conservation of Varroa destructor virus-1 and deformed wing virus in field collected honey bees (Apis mellifera.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available We sequenced small (s RNAs from field collected honeybees (Apis mellifera and bumblebees (Bombuspascuorum using the Illumina technology. The sRNA reads were assembled and resulting contigs were used to search for virus homologues in GenBank. Matches with Varroadestructor virus-1 (VDV1 and Deformed wing virus (DWV genomic sequences were obtained for A. mellifera but not B. pascuorum. Further analyses suggested that the prevalent virus population was composed of VDV-1 and a chimera of 5'-DWV-VDV1-DWV-3'. The recombination junctions in the chimera genomes were confirmed by using RT-PCR, cDNA cloning and Sanger sequencing. We then focused on conserved short fragments (CSF, size > 25 nt in the virus genomes by using GenBank sequences and the deep sequencing data obtained in this study. The majority of CSF sites confirmed conservation at both between-species (GenBank sequences and within-population (dataset of this study levels. However, conserved nucleotide positions in the GenBank sequences might be variable at the within-population level. High mutation rates (Pi>10% were observed at a number of sites using the deep sequencing data, suggesting that sequence conservation might not always be maintained at the population level. Virus-host interactions and strategies for developing RNAi treatments against VDV1/DWV infections are discussed.

  4. Endogenous TasiRNAs mediate non-cell autonomous effects on gene regulation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rebecca Schwab

    Full Text Available BACKGROUND: Different classes of small RNAs (sRNAs refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs, which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs, and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells. PRINCIPAL FINDINGS: We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities. CONCLUSIONS/SIGNIFICANCE: Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.

  5. Identification of microRNA-Like RNAs in the filamentous fungus Trichoderma reesei by solexa sequencing.

    Directory of Open Access Journals (Sweden)

    Kang Kang

    Full Text Available microRNAs (miRNAs are non-coding small RNAs (sRNAs capable of negatively regulating gene expression. Recently, microRNA-like small RNAs (milRNAs were discovered in several filamentous fungi but not yet in Trichoderma reesei, an industrial filamentous fungus that can secrete abundant hydrolases. To explore the presence of milRNA in T. reesei and evaluate their expression under induction of cellulose, two T. reesei sRNA libraries of cellulose induction (IN and non-induction (CON were generated and sequenced using Solexa sequencing technology. A total of 726 and 631 sRNAs were obtained from the IN and CON samples, respectively. Global expression analysis showed an extensively differential expression of sRNAs in T. reesei under the two conditions. Thirteen predicted milRNAs were identified in T. reesei based on the short hairpin structure analysis. The milRNA profiles obtained in deep sequencing were further validated by RT-qPCR assay. Computational analysis predicted a number of potential targets relating to many processes including regulation of enzyme expression. The presence and differential expression of T. reesei milRNAs imply that milRNA might play a role in T. reesei growth and cellulase induction. This work lays foundation for further functional study of fungal milRNAs and their industrial application.

  6. Analysis of sucrose-induced small RNAs in Streptococcus mutans in the presence of different sucrose concentrations.

    Science.gov (United States)

    Liu, Shan Shan; Zhu, Wen Hui; Zhi, Qing Hui; Liu, Jia; Wang, Yan; Lin, Huan Cai

    2017-07-01

    Streptococcus mutans (S. mutans) is the major pathogen contributing to dental caries. Sucrose is an important carbohydrate source for S. mutans and is crucial for dental caries. Small RNAs (sRNAs) are key post-transcriptional regulators of stress adaptation and virulence in bacteria. Here, for the first time, we created three replicate RNA libraries exposed to either 1 or 5% sucrose. The expression levels of sRNAs and target genes (gtfB, gtfC, and spaP) related to virulence were assessed. In addition, some phenotypic traits were evaluated. We obtained 2125 sRNA candidates with at least 100 average reads in 1% sucrose or 5% sucrose. Of these candidates, 2 were upregulated and 20 were downregulated in 1% sucrose. Six of these 22 differentially expressed sRNAs were validated by qRT-PCR. The expression level of target gene gtfB was higher in 1% sucrose. The adherence ratio of S. mutans was higher in 1% sucrose than in 5% sucrose. The synthesis of water-insoluble glucans (WIGs) was significantly higher in 5% sucrose than in 1% sucrose. These data suggest that a series of sRNAs can be induced in response to sucrose, and that some sRNAs might be involved in the regulation of phenotypes, providing new insight into the prevention of caries.

  7. Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Non-coding small RNAs (sRNAs are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.

  8. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

    Directory of Open Access Journals (Sweden)

    Stephanie Pitman

    2015-12-01

    Full Text Available The discovery of small noncoding regulatory RNAs (sRNAs in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described.

  9. Dynamic evolution and biogenesis of small RNAs during sex reversal.

    Science.gov (United States)

    Liu, Jie; Luo, Majing; Sheng, Yue; Hong, Qiang; Cheng, Hanhua; Zhou, Rongjia

    2015-05-06

    Understanding origin, evolution and functions of small RNA (sRNA) genes has been a great challenge in the past decade. Molecular mechanisms underlying sexual reversal in vertebrates, particularly sRNAs involved in this process, are largely unknown. By deep-sequencing of small RNA transcriptomes in combination with genomic analysis, we identified a large amount of piRNAs and miRNAs including over 1,000 novel miRNAs, which were differentially expressed during gonad reversal from ovary to testis via ovotesis. Biogenesis and expressions of miRNAs were dynamically changed during the reversal. Notably, phylogenetic analysis revealed dynamic expansions of miRNAs in vertebrates and an evolutionary trajectory of conserved miR-17-92 cluster in the Eukarya. We showed that the miR-17-92 cluster in vertebrates was generated through multiple duplications from ancestor miR-92 in invertebrates Tetranychus urticae and Daphnia pulex from the Chelicerata around 580 Mya. Moreover, we identified the sexual regulator Dmrt1 as a direct target of the members miR-19a and -19b in the cluster. These data suggested dynamic biogenesis and expressions of small RNAs during sex reversal and revealed multiple expansions and evolutionary trajectory of miRNAs from invertebrates to vertebrates, which implicate small RNAs in sexual reversal and provide new insight into evolutionary and molecular mechanisms underlying sexual reversal.

  10. Diversity of small RNAs expressed in Pseudomonas species

    DEFF Research Database (Denmark)

    Gomez-Lozano, Mara; Marvig, Rasmus Lykke; Molina-Santiago, Carlos

    2015-01-01

    RNA sequencing (RNA-seq) has revealed several hundreds of previously undetected small RNAs (sRNAs) in all bacterial species investigated, including strains of Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas syringae. Nonetheless, only little is known about the extent of conservation...... of expressed sRNAs across strains and species. In this study, we have used RNA-seq to identify sRNAs in P.putidaDOT-T1E and Pseudomonas extremaustralis 14-3b. This is the first strain of P.extremaustralis and the second strain of P.putida to have their transcriptomes analysed for sRNAs, and we identify...... the presence of around 150 novel sRNAs in each strain. Furthermore, we provide a comparison based on sequence conservation of all the sRNAs detected by RNA-seq in the Pseudomonas species investigated so far. Our results show that the extent of sRNA conservation across different species is very limited...

  11. Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

    Science.gov (United States)

    Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

    2014-04-01

    The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

  12. The stationary phase sigma factor, RpoS, regulates the production of a carbapenem antibiotic, a bioactive prodigiosin and virulence in the enterobacterial pathogen Serratia sp. ATCC 39006.

    Science.gov (United States)

    Wilf, Nabil M; Salmond, George P C

    2012-03-01

    Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and invertebrate animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, a prodigiosin and a carbapenem, and the exoenzymes pectate lyase and cellulase. We showed previously that deletion of the RNA chaperone Hfq abolished antibiotic production and attenuated virulence in both animal and plant hosts. Hfq and dependent small RNAs (sRNAs) are known to regulate the post-transcriptional expression of rpoS, which encodes σ(S), the stationary phase sigma factor subunit of RNA polymerase. An S39006 hfq deletion mutant showed decreased transcript levels of rpoS. Therefore, in this study we investigated whether the phenotypes regulated by Hfq were mediated through its control of rpoS. Whereas loss of Hfq abolished prodigiosin and carbapenem production and attenuated virulence in both C. elegans and potato, characterization of an S39006 rpoS mutant showed unexpectedly elevated prodigiosin and carbapenem production. Furthermore, the rpoS mutant exhibited attenuated animal pathogenesis, but not plant pathogenesis. Additionally, a homologue of the Hfq-dependent sRNA, RprA, was identified and shown to regulate prodigiosin production in a manner consistent with its role in positively regulating translation of rpoS mRNA. Combined, these results demonstrate that Hfq regulation of secondary metabolism and plant pathogenesis is independent of RpoS and establishes RpoS and RprA as regulators of antibiotic production.

  13. Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

    Directory of Open Access Journals (Sweden)

    Christopher M Brennan

    Full Text Available The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA, the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

  14. Identification and Expression Analyses of miRNAs from Two Contrasting Flower Color Cultivars of Canna by Deep Sequencing.

    Science.gov (United States)

    Roy, Sribash; Tripathi, Abhinandan Mani; Yadav, Amrita; Mishra, Parneeta; Nautiyal, Chandra Shekhar

    2016-01-01

    miRNAs are endogenous small RNA (sRNA) that play critical roles in plant development processes. Canna is an ornamental plant belonging to family Cannaceae. Here, we report for the first time the identification and differential expression of miRNAs in two contrasting flower color cultivars of Canna, Tropical sunrise and Red president. A total of 313 known miRNAs belonging to 78 miRNA families were identified from both the cultivars. Thirty one miRNAs (17 miRNA families) were specific to Tropical sunrise and 43 miRNAs (10 miRNA families) were specific to Red president. Thirty two and 18 putative new miRNAs were identified from Tropical sunrise and Red president, respectively. One hundred and nine miRNAs were differentially expressed in the two cultivars targeting 1343 genes. Among these, 16 miRNAs families targeting 60 genes were involved in flower development related traits and five miRNA families targeting five genes were involved in phenyl propanoid and pigment metabolic processes. We further validated the expression analysis of a few miRNA and their target genes by qRT-PCR. Transcription factors were the major miRNA targets identified. Target validation of a few randomly selected miRNAs by RLM-RACE was performed but was successful with only miR162. These findings will help in understanding flower development processes, particularly the color development in Canna.

  15. DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.

    Science.gov (United States)

    Lalaouna, David; Morissette, Audrey; Carrier, Marie-Claude; Massé, Eric

    2015-10-01

    The 87 nucleotide long DsrA sRNA has been mostly studied for its translational activation of the transcriptional regulator RpoS. However, it also represses hns mRNA, which encodes H-NS, a major regulator that affects expression of nearly 5% of Escherichia coli genes. A speculative model previously suggested that DsrA would block hns mRNA translation by binding simultaneously to start and stop codon regions of hns mRNA (coaxial model). Here, we show that DsrA efficiently blocked translation of hns mRNA by base-pairing immediately downstream of the start codon. In addition, DsrA induced hns mRNA degradation by actively recruiting the RNA degradosome complex. Data presented here led to a model of DsrA action on hns mRNA, which supports a canonical mechanism of sRNA-induced mRNA degradation by binding to the translation initiation region. Furthermore, using MS2-affinity purification coupled with RNA sequencing technology (MAPS), we also demonstrated that DsrA targets rbsD mRNA, involved in ribose utilization. Surprisingly, DsrA base pairs far downstream of rbsD start codon and induces rapid degradation of the transcript. Thus, our study enables us to draw an extended DsrA targetome. © 2015 John Wiley & Sons Ltd.

  16. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

    Science.gov (United States)

    Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

    2015-02-01

    sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

  17. Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for citrus huanglongbing disease.

    Science.gov (United States)

    Zhao, Hongwei; Sun, Ruobai; Albrecht, Ute; Padmanabhan, Chellappan; Wang, Airong; Coffey, Michael D; Girke, Thomas; Wang, Zonghua; Close, Timothy J; Roose, Mikeal; Yokomi, Raymond K; Folimonova, Svetlana; Vidalakis, Georgios; Rouse, Robert; Bowman, Kim D; Jin, Hailing

    2013-03-01

    Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus 'Candidatus Liberibacter' (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn-a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.

  18. Deep sequencing leads to the identification of eukaryotic translation initiation factor 5A as a key element in Rsv1-mediated lethal systemic hypersensitive response to Soybean mosaic virus infection in soybean.

    Science.gov (United States)

    Chen, Hui; Adam Arsovski, Andrej; Yu, Kangfu; Wang, Aiming

    2017-04-01

    Rsv1, a single dominant resistance locus in soybean, confers extreme resistance to the majority of Soybean mosaic virus (SMV) strains, but is susceptible to the G7 strain. In Rsv1-genotype soybean, G7 infection provokes a lethal systemic hypersensitive response (LSHR), a delayed host defence response. The Rsv1-mediated LSHR signalling pathway remains largely unknown. In this study, we employed a genome-wide investigation to gain an insight into the molecular interplay between SMV G7 and Rsv1-genotype soybean. Small RNA (sRNA), degradome and transcriptome sequencing analyses were used to identify differentially expressed genes (DEGs) and microRNAs (DEMs) in response to G7 infection. A number of DEGs, DEMs and microRNA targets, and the interaction network of DEMs and their target mRNAs responsive to G7 infection, were identified. Knock-down of one of the identified DEGs, the eukaryotic translation initiation factor 5A (eIF5A), diminished the LSHR and enhanced viral accumulation, suggesting the essential role of eIF5A in the G7-induced, Rsv1-mediated LSHR signalling pathway. This work provides an in-depth genome-wide analysis of high-throughput sequencing data, and identifies multiple genes and microRNA signatures that are associated with the Rsv1-mediated LSHR. © 2016 HER MAJESTY THE QUEEN IN RIGHT OF CANADA MOLECULAR PLANT PATHOLOGY © 2016 BSPP AND JOHN WILEY & SONS LTD.

  19. Divergent homologs of the predicted small RNA BpCand697 in Burkholderia spp.

    Science.gov (United States)

    Damiri, Nadzirah; Mohd-Padil, Hirzahida; Firdaus-Raih, Mohd

    2015-09-01

    The small RNA (sRNA) gene candidate, BpCand697 was previously reported to be unique to Burkholderia spp. and is encoded at 3' non-coding region of a putative AraC family transcription regulator gene. This study demonstrates the conservation of BpCand697 sequence across 32 Burkholderia spp. including B. pseudomallei, B. mallei, B. thailandensis and Burkholderia sp. by integrating both sequence homology and secondary structural analyses of BpCand697 within the dataset. The divergent sequence of BpCand697 was also used as a discriminatory power in clustering the dataset according to the potential virulence of Burkholderia spp., showing that B. thailandensis was clearly secluded from the virulent cluster of B. pseudomallei and B. mallei. Finally, the differential co-transcript expression of BpCand697 and its flanking gene, bpsl2391 was detected in Burkholderia pseudomallei D286 after grown under two different culture conditions using nutrient-rich and minimal media. It is hypothesized that the differential expression of BpCand697-bpsl2391 co-transcript between the two standard prepared media might correlate with nutrient availability in the culture media, suggesting that the physical co-localization of BpCand697 in B. pseudomallei D286 might be directly or indirectly involved with the transcript regulation of bpsl2391 under the selected in vitro culture conditions.

  20. A Novel Small RNA Regulates Tolerance and Virulence in Shigella flexneri by Responding to Acidic Environmental Changes

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    Ligui eWang

    2016-03-01

    Full Text Available Shigella flexneri is an important cause of bacillary dysentery in developing countries. Small regulatory RNAs (sRNAs play essential roles in diverse cellular processes. We found a novel sRNA Ssr1 based on RT-PCR, northern blot, and 5´RACE in S. flexneri. Ssr1 responds to acidic environmental changes, as shown by a strong linear correlation between the pH value and Ssr1 expression (R = 0.785, P < 0.05 using the qRT-PCR method. Deletion of Ssr1 results in growth retardation at pH values ranging from 5.0 to 7.0 (P < 0.05, and the survival rate was reduced by 22% in acidic conditions (pH 3.0. Additionally, virulence was significantly increased in an Ssr1 mutant strain, as revealed in a murine lung invasion model and survival model assays. By using the sTarPicker method and proteomic analysis, we considered that DnaK, which is a major factor that confers acidic stress tolerance, may be a direct target of Ssr1. We also found that Ssr1 may enhance virulence by directly targeting OmpA; this leads to altered expression of genes in the type three secretion system. This work provides new insight into the mechanism of adaptation to environmental stress and into the pathogenesis of Shigella.

  1. Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection.

    Science.gov (United States)

    Romanel, Elisson; Silva, Tatiane F; Corrêa, Régis L; Farinelli, Laurent; Hawkins, Jennifer S; Schrago, Carlos E G; Vaslin, Maite F S

    2012-11-01

    Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.

  2. Computational prediction of miRNA genes from small RNA sequencing data

    Directory of Open Access Journals (Sweden)

    Wenjing eKang

    2015-01-01

    Full Text Available Next-generation sequencing now for the first time allows researchers to gauge the depth and variation of entire transcriptomes. However, now as rare transcripts can be detected that are present in cells at single copies, more advanced computational tools are needed to accurately annotate and profile them. miRNAs are 22 nucleotide small RNAs (sRNAs that post-transcriptionally reduce the output of protein coding genes. They have established roles in numerous biological processes, including cancers and other diseases. During miRNA biogenesis, the sRNAs are sequentially cleaved from precursor molecules that have a characteristic hairpin RNA structure. The vast majority of new miRNA genes that are discovered are mined from small RNA sequencing (sRNA-seq, which can detect more than a billion RNAs in a single run. However, given that many of the detected RNAs are degradation products from all types of transcripts, the accurate identification of miRNAs remain a non-trivial computational problem. Here we review the tools available to predict animal miRNAs from sRNA sequencing data. We present tools for generalist and specialist use cases, including prediction from massively pooled data or in species without reference genome. We also present wet-lab methods used to validate predicted miRNAs, and approaches to computationally benchmark prediction accuracy. For each tool, we reference validation experiments and benchmarking efforts. Last, we discuss the future of the field.

  3. Synaptic vesicles isolated from the electric organ of Torpedo californica and from the central nervous system of Mus musculus contain small ribonucleic acids (sRNAs

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    Huinan Li

    2017-06-01

    Full Text Available Synaptic vesicles (SVs are presynaptic organelles that load and release small molecule neurotransmitters at chemical synapses. In addition to classic neurotransmitters, we have demonstrated that SVs isolated from the Peripheral Nervous Systems (PNS of the electric organ of Torpedo californica, a model cholinergic synapse, and SVs isolated from the Central Nervous System (CNS of Mus musculus (mouse contain small ribonucleic acids (sRNAs; ≤50 nucleotides (Scientific Reports, 5:1–14(14918 Li et al. (2015 [1]. Our previous publication provided the five most abundant sequences associated with the T. californica SVs, and the ten most abundant sequences associated with the mouse SVs, representing 59% and 39% of the total sRNA reads sequenced, respectively. We provide here a full repository of the SV sRNAs sequenced from T. californica and the mouse deposited in the NCBI as biosamples. Three data studies are included: SVs isolated from the electric organ of T. californica using standard techniques, SVs isolated from the electric organ of T. californica using standard techniques with an additional affinity purification step, and finally, SVs isolated from the CNS of mouse. The three biosamples are available at https://www.ncbi.nlm.nih.gov/biosample/ SRS1523467, SRS1523466, and SRS1523472 respectively.

  4. The origin and effect of small RNA signaling in plants

    Directory of Open Access Journals (Sweden)

    Jean-Sébastien eParent

    2012-08-01

    Full Text Available Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute and other proteins to form the RNA-induced silencing complex (RISC that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the Argonaute protein involved, RISC triggers either DNA methylation and chromatin modification (leading to transcriptional gene silencing, TGS or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS. In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of small RNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal.

  5. Sibling sRNA RyfA1 Influences Shigella dysenteriae Pathogenesis

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    Megan E. Fris

    2017-01-01

    Full Text Available Small regulatory RNAs (sRNAs of Shigella dysenteriae and other pathogens are vital for the regulation of virulence-associated genes and processes. Here, we characterize RyfA1, one member of a sibling pair of sRNAs produced by S. dysenteriae. Unlike its nearly identical sibling molecule, RyfA2, predicted to be encoded almost exclusively by non-pathogenic species, the presence of a gene encoding RyfA1, or a RyfA1-like molecule, is strongly correlated with virulence in a variety of enteropathogens. In S. dysenteriae, the overproduction of RyfA1 negatively impacts the virulence-associated process of cell-to-cell spread as well as the expression of ompC, a gene encoding a major outer membrane protein important for the pathogenesis of Shigella. Interestingly, the production of RyfA1 is controlled by a second sRNA, here termed RyfB1, the first incidence of one regulatory small RNA controlling another in S. dysenteriae or any Shigella species.

  6. Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer.

    Science.gov (United States)

    Baba, Seiki; Someya, Tatsuhiko; Kawai, Gota; Nakamura, Kouji; Kumasaka, Takashi

    2010-05-01

    The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq-RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq-RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 A, while the type 2 Hfq-RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 A. Diffraction data were collected to a resolution of 2.20 A from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis.

  7. Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer

    International Nuclear Information System (INIS)

    Baba, Seiki; Someya, Tatsuhiko; Kawai, Gota; Nakamura, Kouji; Kumasaka, Takashi

    2010-01-01

    The RNA-binding protein Hfq from B. subtilis was crystallized using the hanging-drop vapour-diffusion method in two crystal forms that belonged to space groups I422 and F222; diffraction data were collected to 2.2 Å resolution from both forms. The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq–RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq–RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 Å, while the type 2 Hfq–RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 Å. Diffraction data were collected to a resolution of 2.20 Å from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis

  8. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

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    Carlos A Santiviago

    2009-07-01

    Full Text Available Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS.

  9. Conceptual model of fractured aquifer of Uranium Deposit in Caetité, Bahia: implications for groundwater flow

    International Nuclear Information System (INIS)

    Silva, Liliane Ferreira da

    2015-01-01

    The studied area is represented by the uraniferous district of Lagoa Real, located in the center-south of Bahia State, Brazil. The region is set in a semiarid climate context, with hot and dry weather parameters, with hydric deficit along all months of the year and high aridity index. Rural population is affected on drought periods since small agriculture and animal rearing are the main economic activities which are vulnerable in dry seasons. Groundwater represents the main supply source considering that most surface water sources are temporary and only exhibit flow in rainy periods. The main aquifer system present on the region is fractured, and the presence of groundwater flow occurs through the discontinuities of the rock considering that the rock mass corresponds to the set formed by the rock matrix and all its discontinuities (fractures, foliations, discordance, etc). In this sense, the main purpose of this Master Dissertation was to develop a conceptual model for the aquifer system, through the geotechnical characterization of discontinuities, once these structures allow the secondary porosity of the medium. Hydrochemical data hand out as complement for physical characterization for the behavioral interpretation of the aquifer. The aquifer system is unconfined, however, presents points of stagnation of flow forming compartments without communication with the surrounding areas. According to the International Society of Rock Mechanics ISRM method, which consist on qualitative and quantitative characterization of discontinuities of rock mass scanlines were constructed, systematically, describing, the following structure parameters: attitude, spacing, persistence, openness, infilling and roughness. From the results analysis it could be concluded that the aquifer system is composed of three discontinuities sets: one set which dips to NE, second set dipping to SW-W-NW and the last set sub-horizontal. The first and second sets are responsible for the aquifer

  10. Initial report on drilling into seismogenic zones of M2.0 - M5.5 earthquakes from deep South African gold mines (DSeis)

    Science.gov (United States)

    Ogasawara, Hiroshi; Durrheim, Raymond; Yabe, Yasuo; Ito, Takatoshi; van Aswegen, Gerrie; Grobbelaar, Michelle; Funato, Akio; Ishida, Akimasa; Ogasawara, Hiroyuki; Mngadi, Siyanda; Manzi, Musa; Ziegler, Martin; Ward, Tony; Moyer, Pamela; Boettcher, Margaret; Ellsworth, Bill; Liebenberg, Bennie; Wechsler, Neta; Onstott, Tullis; Berset, Nicolas

    2017-04-01

    The International Continental Scientific Drilling Program (ICDP) approved our proposal (Ogasawara et al., EGU 2016) to drill into and around seismogenic zones where critically stressed faults initiated ruptures at depth. The drilling targets include four ruptures equivalent to M2.0, 2.8, 3.5, and 5.5 that dynamically and quasi-statically evolved in 2.9 Ga hard rock in the Witwatersrand basin, South Africa. Major advantages of our drilling locations are the large quantity and high-quality of existing data from dense seismic arrays both on surface and near-field underground in three deep South African gold mines. Additionally, the great depths (1.0 to 3.3 km from surface) to collar holes reduce drilling costs significantly and enable a larger number of holes to be drilled. Flexibility in drilling direction will also allow us to minimize damage in borehole or drilled cores. With the ICDP funds, we will conduct full-core drilling of 16 holes with drilling ranges from 50 to 750 m to recover both materials and fractures in and around the seismogenic zones, followed by core and borehole logging. Additional in-hole monitoring at close proximity will be supported by co-mingled funds and will follow the ICDP drilling. Expected magnitudes of maximum shear stress are several tens of MPa. We have established an overcoring procedure to measure 3D-stress state for adverse underground working conditions so as not to interfere with mining operations. This procedure was optimized based on the Compact Conic-ended Borehole Overcoring (CCBO) technique (ISRM suggested; Sugawara and Obara, 1999). Funato and Ito (2016 IJRMMS) developed a diametrical core deformation analysis (DCDA) method to measure differential stress using only drilled core by assuming diametrical change with roll angles caused by elastic in-axisymmetrical expansion during drilling. A gold mine has already drilled a hole to intersect the hypocenter of a 2016 M3.5 earthquake and carried out the CCBO stress measurement in

  11. Long-Term Mechanical Behavior of Yucca Mountain Tuff and its Variability, Final Technical Report for Task ORD-FY04-021

    International Nuclear Information System (INIS)

    Daemen, Jaak J.K.; Ma, Lumin; Zhao, Guohua

    2006-01-01

    The study of the long term mechanical behavior of Yucca Mountain tuffs is important for several reasons. Long term stability of excavations will affect accessibility (e.g. for inspection purposes), and retrievability. Long term instabilities may induce loading of drip shields and/or emplaced waste, thus affecting drip shield and/or waste package corrosion. Failure of excavations will affect airflow, may affect water flow, and may affect temperature distributions. The long term mechanical behavior of rocks remains an elusive topic, loaded with uncertainties. A variety of approaches have been used to improve the understanding of this complex subject, but it is doubtful that it has reached a stage where firm predictions can be considered feasible. The long term mechanical behavior of ''soft'' rocks, especially evaporites, and in particular rock salt, has been the subject of numerous investigations (e.g. Cristescu and Hunsche, 1998, Cristescu et al, 2002), and basic approaches towards engineering taking into account the long term behavior of such materials have long been well established (e.g. Dreyer, 1972, 1982). The same is certainly not true of ''hard'' rocks. While it long has been recognized that the long term strength of ''hard'' rocks almost certainly is significantly less than that measured during ''short'', i.e. standard (ASTM D 2938), ISRM suggested (Bieniawski et al, 1978) and conventionally used test procedures (e.g. Bieniawski, 1970, Wawersik, 1972, Hoek and Brown, 1980, p. 150), what limited approaches have been taken to develop strategies toward determining the long term mechanical behavior of ''hard'' rock remain in the early research and investigation stage, at best. One early model developed specifically for time dependent analysis of underground ''hard'' rock structures is the phenomenological model by Kaiser and Morgenstern (1981). Brady and Brown (1985, p. 93) state that over a wide range of strain rates, from 10 -8 to 10 2 /s the difference in

  12. Small RNAs in plants: recent development and application for crop improvement.

    Science.gov (United States)

    Kamthan, Ayushi; Chaudhuri, Abira; Kamthan, Mohan; Datta, Asis

    2015-01-01

    The phenomenon of RNA interference (RNAi) which involves sequence-specific gene regulation by small non-coding RNAs, i.e., small interfering RNA (siRNA) and microRNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits and vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold, etc.). Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. microRNAs are key regulators of important plant processes like growth, development, and response to various stresses. In spite of similarity in size (20-24 nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. microRNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA (sRNA) based transgenics are much safer for consumption than those over-expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of sRNAs and its application for crop improvement.

  13. Gravity-regulated gene expression in Arabidopsis thaliana

    Science.gov (United States)

    Sederoff, Heike; Brown, Christopher S.; Heber, Steffen; Kajla, Jyoti D.; Kumar, Sandeep; Lomax, Terri L.; Wheeler, Benjamin; Yalamanchili, Roopa

    Plant growth and development is regulated by changes in environmental signals. Plants sense environmental changes and respond to them by modifying gene expression programs to ad-just cell growth, differentiation, and metabolism. Functional expression of genes comprises many different processes including transcription, translation, post-transcriptional and post-translational modifications, as well as the degradation of RNA and proteins. Recently, it was discovered that small RNAs (sRNA, 18-24 nucleotides long), which are heritable and systemic, are key elements in regulating gene expression in response to biotic and abiotic changes. Sev-eral different classes of sRNAs have been identified that are part of a non-cell autonomous and phloem-mobile network of regulators affecting transcript stability, translational kinetics, and DNA methylation patterns responsible for heritable transcriptional silencing (epigenetics). Our research has focused on gene expression changes in response to gravistimulation of Arabidopsis roots. Using high-throughput technologies including microarrays and 454 sequencing, we iden-tified rapid changes in transcript abundance of genes as well as differential expression of small RNA in Arabidopsis root apices after minutes of reorientation. Some of the differentially regu-lated transcripts are encoded by genes that are important for the bending response. Functional mutants of those genes respond faster to reorientation than the respective wild type plants, indicating that these proteins are repressors of differential cell elongation. We compared the gravity responsive sRNAs to the changes in transcript abundances of their putative targets and identified several potential miRNA: target pairs. Currently, we are using mutant and transgenic Arabidopsis plants to characterize the function of those miRNAs and their putative targets in gravitropic and phototropic responses in Arabidopsis.

  14. RpoS induces expression of the Vibrio anguillarum quorum-sensing regulator VanT.

    Science.gov (United States)

    Weber, Barbara; Croxatto, Antony; Chen, Chang; Milton, Debra L

    2008-03-01

    In vibrios, regulation of the Vibrio harveyi-like LuxR transcriptional activators occurs post-transcriptionally via small regulatory RNAs (sRNAs) that destabilize the luxR mRNA at a low cell population, eliminating expression of LuxR. Expression of the sRNAs is modulated by the vibrio quorum-sensing phosphorelay systems. However, vanT mRNA, which encodes a LuxR homologue in Vibrio anguillarum, is abundant at low and high cell density, indicating that VanT expression may be regulated via additional mechanisms. In this study, Western analyses showed that VanT was expressed throughout growth with a peak of expression during late exponential growth. VanO induced partial destabilization of vanT mRNA via activation of at least one Qrr sRNA. Interestingly, the sigma factor RpoS significantly stabilized vanT mRNA and induced VanT expression during late exponential growth. This induction was in part due to RpoS repressing expression of Hfq, an RNA chaperone. RpoS is not part of the quorum-sensing regulatory cascade since RpoS did not regulate expression or activity of VanO, and RpoS was not regulated by VanO or VanT. VanT and RpoS were needed for survival following UV irradiation and for pigment and metalloprotease production, suggesting that RpoS works with the quorum-sensing systems to modulate expression of VanT, which regulates survival and stress responses.

  15. Roles of Non-Coding RNA in Sugarcane-Microbe Interaction.

    Science.gov (United States)

    Thiebaut, Flávia; Rojas, Cristian A; Grativol, Clícia; Calixto, Edmundo P da R; Motta, Mariana R; Ballesteros, Helkin G F; Peixoto, Barbara; de Lima, Berenice N S; Vieira, Lucas M; Walter, Maria Emilia; de Armas, Elvismary M; Entenza, Júlio O P; Lifschitz, Sergio; Farinelli, Laurent; Hemerly, Adriana S; Ferreira, Paulo C G

    2017-12-20

    Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs) in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae . Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs) were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a copper-transporter gene whose expression was induced in the presence of A. avenae , while the siRNAs were repressed in the presence of A. avenae . Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR). Among these miRNAs, miR408-a copper-microRNA-was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 5'RACE (rapid amplification of cDNA ends) assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly.

  16. Deciphering the role of a miRNA in rice domestication

    Directory of Open Access Journals (Sweden)

    Swetha Chenna

    2017-10-01

    Full Text Available MicroRNAs (miRNAs are a class of 21 nt non-coding small RNAs (sRNAs produced from endogenously expressed MIR genes. miRNAs are mostly involved in development and disease resistance. We are interested in identifying key miRNAs that are differentially expressed among wild and cultivated rice species. Analysis of sRNA datasets from two wild species (O. nivara and O. rufipogon and one cultivated species of rice (O. sativa var. indica Pusa Basmati-1, revealed a surprisingly higher abundance of small RNAs originating from Chromosome 2 in wild rice species. This locus codes for a novel 22 nt miRNA. This novel miRNA was found to be highly abundant in flag leaf of wild species, a tissue that usually provides 70% of energy required for grain filling. This miRNA targets a group of proteins (Os03g0273200, Os01g0827300, Os01g0850700, Os11g0708100 and Os01g0842500 which are involved in secondary metabolite production, although a functional significance of this interaction has not been understood. The expression of these targets also differs across the species. Typical of 22 nt miRNAs, the identified miRNA also triggers a secondary cascade silencing by producing small interfering RNAs (siRNAs from target mRNAs in O. nivara. These secondary siRNAs are observed only among wild rice species but not in cultivated rice. Currently we are using a range of genetic, biochemical and molecular techniques to understand role of this novel miRNA in domestication of rice.

  17. Computational identification and analysis of novel sugarcane microRNAs

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    Thiebaut Flávia

    2012-07-01

    Full Text Available Abstract Background MicroRNA-regulation of gene expression plays a key role in the development and response to biotic and abiotic stresses. Deep sequencing analyses accelerate the process of small RNA discovery in many plants and expand our understanding of miRNA-regulated processes. We therefore undertook small RNA sequencing of sugarcane miRNAs in order to understand their complexity and to explore their role in sugarcane biology. Results A bioinformatics search was carried out to discover novel miRNAs that can be regulated in sugarcane plants submitted to drought and salt stresses, and under pathogen infection. By means of the presence of miRNA precursors in the related sorghum genome, we identified 623 candidates of new mature miRNAs in sugarcane. Of these, 44 were classified as high confidence miRNAs. The biological function of the new miRNAs candidates was assessed by analyzing their putative targets. The set of bona fide sugarcane miRNA includes those likely targeting serine/threonine kinases, Myb and zinc finger proteins. Additionally, a MADS-box transcription factor and an RPP2B protein, which act in development and disease resistant processes, could be regulated by cleavage (21-nt-species and DNA methylation (24-nt-species, respectively. Conclusions A large scale investigation of sRNA in sugarcane using a computational approach has identified a substantial number of new miRNAs and provides detailed genotype-tissue-culture miRNA expression profiles. Comparative analysis between monocots was valuable to clarify aspects about conservation of miRNA and their targets in a plant whose genome has not yet been sequenced. Our findings contribute to knowledge of miRNA roles in regulatory pathways in the complex, polyploidy sugarcane genome.

  18. Autoinducer-2 Quorum Sensing Contributes to Regulation of Microcin PDI in Escherichia coli

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    Shao-Yeh Lu

    2017-12-01

    Full Text Available The Escherichia coli quorum sensing (QS signal molecule, autoinducer-2 (AI-2, reaches its maximum concentration during mid-to-late growth phase after which it quickly degrades during stationary phase. This pattern of AI-2 concentration coincides with the up- then down-regulation of a recently described microcin PDI (mccPDI effector protein (McpM. To determine if there is a functional relationship between these systems, a prototypical mccPDI-expressing strain of E. coli 25 was used to generate ΔluxS, ΔlsrACDBFG (Δlsr, and ΔlsrR mutant strains that are deficient in AI-2 production, transportation, and AI-2 transport regulation, respectively. Trans-complementation, RT-qPCR, and western blot assays were used to detect changes of microcin expression and synthesis under co-culture and monoculture conditions. Compared to the wild-type strain, the AI-2-deficient strain (ΔluxS and -uptake negative strain (Δlsr were >1,000-fold less inhibitory to susceptible bacteria (P < 0.05. With in trans complementation of luxS, the AI-2 deficient mutant reduced the susceptible E. coli population by 4-log, which was within 1-log of the wild-type phenotype. RT-qPCR and western blot results for the AI-2 deficient E. coli 25 showed a 5-fold reduction in mcpM transcription with an average 2-h delay in McpM synthesis. Furthermore, overexpression of sRNA micC and micF (both involved in porin protein regulation was correlated with mcpM regulation, consistent with a possible link between QS and mcpM regulation. This is the direct first evidence that microcin regulation can be linked to quorum sensing in a Gram-negative bacterium.

  19. Genome-wide discovery of putative sRNAs in Paracoccus denitrificans expressed under nitrous oxide emitting conditions.

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    Hannah Gaimster

    2016-11-01

    Full Text Available Nitrous oxide (N2O is a stable, ozone depleting greenhouse gas. Emissions of N2O into the atmosphere continue to rise, primarily due to the use of nitrogen-containing fertilizers by soil denitrifying microbes. It is clear more effective mitigation strategies are required to reduce emissions. One way to help develop future mitigation strategies is to address the currently poor understanding of transcriptional regulation of the enzymes used to produce and consume N2O. With this ultimate aim in mind we performed RNA-seq on a model soil denitrifier, Paracoccus denitrificans, cultured anaerobically under high N2O and low N2O emitting conditions, and aerobically under zero N2O emitting conditions to identify small RNAs (sRNAs with potential regulatory functions transcribed under these conditions. sRNAs are short (∼40–500 nucleotides non-coding RNAs that regulate a wide range of activities in many bacteria. 167 sRNAs were identified throughout the P. denitrificans genome which are either present in intergenic regions or located antisense to ORFs. Furthermore, many of these sRNAs are differentially expressed under high N2O and low N2O emitting conditions respectively, suggesting they may play a role in production or reduction of N2O. Expression of 16 of these sRNAs have been confirmed by RT-PCR. 90% of the sRNAs are predicted to form secondary structures. Predicted targets include transporters and a number of transcriptional regulators. A number of sRNAs were conserved in other members of the α-proteobacteria. Better understanding of the sRNA factors which contribute to expression of the machinery required to reduce N2O will, in turn, help to inform strategies for mitigation of N2O emissions.

  20. Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

    International Nuclear Information System (INIS)

    Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R.

    2014-01-01

    In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria.

  1. Alkane biosynthesis genes in cyanobacteria and their transcriptional organization

    Directory of Open Access Journals (Sweden)

    Stephan eKlähn

    2014-07-01

    Full Text Available In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl-acyl carrier protein reductase (AAR and aldehyde deformylating oxygenase (ADO. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado and sll0209 (aar, that give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313 and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in

  2. Global Analysis of Photosynthesis Transcriptional Regulatory Networks

    Science.gov (United States)

    Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

    2014-01-01

    Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

  3. Crystal structure and optical property of complex perovskite oxynitrides ALi0.2Nb0.8O2.8N0.2, ANa0.2Nb0.8O2.8N0.2, and AMg0.2Nb0.8O2.6N0.4 (A = Sr, Ba)

    Science.gov (United States)

    Moon, Keon Ho; Avdeev, Maxim; Kim, Young-Il

    2017-10-01

    Oxynitride type complex perovskites AM0.2Nb0.8O3-xNx (A = Sr, Ba; M = Li, Na, Mg) were newly synthesized by the solid state diffusion of Li+, Na+, or Mg2+ into the layered oxide, A5Nb4O15, with concurrent O/N substitution. Neutron and synchrotron X-ray Rietveld refinement showed that SrLi0.2Nb0.8O2.8N0.2, SrNa0.2Nb0.8O2.8N0.2, and SrMg0.2Nb0.8O2.6N0.4 had body-centered tetragonal symmetry (I4/mcm), while those with A = Ba had simple cubic symmetry (Pm 3 ̅ m). In the tetragonal Sr-compounds, the nitrogen atoms were localized on the c-axial 4a site. However, the octahedral cations, M/Nb (M = Li, Na, Mg) were distributed randomly in all six compounds. The lattice volume of AM0.2Nb0.8O3-xNx was dependent on various factors including the type of A and the electronegativity of M. Compared to the simple perovskites, ANbO2N (A = Sr, Ba), AM0.2Nb0.8O3-xNx had wider band gaps (1.76-2.15 eV for A = Sr and 1.65-2.10 eV for A = Ba), but significantly lower sub-gap absorption.

  4. Identification of novel miRNAs and miRNA dependent developmental shifts of gene expression in Arabidopsis thaliana.

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    Shuhua Zhan

    Full Text Available microRNAs (miRNAs are small, endogenous RNAs of 20 approximately 25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.

  5. Transcriptome and Degradome of microRNAs and Their Targets in Response to Drought Stress in the Plants of a Diploid and Its Autotetraploid Paulownia australis.

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    Suyan Niu

    Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs that play vital roles in plant growth, development, and stress response. Increasing numbers of studies aimed at discovering miRNAs and analyzing their functions in plants are being reported. In this study, we investigated the effect of drought stress on the expression of miRNAs and their targets in plants of a diploid and derived autotetraploid Paulownia australis. Four small RNA (sRNA libraries and four degradome libraries were constructed from diploid and autotetraploid P. australis plants treated with either 75% or 25% relative soil water content. A total of 33 conserved and 104 novel miRNAs (processing precision value > 0.1 were identified, and 125 target genes were identified for 36 of the miRNAs by using the degradome sequencing. Among the identified miRNAs, 54 and 68 were differentially expressed in diploid and autotetraploid plants under drought stress (25% relative soil water content, respectively. The expressions of miRNAs and target genes were also validated by quantitative real-time PCR. The results showed that the relative expression trends of the randomly selected miRNAs were similar to the trends predicted by Illumina sequencing. And the correlations between miRNAs and their target genes were also analyzed. Furthermore, the functional analysis showed that most of these miRNAs and target genes were associated with plant development and environmental stress response. This study provided molecular evidence for the possible involvement of certain miRNAs in the drought response and/or tolerance in P. australis, and certain level of differential expression between diploid and autotetraploid plants.

  6. Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi.

    Science.gov (United States)

    Nerva, L; Ciuffo, M; Vallino, M; Margaria, P; Varese, G C; Gnavi, G; Turina, M

    2016-07-02

    The number of reported mycoviruses is increasing exponentially due to the current ability to detect mycoviruses using next-generation sequencing (NGS) approaches, with a large number of viral genomes built in-silico using data from fungal transcriptome projects. We decided to screen a collection of fungi originating from a specific marine environment (associated with the seagrass Posidonia oceanica) for the presence of mycoviruses: our findings reveal a wealth of diversity among these symbionts and this complexity will require further studies to address their specific role in this ecological niche. In specific, we identified twelve new virus species belonging to nine distinct lineages: they are members of megabirnavirus, totivirus, chrysovirus, partitivirus and five still undefined clades. We showed evidence of an endogenized virus ORF, and evidence of accumulation of dsRNA from metaviridae retroviral elements. We applied different techniques for detecting the presence of mycoviruses including (i) dsRNA extraction and cDNA cloning, (ii) small and total RNA sequencing through NGS techniques, (iii) rolling circle amplification (RCA) and total DNA extraction analyses, (iv) virus purifications and electron microscopy. We tried also to critically evaluate the intrinsic value and limitations of each of these techniques. Based on the samples we could compare directly, RNAseq analysis is superior to sRNA for de novo assembly of mycoviruses. To our knowledge this is the first report on the virome of fungi isolated from marine environment. The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: KT601099-KT601110; KT601114-KT601120; KT592305; KT950836-KT950841. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns

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    Domingues Douglas S

    2012-04-01

    Full Text Available Abstract Background Sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out. Results Sixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. FISH analysis resulted in the expected pattern of Gypsy/heterochromatin, Copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. Analysis of related ESTs and RT-PCR showed transcriptional variation between tissues and families. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements. Conclusions Individual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNA's that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome.

  8. C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response.

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    Doug E Brackney

    2010-10-01

    Full Text Available Mosquitoes rely on RNA interference (RNAi as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV infection in C6/36 (Aedes albopictus cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses. Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae, Sindbis virus (SINV, Togaviridae and La Crosse virus (LACV, Bunyaviridae and total RNA recovered from cell lysates. Small RNA (sRNA libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand and distribution (position along viral genome of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.

  9. Identification of miRNAs Responsive to Botrytis cinerea in Herbaceous Peony (Paeonia lactiflora Pall. by High-Throughput Sequencing

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    Daqiu Zhao

    2015-09-01

    Full Text Available Herbaceous peony (Paeonia lactiflora Pall., one of the world’s most important ornamental plants, is highly susceptible to Botrytis cinerea, and improving resistance to this pathogenic fungus is a problem yet to be solved. MicroRNAs (miRNAs play an essential role in resistance to B. cinerea, but until now, no studies have been reported concerning miRNAs induction in P. lactiflora. Here, we constructed and sequenced two small RNA (sRNA libraries from two B. cinerea-infected P. lactiflora cultivars (“Zifengyu” and “Dafugui” with significantly different levels of resistance to B. cinerea, using the Illumina HiSeq 2000 platform. From the raw reads generated, 4,592,881 and 5,809,796 sRNAs were obtained, and 280 and 306 miRNAs were identified from “Zifengyu” and “Dafugui”, respectively. A total of 237 conserved and 7 novel sequences of miRNAs were differentially expressed between the two cultivars, and we predicted and annotated their potential target genes. Subsequently, 7 differentially expressed candidate miRNAs were screened according to their target genes annotated in KEGG pathways, and the expression patterns of miRNAs and corresponding target genes were elucidated. We found that miR5254, miR165a-3p, miR3897-3p and miR6450a might be involved in the P. lactiflora response to B. cinerea infection. These results provide insight into the molecular mechanisms responsible for resistance to B. cinerea in P. lactiflora.

  10. High-throughput sequencing identification and characterization of potentially adhesion-related small RNAs in Streptococcus mutans.

    Science.gov (United States)

    Zhu, Wenhui; Liu, Shanshan; Liu, Jia; Zhou, Yan; Lin, Huancai

    2018-05-01

    Adherence capacity is one of the principal virulence factors of Streptococcus mutans, and adhesion virulence factors are controlled by small RNAs (sRNAs) at the post-transcriptional level in various bacteria. Here, we aimed to identify and decipher putative adhesion-related sRNAs in clinical strains of S. mutans. RNA deep-sequencing was performed to identify potential sRNAs under different adhesion conditions. The expression of sRNAs was analysed by quantitative real-time PCR (qRT-PCR), and bioinformatic methods were used to predict the functional characteristics of sRNAs. A total of 736 differentially expressed candidate sRNAs were predicted, and these included 352 sRNAs located on the antisense to mRNA (AM) and 384 sRNAs in intergenic regions (IGRs). The top 7 differentially expressed sRNAs were successfully validated by qRT-PCR in UA159, and 2 of these were further confirmed in 100 clinical isolates. Moreover, the sequences of two sRNAs were conserved in other Streptococcus species, indicating a conserved role in such closely related species. A good correlation between the expression of sRNAs and the adhesion of 100 clinical strains was observed, which, combined with GO and KEGG, provides a perspective for the comprehension of sRNA function annotation. This study revealed a multitude of novel putative adhesion-related sRNAs in S. mutans and contributed to a better understanding of information concerning the transcriptional regulation of adhesion in S. mutans.

  11. The Metabolic Effects of Traditional Chinese Medication Qiliqiangxin on H9C2 Cardiomyocytes

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    Shenghui Lin

    2015-11-01

    Full Text Available Background/Aims: A traditional Chinese medicine, Qiliqiangxin (QLQX has been identified to perform protective effects on myocardium energy metabolism in mice with acute myocardial infarction, though the effects of QLQX on myocardial mitochondrial biogenesis under physiological condition is still largely elusive. Methods: H9C2 cells were treated with different concentrations of QLQX (0.25, 0.5, and 1.0 µg/mL from 6 to 48 hours. Oxidative metabolism and glycolysis were measured by oxygen consumption and extracellular acidification with XF96 analyzer (SeaHorse. Mitochondrial content and ultrastructure were assessed by Mitotracker staining, confocal microscopy, flow cytometry, and transmission electron microscopy. Mitochondrial biogenesis-related genes were measured by qRT-PCR and Western blot. Results: H9C2 cells treated with QLQX exhibited increased glycolysis at earlier time points (6, 12, and 24 hours, while QLQX could enhance oxidative metabolism and mitochondrial uncoupling in H9C2 cells with longer duration of treatment (48 hours. QLQX also increased mitochondrial content and mitochondrial biogenesis-related gene expression levels, including 16sRNA, SSBP1, TWINKLE, TOP1MT and PLOG, with an activation of peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α and its downstream effectors. Silencing PGC-1α could abolish the increased mitochondrial content in H9C2 cells treated with QLQX. Conclusion: Our study is the first to document enhanced metabolism in cardiomyocytes treated with QLQX, which is linked to increased mitochondrial content and mitochondrial biogenesis via activation of PGC-1α.

  12. Global analysis of photosynthesis transcriptional regulatory networks.

    Directory of Open Access Journals (Sweden)

    Saheed Imam

    2014-12-01

    Full Text Available Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888, which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

  13. Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

    Energy Technology Data Exchange (ETDEWEB)

    Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R., E-mail: wolfgang.hess@biologie.uni-freiburg.de [Genetics and Experimental Bioinformatics, Institute of Biology 3, Faculty of Biology, University of Freiburg, Freiburg (Germany)

    2014-07-14

    In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria.

  14. A Natural Light/Dark Cycle Regulation of Carbon-Nitrogen Metabolism and Gene Expression in Rice Shoots.

    Science.gov (United States)

    Li, Haixing; Liang, Zhijun; Ding, Guangda; Shi, Lei; Xu, Fangsen; Cai, Hongmei

    2016-01-01

    Light and temperature are two particularly important environmental cues for plant survival. Carbon and nitrogen are two essential macronutrients required for plant growth and development, and cellular carbon and nitrogen metabolism must be tightly coordinated. In order to understand how the natural light/dark cycle regulates carbon and nitrogen metabolism in rice plants, we analyzed the photosynthesis, key carbon-nitrogen metabolites, and enzyme activities, and differentially expressed genes and miRNAs involved in the carbon and nitrogen metabolic pathway in rice shoots at the following times: 2:00, 6:00, 10:00, 14:00, 18:00, and 22:00. Our results indicated that more CO2 was fixed into carbohydrates by a high net photosynthetic rate, respiratory rate, and stomatal conductance in the daytime. Although high levels of the nitrate reductase activity, free ammonium and carbohydrates were exhibited in the daytime, the protein synthesis was not significantly facilitated by the light and temperature. In mRNA sequencing, the carbon and nitrogen metabolism-related differentially expressed genes were obtained, which could be divided into eight groups: photosynthesis, TCA cycle, sugar transport, sugar metabolism, nitrogen transport, nitrogen reduction, amino acid metabolism, and nitrogen regulation. Additionally, a total of 78,306 alternative splicing events have been identified, which primarily belong to alternative 5' donor sites, alternative 3' acceptor sites, intron retention, and exon skipping. In sRNA sequencing, four carbon and nitrogen metabolism-related miRNAs (osa-miR1440b, osa-miR2876-5p, osa-miR1877 and osa-miR5799) were determined to be regulated by natural light/dark cycle. The expression level analysis showed that the four carbon and nitrogen metabolism-related miRNAs negatively regulated their target genes. These results may provide a good strategy to study how natural light/dark cycle regulates carbon and nitrogen metabolism to ensure plant growth and

  15. Influence of manure age and sunlight on the community structure of cattle fecal bacteria as revealed by Illumina sequencing

    Science.gov (United States)

    Wong, K.; Shaw, T. I.; Oladeinde, A.; Molina, M.

    2013-12-01

    Fecal pollution of environmental waters is a major concern for the general public because exposure to fecal-associated pathogens can have severe impacts on human health. Stream and river impairment due to fecal pollution is largely the result of agricultural activities in the United States. In the last few years, numerous metagenomic studies utilized next generation sequencing to develop microbial community profiles by massively sequencing the 16sRNA hypervariable region. This technology supports the application of water quality assessment such as pathogen detection and fecal source tracking. The bacteria communities of samples in these studies were determined when they were freshly collected; therefore, little is known about how feces age or how environmental stress influences the microbial ecology of fecal materials. In this study we monitored bacteria community changes in cattle feces for 57 days after excretion (day 0, 2, 4 8, 15, 22, 29, 43, 57) by sequencing the 16s variable region 4, using Illumnia MiSeq. Twelve cattle feces were studied; half of the samples were directly exposed to sunlight (unshaded) and half were shaded. Results indicate that the relative abundance (RA) profile in both shaded and unshaded samples rapidly changed from day 0 to 15, but stabilized from day 22 to 57. Firmcutes were the most abundant phylum (~40%) at day 0, but were reduced to rarefaction curve analysis, richness of bacteria diversity in feces decreased as time progressed. Some pathogens such as Campylobacter were detected only at the beginning, meaning they substantially decayed during the course of our study. Overall, this study indicated: (1) sunlight can influence the community structure and (2) after excretion the fecal bacteria diversity can be significantly changed over time. Future studies should therefore use not only the microbial signature of fresh but also moderately aged fecal samples to develop more accurate community profiles for fecal source tracking.

  16. Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli

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    Sushovan Dam

    2017-12-01

    Full Text Available Antibiotic resistant Gram-negative bacteria are a serious threat for public health. The permeation of antibiotics through their outer membrane is largely dependent on porin, changes in which cause reduced drug uptake and efficacy. Escherichia coli produces two major porins, OmpF and OmpC. MicF and MicC are small non-coding RNAs (sRNAs that modulate the expression of OmpF and OmpC, respectively. In this work, we investigated factors that lead to increased production of MicC. micC promoter region was fused to lacZ, and the reporter plasmid was transformed into E. coli MC4100 and derivative mutants. The response of micC–lacZ to antimicrobials was measured during growth over a 6 h time period. The data showed that the expression of micC was increased in the presence of β-lactam antibiotics and in an rpoE depleted mutant. Interestingly, the same conditions enhanced the activity of an ompN–lacZ fusion, suggesting a dual transcriptional regulation of micC and the quiescent adjacent ompN. Increased levels of OmpN in the presence of sub-inhibitory concentrations of chemicals could not be confirmed by Western blot analysis, except when analyzed in the absence of the sigma factor σE. We suggest that the MicC sRNA acts together with the σE envelope stress response pathway to control the OmpC/N levels in response to β-lactam antibiotics.

  17. Analysis of integrated multiple 'omics' datasets reveals the mechanisms of initiation and determination in the formation of tuberous roots in Rehmannia glutinosa.

    Science.gov (United States)

    Li, Mingjie; Yang, Yanhui; Li, Xinyu; Gu, Li; Wang, Fengji; Feng, Fajie; Tian, Yunhe; Wang, Fengqing; Wang, Xiaoran; Lin, Wenxiong; Chen, Xinjian; Zhang, Zhongyi

    2015-09-01

    All tuberous roots in Rehmannia glutinosa originate from the expansion of fibrous roots (FRs), but not all FRs can successfully transform into tuberous roots. This study identified differentially expressed genes and proteins associated with the expansion of FRs, by comparing the tuberous root at expansion stages (initiated tuberous root, ITRs) and FRs at the seedling stage (initiated FRs, IFRs). The role of miRNAs in the expansion of FRs was also explored using the sRNA transcriptome and degradome to identify miRNAs and their target genes that were differentially expressed between ITRs and FRs at the mature stage (unexpanded FRs, UFRs, which are unable to expand into ITRs). A total of 6032 genes and 450 proteins were differentially expressed between ITRs and IFRs. Integrated analyses of these data revealed several genes and proteins involved in light signalling, hormone response, and signal transduction that might participate in the induction of tuberous root formation. Several genes related to cell division and cell wall metabolism were involved in initiating the expansion of IFRs. Of 135 miRNAs differentially expressed between ITRs and UFRs, there were 27 miRNAs whose targets were specifically identified in the degradome. Analysis of target genes showed that several miRNAs specifically expressed in UFRs were involved in the degradation of key genes required for the formation of tuberous roots. As far as could be ascertained, this is the first time that the miRNAs that control the transition of FRs to tuberous roots in R. glutinosa have been identified. This comprehensive analysis of 'omics' data sheds new light on the mechanisms involved in the regulation of tuberous roots formation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Roles of Non-Coding RNA in Sugarcane-Microbe Interaction

    Science.gov (United States)

    Grativol, Clícia; Motta, Mariana R.; Ballesteros, Helkin G. F.; Peixoto, Barbara; Vieira, Lucas M.; Walter, Maria Emilia; de Armas, Elvismary M.; Entenza, Júlio O. P.; Lifschitz, Sergio; Farinelli, Laurent; Hemerly, Adriana S.

    2017-01-01

    Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs) in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae. Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs) were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a copper-transporter gene whose expression was induced in the presence of A. avenae, while the siRNAs were repressed in the presence of A. avenae. Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR). Among these miRNAs, miR408—a copper-microRNA—was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 5′RACE (rapid amplification of cDNA ends) assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly. PMID:29657296

  19. Analysis of physiological and miRNA responses to Pi deficiency in alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Li, Zhenyi; Xu, Hongyu; Li, Yue; Wan, Xiufu; Ma, Zhao; Cao, Jing; Li, Zhensong; He, Feng; Wang, Yufei; Wan, Liqiang; Tong, Zongyong; Li, Xianglin

    2018-03-01

    The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake. Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.

  20. Evolutionary Dynamics of Small RNAs in 27 Escherichia coli and Shigella Genomes

    Science.gov (United States)

    Skippington, Elizabeth; Ragan, Mark A.

    2012-01-01

    Small RNAs (sRNAs) are widespread in bacteria and play critical roles in regulating physiological processes. They are best characterized in Escherichia coli K-12 MG1655, where 83 sRNAs constitute nearly 2% of the gene complement. Most sRNAs act by base pairing with a target mRNA, modulating its translation and/or stability; many of these RNAs share only limited complementarity to their mRNA target, and require the chaperone Hfq to facilitate base pairing. Little is known about the evolutionary dynamics of bacterial sRNAs. Here, we apply phylogenetic and network analyses to investigate the evolutionary processes and principles that govern sRNA gene distribution in 27 E. coli and Shigella genomes. We identify core (encoded in all 27 genomes) and variable sRNAs; more than two-thirds of the E. coli K-12 MG1655 sRNAs are core, whereas the others show patterns of presence and absence that are principally due to genetic loss, not duplication or lateral genetic transfer. We present evidence that variable sRNAs are less tightly integrated into cellular genetic regulatory networks than are the core sRNAs, and that Hfq facilitates posttranscriptional cross talk between the E. coli–Shigella core and variable genomes. Finally, we present evidence that more than 80% of genes targeted by Hfq-associated core sRNAs have been transferred within the E. coli–Shigella clade, and that most of these genes have been transferred intact. These results suggest that Hfq and sRNAs help integrate laterally acquired genes into established regulatory networks. PMID:22223756

  1. Roles of Non-Coding RNA in Sugarcane-Microbe Interaction

    Directory of Open Access Journals (Sweden)

    Flávia Thiebaut

    2017-12-01

    Full Text Available Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae. Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a copper-transporter gene whose expression was induced in the presence of A. avenae, while the siRNAs were repressed in the presence of A. avenae. Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR. Among these miRNAs, miR408—a copper-microRNA—was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 5′RACE (rapid amplification of cDNA ends assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly.

  2. An integrated approach for finding overlooked genes in Shigella.

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    Junping Peng

    Full Text Available BACKGROUND: The completion of numerous genome sequences introduced an era of whole-genome study. However, many genes are missed during genome annotation, including small RNAs (sRNAs and small open reading frames (sORFs. In order to improve genome annotation, we aimed to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery. METHODOLOGY/PRINCIPAL FINDINGS: We identified 64 sRNAs in Shigella, which were experimentally validated in other bacteria based on sequence conservation. We employed computer-based and tiling array-based methods to search for sRNAs, followed by RT-PCR and northern blots, to identify nine sRNAs in Shigella flexneri strain 301 (Sf301 and 256 regions containing possible sRNA genes. We found 29 candidate sORFs using bioinformatic prediction, array hybridization and RT-PCR verification. We experimentally validated 557 (57.9% DOOR operon predictions in the chromosomes of Sf301 and 46 (76.7% in virulence plasmid.We found 40 additional co-expressed gene pairs that were not predicted by DOOR. CONCLUSIONS/SIGNIFICANCE: We provide an updated and comprehensive annotation of the Shigella genome. Our study increased the expected numbers of sORFs and sRNAs, which will impact on future functional genomics and proteomics studies. Our method can be used for large scale reannotation of sRNAs and sORFs in any microbe with a known genome sequence.

  3. Identification of arbuscular mycorrhiza (AM-responsive microRNAs in tomato

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    Ping eWu

    2016-03-01

    Full Text Available A majority of land plants can form symbiosis with arbuscular mycorrhizal (AM fungi. MicroRNAs (miRNAs have been implicated to regulate this process in legumes, but their involvement in non-legume species is largely unknown. In this study, by performing deep sequencing of sRNA libraries in tomato roots and comparing with tomato genome, a total of 700 potential miRNAs were predicted, among them, 187 are known plant miRNAs that have been previously deposited in miRBase. Unlike the profiles in other plants such as rice and Arabidopsis, a large proportion of predicted tomato miRNAs was 24 nt in length. A similar pattern was observed in the potato genome but not in tobacco, indicating a Solanum genus-specific expansion of 24-nt miRNAs. About 40% identified tomato miRNAs showed significantly altered expressions upon Rhizophagus irregularis inoculation, suggesting the potential roles of these novel miRNAs in AM symbiosis. The differential expression of five known and six novel miRNAs were further validated using qPCR analysis. Interestingly, three up-regulated known tomato miRNAs belong to a known miR171 family, a member of which has been reported in Medicago truncatula to regulate AM symbiosis. Thus, the miR171 family likely regulates AM symbiosis conservatively across different plant lineages. More than 1000 genes targeted by potential AM-responsive miRNAs were provided and their roles in AM symbiosis are worth further exploring.

  4. Domestication of transposable elements into MicroRNA genes in plants.

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    Yang Li

    Full Text Available Transposable elements (TE usually take up a substantial portion of eukaryotic genome. Activities of TEs can cause genome instability or gene mutations that are harmful or even disastrous to the host. TEs also contribute to gene and genome evolution at many aspects. Part of miRNA genes in mammals have been found to derive from transposons while convincing evidences are absent for plants. We found that a considerable number of previously annotated plant miRNAs are identical or homologous to transposons (TE-MIR, which include a small number of bona fide miRNA genes that conform to generally accepted plant miRNA annotation rules, and hairpin derived siRNAs likely to be pre-evolved miRNAs. Analysis of these TE-MIRs indicate that transitions from the medium to high copy TEs into miRNA genes may undergo steps such as inverted repeat formation, sequence speciation and adaptation to miRNA biogenesis. We also identified initial target genes of the TE-MIRs, which contain homologous sequences in their CDS as consequence of cognate TE insertions. About one-third of the initial target mRNAs are supported by publicly available degradome sequencing data for TE-MIR sRNA induced cleavages. Targets of the TE-MIRs are biased to non-TE related genes indicating their penchant to acquire cellular functions during evolution. Interestingly, most of these TE insertions span boundaries between coding and non-coding sequences indicating their incorporation into CDS through alteration of splicing or translation start or stop signals. Taken together, our findings suggest that TEs in gene rich regions can form foldbacks in non-coding part of transcripts that may eventually evolve into miRNA genes or be integrated into protein coding sequences to form potential targets in a "temperate" manner. Thus, transposons may supply as resources for the evolution of miRNA-target interactions in plants.

  5. Comparative Genomic Analysis Reveals a Diverse Repertoire of Genes Involved in Prokaryote-Eukaryote Interactions within the Pseudovibrio Genus.

    Science.gov (United States)

    Romano, Stefano; Fernàndez-Guerra, Antonio; Reen, F Jerry; Glöckner, Frank O; Crowley, Susan P; O'Sullivan, Orla; Cotter, Paul D; Adams, Claire; Dobson, Alan D W; O'Gara, Fergal

    2016-01-01

    Strains of the Pseudovibrio genus have been detected worldwide, mainly as part of bacterial communities associated with marine invertebrates, particularly sponges. This recurrent association has been considered as an indication of a symbiotic relationship between these microbes and their host. Until recently, the availability of only two genomes, belonging to closely related strains, has limited the knowledge on the genomic and physiological features of the genus to a single phylogenetic lineage. Here we present 10 newly sequenced genomes of Pseudovibrio strains isolated from marine sponges from the west coast of Ireland, and including the other two publicly available genomes we performed an extensive comparative genomic analysis. Homogeneity was apparent in terms of both the orthologous genes and the metabolic features shared amongst the 12 strains. At the genomic level, a key physiological difference observed amongst the isolates was the presence only in strain P. axinellae AD2 of genes encoding proteins involved in assimilatory nitrate reduction, which was then proved experimentally. We then focused on studying those systems known to be involved in the interactions with eukaryotic and prokaryotic cells. This analysis revealed that the genus harbors a large diversity of toxin-like proteins, secretion systems and their potential effectors. Their distribution in the genus was not always consistent with the phylogenetic relationship of the strains. Finally, our analyses identified new genomic islands encoding potential toxin-immunity systems, previously unknown in the genus. Our analyses shed new light on the Pseudovibrio genus, indicating a large diversity of both metabolic features and systems for interacting with the host. The diversity in both distribution and abundance of these systems amongst the strains underlines how metabolically and phylogenetically similar bacteria may use different strategies to interact with the host and find a niche within its

  6. In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

    Science.gov (United States)

    Yakovlev, Igor A; Fossdal, Carl G

    2017-01-01

    Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21-22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21-22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic

  7. In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce

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    Igor A. Yakovlev

    2017-09-01

    Full Text Available Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs and other small non-coding RNAs (sRNAs play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C. We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21–22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21–22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be

  8. InvS Coordinates Expression of PrgH and FimZ and Is Required for Invasion of Epithelial Cells by Salmonella enterica serovar Typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lu; Cai, Xia; Wu, Shuyan; Bomjan, Rajdeep; Nakayasu, Ernesto S.; Händler, Kristian; Hinton, Jay C. D.; Zhou, Daoguo; DiRita, Victor J.

    2017-04-24

    about the role played by small RNAs (sRNAs) in this process. We have identified a novel sRNA, InvS, that is involved inSalmonellainvasion. Our result will likely provide an opportunity to better understand the fundamental question of howSalmonellaregulates invasion gene expression and may inform strategies for therapeutic intervention.

  9. A natural light/dark cycle regulation of carbon-nitrogen metabolism and gene expression in rice shoots

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    Haixing Li

    2016-08-01

    Full Text Available Light and temperature are two particularly important environmental cues for plant survival. Carbon and nitrogen are two essential macronutrients required for plant growth and development, and cellular carbon and nitrogen metabolism must be tightly coordinated. In order to understand how the natural light/dark cycle regulates carbon and nitrogen metabolism in rice plants, we analyzed the photosynthesis, key carbon-nitrogen metabolites and enzyme activities, and differentially expressed genes and miRNAs involved in the carbon and nitrogen metabolic pathway in rice shoots at the following times: 2:00, 6:00, 10:00, 14:00, 18:00 and 22:00. Our results indicated that more CO2 was fixed into carbohydrates by a high net photosynthetic rate, respiratory rate and stomatal conductance in the daytime. Although high levels of the nitrate reductase activity, free ammonium and carbohydrates were exhibited in the daytime, the protein synthesis was not significantly facilitated by the light and temperature. In mRNA sequencing, the carbon and nitrogen metabolism-related differentially expressed genes were obtained, which could be divided into eight groups: photosynthesis, TCA cycle, sugar transport, sugar metabolism, nitrogen transport, nitrogen reduction, amino acid metabolism and nitrogen regulation. Additionally, a total of 78,306 alternative splicing events have been identified, which primarily belong to alternative 5' donor sites, alternative 3' acceptor sites, intron retention and exon skipping. In sRNA sequencing, four carbon and nitrogen metabolism-related miRNAs (osa-miR1440b, osa-miR2876-5p, osa-miR1877 and osa-miR5799 were determined to be regulated by natural light/dark cycle. The expression level analysis showed that the four carbon and nitrogen metabolism-related miRNAs negatively regulated their target genes. These results may provide a good strategy to study how natural light/dark cycle regulates carbon and nitrogen metabolism to ensure plant

  10. [Effects of oral interventions on carotid artery in rats with chronic periodontitis for the detection of Porphyromonas gingivalis and the expression of C-reactive protein].

    Science.gov (United States)

    Xiuyun, Ren; Chong, Wang; Xin, Liu; Hao, Li; Qianhui, Ma; Mu, Lin; Xuexue, Shi; Jinhua, Gao

    2017-04-01

    This study aimed to establish a SD rat model of chronic periodontitis (CP) merged with hyperlipidemia (HL), perform periodontal treatment, detect the expression of partial C-reactive protein (CRP) and Porphyromonas gingivalis (P. gingivalis) in the rat carotid artery, and explore the relationship between periodontitis and atherosclerosis. SD rats were randomly divided into three groups: control group (A), HL group (B), and CP+HL group (C). Group C rats were divided into natural process group (C1), scaling and root planning group (C2), and tooth extraction group (C3). Group C2 rats were randomly divided into C2-1 (scaling and root planning group) and C2-2 (scaling and root planning+minocyline+systemic antibiotics group). Group C3 rats were randomly divided into C3-1 (tooth extraction group) and C3-2 (tooth extraction+systemic antibiotic group). One rat from group B was randomly selected and sacrificed after 15 weeks. Subsequently, the carotid vascular tissue was collected for oil red O staining. Modeling was successful when foam cell formation was observed. Periodontal treatments were conducted twice, and euthanasia was performed after the experiment. Moreover, double-carotid artery bifurcation was carried out to detect the expression of CRP and P. gingivalis. Immunohistochemical and 16sRNA semiquantitative methods were used to detect the CRP expression and the relative contents of P. gingivalis, respectively. Immunohistochemical results showed that the CRP-positive expression in groups B and C was significantly higher than that in group A (PC rats were significantly lower than that in group C1 (PC2-2 was the lowest among the groups (PC1 was the highest and significantly higher than that in groups A and B (PC2-1, C2-2, C3-1, and C3-2 were significantly lower than that in group C1 (PC3-2 was the lowest (Pperiodontal basic treatment and tooth extraction, could improve carotid artery lesions. The basic treatment with local and systemic anti-inflammatory drugs exerts

  11. Coupled S and Sr isotope evidences for elevated arsenic concentrations in groundwater from the world's largest antimony mine, Central China

    Science.gov (United States)

    Wen, Bing; Zhou, Aiguo; Zhou, Jianwei; Liu, Cunfu; Huang, Yuliu; Li, Ligang

    2018-02-01

    The Xikuangshan(XKS) mine, the world's largest antimony mine, was chosen for a detailed arsenic hydrogeochemical study because of the elevated arsenic in bedrock aquifers used by local residents. Hydrochemical data, δ34S values of dissolved SO42- and 87Sr/86Sr ratios have been analyzed to identify the predominant geochemical processes that control the arsenic mobilization within the aquifers. Groundwater samples can be divided into three major types: low arsenic groundwater (0-50 μg/L), high arsenic groundwater (50-1000 μg/L) and anomalous high arsenic groundwater (>1000 μg/L). Arsenic occurs under oxidizing conditions at the XKS Sb mine as the HAsO42- anion. The Ca/Na ratio correlates significantly with HCO3-/Na and Sr/Na ratios, indicating that carbonate dissolution and silicate weathering are the dominant processes controlling groundwater hydrochemistry. The δ34S values of the groundwater indicate that dissolved SO42- in groundwater is mainly sourced from the oxidation of sulfide minerals, and elevated As concentrations in groundwater are influenced by the mixing of mine water and surface water. Furthermore, the δ34S values are not correlated with dissolved As concentrations and Fe concentrations, suggesting that the reduction dissolution of Fe(III) hydroxides is not the dominant process controlling As mobilization. The 87Sr/86Sr ratios imply that elevated As concentrations in groundwater are primarily derived from the interaction with the stibnite and silicified limestone. More specifically, the excess-Na ion, the feature of Ca/Na ratio, and the spatial association of elevated As concentrations in groundwater collectively suggest that high and anomalous high arsenic groundwater are associated with smelting slags and, in particular, the arsenic alkali residue. In general, the hydrochemistry analysis, especially the S and Sr isotope evidences elucidate that elevated As concentrations and As mobilization are influenced by several geochemical processes

  12. Identification of drought-responsive miRNAs and physiological characterization of tea plant (Camellia sinensis L.) under drought stress.

    Science.gov (United States)

    Guo, Yuqiong; Zhao, Shanshan; Zhu, Chen; Chang, Xiaojun; Yue, Chuan; Wang, Zhong; Lin, Yuling; Lai, Zhongxiong

    2017-11-21

    Drought stress is one of the major natural challenges in the main tea-producing regions of China. The tea plant (Camellia sinensis) is a traditional beverage plant whose growth status directly affects tea quality. Recent studies have revealed that microRNAs (miRNAs) play key functions in plant growth and development. Although some miRNAs have been identified in C. sinensis, little is known about their roles in the drought stress response of tea plants. Physiological characterization of Camellia sinensis 'Tieguanyin' under drought stress showed that the malondialdehyde concentration and electrical conductivity of leaves of drought-stressed plants increased when the chlorophyll concentration decreased under severe drought stress. We sequenced four small-RNA (sRNA) libraries constructed from leaves of plants subjected to four different treatments, normal water supply (CK); mild drought stress (T1); moderate drought stress (T2) and severe drought stress (T3). A total of 299 known mature miRNA sequences and 46 novel miRNAs were identified. Gene Ontology enrichment analysis revealed that most of the differentially expressed-miRNA target genes were related to regulation of transcription. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the most highly enriched pathways under drought stress were D-alanine metabolism, sulfur metabolism, and mineral absorption pathways. Real-time quantitative PCR (qPCR) was used to validate the expression patterns of 21 miRNAs (2 up-regulated and 19 down-regulated under drought stress). The observed co-regulation of the miR166 family and their targets ATHB-14-like and ATHB-15-like indicate the presence of negative feedback regulation in miRNA pathways. Analyses of drought-responsive miRNAs in tea plants showed that most of differentially expressed-miRNA target genes were related to regulation of transcription. The results of study revealed that the expressions of phase-specific miRNAs vary with morphological, physiological, and

  13. Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

    Science.gov (United States)

    de Vega-Bartol, José J; Simões, Marta; Lorenz, W Walter; Rodrigues, Andreia S; Alba, Rob; Dean, Jeffrey F D; Miguel, Célia M

    2013-08-30

    It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in

  14. The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

    Directory of Open Access Journals (Sweden)

    Hongtao Hu

    of a second round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.

  15. Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis.

    Science.gov (United States)

    Chen, Jingli; Zheng, Yi; Qin, Li; Wang, Yan; Chen, Lifei; He, Yanjun; Fei, Zhangjun; Lu, Gang

    2016-04-12

    MicroRNAs (miRNAs), a class of non-coding small RNAs (sRNAs), regulate various biological processes. Although miRNAs have been identified and characterized in several plant species, miRNAs in Asparagus officinalis have not been reported. As a dioecious plant with homomorphic sex chromosomes, asparagus is regarded as an important model system for studying mechanisms of plant sex determination. Two independent sRNA libraries from male and female asparagus plants were sequenced with Illumina sequencing, thereby generating 4.13 and 5.88 million final clean reads, respectively. Both libraries predominantly contained 24-nt sRNAs, followed by 21-nt sRNAs. Further analysis identified 154 conserved miRNAs, which belong to 26 families, and 39 novel miRNA candidates seemed to be specific to asparagus. Comparative profiling revealed that 63 miRNAs exhibited significant differential expression between male and female plants, which was confirmed by real-time quantitative PCR analysis. Among them, 37 miRNAs were significantly up-regulated in the female library, whereas the others were preferentially expressed in the male library. Furthermore, 40 target mRNAs representing 44 conserved and seven novel miRNAs were identified in asparagus through high-throughput degradome sequencing. Functional annotation showed that these target mRNAs were involved in a wide range of developmental and metabolic processes. We identified a large set of conserved and specific miRNAs and compared their expression levels between male and female asparagus plants. Several asparagus miRNAs, which belong to the miR159, miR167, and miR172 families involved in reproductive organ development, were differentially expressed between male and female plants, as well as during flower development. Consistently, several predicted targets of asparagus miRNAs were associated with floral organ development. These findings suggest the potential roles of miRNAs in sex determination and reproductive developmental processes in

  16. The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

    Science.gov (United States)

    Lloyd, Chelsea R; Park, Seongjin; Fei, Jingyi; Vanderpool, Carin K

    2017-06-01

    The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria. IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic

  17. Diversity of cultivable vaginal microbiota in asymptomatic women of reproductive age in Mumbai, India

    Directory of Open Access Journals (Sweden)

    Rinku Pramanick

    2017-10-01

    Full Text Available Microbes in the vaginal microbiota form a mutual relation with its constituent members and its host. In recent years our acquaintance with vaginal microbiota has widened, however, insufficient knowledge is available in Indian scenario. In the present study, the diversity of cultivable vaginal microbiota in asymptomatic women of the reproductive age group from Mumbai was investigated using multiplex PCR and species specific PCR, validated by 16sRNA Sanger sequencing. Vaginal samples taken from 199 women were classified according to Nugent score as normal (n=147, intermediate (n=23 and bacterial vaginosis (n=29 indicating 14.5% asymptomatic BV. Cultivable Lactobacilli were recovered from 97.9% (195 participants. The abundance of vaginal Lactobacilli was reduced in women with BV. Of 147 women, 110 were considered healthy, as 37 women colonized vaginal Candida. The most predominant vaginal Lactobacillus spp. in healthy women were L. iners (70.9%, L. crispatus (26.4%, L. reuteri (20.9%, L. gasseri (18.2%, and L. jensenii (15.5%. Our data demonstrated a profound shift in the prevalent vaginal Lactobacillus spp. when comparing women with healthy and diseased conditions. In women with normal flora colonizing Candida, L. rhamnosus (24.3% was one of the prevalent Lactobacilli. L. crispatus was identified as a specific species present only in the healthy state. L. iners was fund to be the most frequent vaginal Lactobacillus irrespective of the vaginal health. Majority of the women harbored heterogeneous population of Lactobacillus indicating their cumulative effect in maintaining the vaginal niche. Among the single species population, distinct diversity of Lactobacilli were found in women with Normal, Intermediate and BV microflora. Though most frequently identified, L. iners, significantly coexisted with other Lactobacillus spp., suggesting its minimal protective role alone in the vaginal niche. About one third of study population colonized Candida, most

  18. The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

    Science.gov (United States)

    Hu, Hongtao; Rashotte, Aaron M; Singh, Narendra K; Weaver, David B; Goertzen, Leslie R; Singh, Shree R; Locy, Robert D

    2015-01-01

    round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.

  19. Genomic Targets and Features of BarA-UvrY (-SirA Signal Transduction Systems.

    Directory of Open Access Journals (Sweden)

    Tesfalem R Zere

    Full Text Available The two-component signal transduction system BarA-UvrY of Escherichia coli and its orthologs globally regulate metabolism, motility, biofilm formation, stress resistance, virulence of pathogens and quorum sensing by activating the transcription of genes for regulatory sRNAs, e.g. CsrB and CsrC in E. coli. These sRNAs act by sequestering the RNA binding protein CsrA (RsmA away from lower affinity mRNA targets. In this study, we used ChIP-exo to identify, at single nucleotide resolution, genomic sites for UvrY (SirA binding in E. coli and Salmonella enterica. The csrB and csrC genes were the strongest targets of crosslinking, which required UvrY phosphorylation by the BarA sensor kinase. Crosslinking occurred at two sites, an inverted repeat sequence far upstream of the promoter and a site near the -35 sequence. DNAse I footprinting revealed specific binding of UvrY in vitro only to the upstream site, indicative of additional binding requirements and/or indirect binding to the downstream site. Additional genes, including cspA, encoding the cold-shock RNA-binding protein CspA, showed weaker crosslinking and modest or negligible regulation by UvrY. We conclude that the global effects of UvrY/SirA on gene expression are primarily mediated by activating csrB and csrC transcription. We also used in vivo crosslinking and other experimental approaches to reveal new features of csrB/csrC regulation by the DeaD and SrmB RNA helicases, IHF, ppGpp and DksA. Finally, the phylogenetic distribution of BarA-UvrY was analyzed and found to be uniquely characteristic of γ-Proteobacteria and strongly anti-correlated with fliW, which encodes a protein that binds to CsrA and antagonizes its activity in Bacillus subtilis. We propose that BarA-UvrY and orthologous TCS transcribe sRNA antagonists of CsrA throughout the γ-Proteobacteria, but rarely or never perform this function in other species.

  20. Rock stability considerations for siting and constructing a KBS-3 repository. Based on experiences from Aespoe HRL, AECL's URL, tunnelling and mining

    Energy Technology Data Exchange (ETDEWEB)

    Martin, C.D. [Univ. of Alberta, Edmonton (Canada); Christiansson, Rolf [Swedish Nuclear Fuel and Waste Management Co., Stockholm (Sweden); Soederhaell, J. [VBB VIAK AB, Stockholm (Sweden)

    2001-12-01

    mapping of the borehole walls. Two major tasks must be accomplished during this period: 1. an assessment of the quality of the rock mass and 2. an assessment of the state of stress within the volume of rock containing the repository. Empirical methods such as the Q system can be used to establish the domains of rock mass quality and to assess tunnel support requirements during the preliminary design phase. The laboratory testing should be carried out to determine the crack initiation stress, the long-term strength, peak strength and post-peak response. The determination of these parameters should be determined from stress-strain data, as well acoustic emission testing techniques, using testing methods based on accepted national standards, such as the ISRM suggested methods or ASTM. The in-situ stress state must be measured with confidence. The number of measurements and the method(s) used will be a function of the geology of the site. Practical experience indicates that stress-induced failure (spalling) will occur on the boundary of an underground opening in hard rocks when the maximum tangential stresses on the boundary of the opening exceed approximately 0.3 to 0.4 of the laboratory uniaxial compressive strength. Hence to assess the potential for spalling, numerical analysis will be required for the various shaped openings planned for the repository. These numerical analysis can be used to optimize the shape of the tunnels, the orientation of the tunnels relative to the far-field stress state, intersection support, and deposition tunnel/borehole spacing. The support for the tunnels in a repository is expected to range from light support pressure equivalent to standard spot-bolting to local bolts with mesh and fibre-reinforced shotcrete. At major intersections medium to heavy support pressure may be required. The layout of a repository will be similar to a mine using a room-and-pillar mining method but the extraction ratio will be of the order of <30%. A drill

  1. Rock stability considerations for siting and constructing a KBS-3 repository. Based on experiences from Aespoe HRL, AECL's URL, tunnelling and mining

    International Nuclear Information System (INIS)

    Martin, C.D.; Christiansson, Rolf; Soederhaell, J.

    2001-12-01

    borehole walls. Two major tasks must be accomplished during this period: 1. an assessment of the quality of the rock mass and 2. an assessment of the state of stress within the volume of rock containing the repository. Empirical methods such as the Q system can be used to establish the domains of rock mass quality and to assess tunnel support requirements during the preliminary design phase. The laboratory testing should be carried out to determine the crack initiation stress, the long-term strength, peak strength and post-peak response. The determination of these parameters should be determined from stress-strain data, as well acoustic emission testing techniques, using testing methods based on accepted national standards, such as the ISRM suggested methods or ASTM. The in-situ stress state must be measured with confidence. The number of measurements and the method(s) used will be a function of the geology of the site. Practical experience indicates that stress-induced failure (spalling) will occur on the boundary of an underground opening in hard rocks when the maximum tangential stresses on the boundary of the opening exceed approximately 0.3 to 0.4 of the laboratory uniaxial compressive strength. Hence to assess the potential for spalling, numerical analysis will be required for the various shaped openings planned for the repository. These numerical analysis can be used to optimize the shape of the tunnels, the orientation of the tunnels relative to the far-field stress state, intersection support, and deposition tunnel/borehole spacing. The support for the tunnels in a repository is expected to range from light support pressure equivalent to standard spot-bolting to local bolts with mesh and fibre-reinforced shotcrete. At major intersections medium to heavy support pressure may be required. The layout of a repository will be similar to a mine using a room-and-pillar mining method but the extraction ratio will be of the order of <30%. A drill-and-blast excavation

  2. Conceptual model of fractured aquifer of Uranium Deposit in Caetité, Bahia: implications for groundwater flow; Modelo conceitual do aquífero fraturado da área da jazida de urânio de Caetité, Bahia: implicações para o fluxo subterrâneo

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Liliane Ferreira da

    2015-07-01

    The studied area is represented by the uraniferous district of Lagoa Real, located in the center-south of Bahia State, Brazil. The region is set in a semiarid climate context, with hot and dry weather parameters, with hydric deficit along all months of the year and high aridity index. Rural population is affected on drought periods since small agriculture and animal rearing are the main economic activities which are vulnerable in dry seasons. Groundwater represents the main supply source considering that most surface water sources are temporary and only exhibit flow in rainy periods. The main aquifer system present on the region is fractured, and the presence of groundwater flow occurs through the discontinuities of the rock considering that the rock mass corresponds to the set formed by the rock matrix and all its discontinuities (fractures, foliations, discordance, etc). In this sense, the main purpose of this Master Dissertation was to develop a conceptual model for the aquifer system, through the geotechnical characterization of discontinuities, once these structures allow the secondary porosity of the medium. Hydrochemical data hand out as complement for physical characterization for the behavioral interpretation of the aquifer. The aquifer system is unconfined, however, presents points of stagnation of flow forming compartments without communication with the surrounding areas. According to the International Society of Rock Mechanics ISRM method, which consist on qualitative and quantitative characterization of discontinuities of rock mass scanlines were constructed, systematically, describing, the following structure parameters: attitude, spacing, persistence, openness, infilling and roughness. From the results analysis it could be concluded that the aquifer system is composed of three discontinuities sets: one set which dips to NE, second set dipping to SW-W-NW and the last set sub-horizontal. The first and second sets are responsible for the aquifer

  3. Pegasus Workflow Management System: Helping Applications From Earth and Space

    Science.gov (United States)

    Mehta, G.; Deelman, E.; Vahi, K.; Silva, F.

    2010-12-01

    . Astrophysics: The Laser Interferometer Gravitational-Wave Observatory (LIGO) uses Pegasus WMS to search for binary inspiral gravitational waves. A month of LIGO data requires many thousands of jobs, running for days on hundreds of CPUs on the LIGO Data Grid (LDG) and Open Science Grid (OSG). Ocean Temperature Forecast: Researchers at the Jet Propulsion Laboratory are exploring Pegasus WMS to run ocean forecast ensembles of the California coastal region. These models produce a number of daily forecasts for water temperature, salinity, and other measures. Helioseismology: The Solar Dynamics Observatory (SDO) is NASA's most important solar physics mission of this coming decade. Pegasus WMS is being used to analyze the data from SDO, which will be predominantly used to learn about solar magnetic activity and to probe the internal structure and dynamics of the Sun with helioseismology. Bacterial RNA studies: SIPHT is an application in bacterial genomics, which predicts sRNA (small non-coding RNAs)-encoding genes in bacteria. This project currently provides a web-based interface using Pegasus WMS at the backend to facilitate large-scale execution of the workflows on varied resources and provide better notifications of task/workflow completion.

  4. Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli.

    Science.gov (United States)

    Pannuri, Archana; Vakulskas, Christopher A; Zere, Tesfalem; McGibbon, Louise C; Edwards, Adrianne N; Georgellis, Dimitris; Babitzke, Paul; Romeo, Tony

    2016-11-01

    -affinity mRNA targets, thus eliciting major shifts in the bacterial lifestyle. CsrB/C transcription and turnover are activated by carbon metabolism products (e.g., formate and acetate) and by a preferred carbon source (glucose), respectively. We show that cAMP-CRP, a mediator of classical catabolite repression, inhibits csrC transcription by binding to the upstream region of this gene and also inhibits csrB transcription, apparently indirectly. We propose that glucose availability activates pathways for both synthesis and turnover of CsrB/C, thus shaping the dynamics of global signaling in response to the nutritional environment by poising CsrB/C sRNA levels for rapid response. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. RsmV a small non-coding regulatory RNA in Pseudomonas aeruginosa that sequesters RsmA and RsmF from target mRNAs.

    Science.gov (United States)

    Janssen, Kayley H; Diaz, Manisha R; Gode, Cindy J; Wolfgang, Matthew C; Yahr, Timothy L

    2018-06-04

    The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm post-transcriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the post-transcriptional level. Previous work found that RsmA activity is controlled by at least three small, non-coding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in-silico approach to identify additional sRNAs that might function in the sequestration of RsmA and/or RsmF and identified RsmV, a 192 nt transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1 , a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contribute to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play distinct roles in controlling RsmA and RsmF activity. IMPORTANCE The CsrA/RsmA family of RNA-binding proteins play important roles in post-transcriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small non-coding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two Csr

  6. PREPARATION AND APPLICATIONS OF MONOCLONAL ANTIBODY AGAINST THE PATHOGENIC HARVEYI FROM PSEUDOSCIAENA CROCEA%大黄鱼病原哈维氏弧菌单克隆抗体的制备及其应用

    Institute of Scientific and Technical Information of China (English)

    郝贵杰; 沈锦玉; 徐洋; 姚嘉赟; 潘晓艺; 尹文林

    2009-01-01

    用甲醛灭活哈维氏弧菌(Vibrio harveyi)GYC1108-1制备免疫原免疫8周龄雌性BALB/c小鼠,利用淋巴细胞杂交瘤技术,用间接酶联免疫吸附试验(ELISA)对杂交瘤进行筛选,阳性克隆经2-3次亚克隆后,共获得5株针对GYC1108-1的单克隆抗体.选取1B7制备小鼠腹水抗体,其细胞上清及腹水效价分别为1∶3200和1∶32000.同样用灭活的哈维氏弧菌免疫新西兰大白兔,5次免疫后颈动脉采血,离心取血清,并用饱和硫酸铵法进行纯化,制备了兔抗哈维氏弧菌多克隆抗体,其ELISA效价为1∶51200.利用1B7单克隆抗体和兔抗哈维氏弧菌多克隆抗体以及山羊抗小鼠HRP酶标二抗,建立了检测哈维氏弧菌的三抗体夹心酶联免疫吸附试验(TAS-ELISA)方法.该方法对哈维氏弧菌的最小检出浓度为1×104个/mL.用该TAS-ELISA方法检测大黄鱼(Pseudosciaena crocea)样品,54尾患病大黄鱼中有45尾检出哈维氏弧菌,而14尾健康大黄鱼都没有检出哈维氏弧菌.由此可见,本试验建立的TAS-ELISA方法,可以用于患病大黄鱼哈维氏弧菌的快速诊断.%Vibrio harveyi is a kind of important pathogenic bacteria for seawater animals, such as prawn, crab, calm and ormer. Along with the development of seawater fish cage-culture in China, Vibrio harveyi also becomes a main cause of disease of cage-culture fish, including Lutianus erythopterus, Seriola dumerili , Epinephelus coioides and Pseudosciaena cro-cea. One pathogenic V. harveyi strain, GYC1108-1, which was isolated from diseased Pseudosciaena crocea in Zhejiang Province, 2003, was identified after morphological observation, biochemical characteristic analysis, 16sRNA and HSP60 gene sequence detection.

  7. Programa de Pós-Graduação em Geologia - Dissertações Defendidas 1999 - Mestrado - Instituto de Geociências - Universidade Federal do Rio de Janeiro

    Directory of Open Access Journals (Sweden)

    1999-01-01

    Full Text Available 105 Anuário do Instituto de Geociências - UFRJ Volume 22 / 1999 Nome: Luiz Carlos Rodrigues de Almeida Orientador: Eurípedes Do Amaral Vargas Jr. Título: Estudo Experimental Da Anisotropia De Resistência De Rochas Graníticas E Sua Aplicação A Processos de Desmontes Resumo: Nesta dissertação apresenta-se um estudo experimental da anisotropia de resistência mecânica de rochas graníticas e sua aplicação à processos de desmonte. Estas anisotropias estão relacionadas a três superfícies preferenciais de corte, usualmente adotadas na prática de desmontes controlados com o objetivo de otimizar a operação. A caracterização das anisotropias abordou uma das principais características envolvidas no processo de desmonte controlado: a energia necessária para se criar uma nova superfície segundo uma orientação pré-determinada. A avaliação dos resultados de ensaios indiretos de resistência à tração e tenacidade à fratura, realizados para dois grupos de amostras de rochas graníticas com texturas distintas, permitiu que algumas conclusões fossem obtidas. A determinação da resistência à tração através do método indireto sugerido pela I.S.R.M. (1981 não se mostrou adequada. Os resultados apresentaram acentuada variabilidade, o que inviabilizou qualquer possibilidade de caracterização de um comportamento anisotrópico. Por outro lado, a tenacidade à fratura das rochas se confirmou como a propriedade mecânica com maior influência no comportamento anisotrópico das rochas ensaiadas. Os resultados mostraram-se bastante conclusivos, apontando claramente um comportamento mecânico diferenciado, segundo cada uma das três superfícies de corte investigadas. Entre as duas metodologias empregadas neste estudo, para a caracterização da energia necessária para criação de uma nova superfície, a metodologia proposta pela I.S.R.M. (1994 para a determinação da tenacidade à fratura usando discos semelhantes aos adotados